Molecular Biology of B Cells Edited by
Tasuku Honjo Department of Medical Chemistry Kyoto University Faculty of Medicine Kyoto, Japan
Frederick W. Alt Howard Hughes Medical Institute The Center for Blood Research The Children’s Hospital, Boston, Massachusetts
Michael S. Neuberger MRC Laboratory of Molecular Biology Protein and Nucleic Acid Chemistry Division Cambridge, United Kingdom
AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Molecular Biology of B Cells
Elsevier Academic Press 84 Theobald’s Road, London WC1X 8RR, UK 525 B Street, Suite 1900, San Diego, California 92101-4495, USA Copyright © 2004, Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail:
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Contributors
Dr. Frederick W. Alt Howard Hughes Medical Institute, The Children’s Hospital, The Center for Blood Research, Boston, MA, USA
Dr. Adolfo Ferrando Department of Pediatrics, Children’s Hospital, DanaFarber Cancer Institute, Boston, MA, USA
Dr. Barbara Birshtein Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA
Dr. Martin F. Flajnik Department of Microbiology and Immunology, University of Maryland, Baltimore, MD, USA
Dr. Constantin A. Bona Department of Microbiology, The Mount Sinai School of Medicine, New York, NY, USA
Dr. Raif S. Geha Department of Pediatrics, Harvard Medical School, Boston, MA, USA
Dr. Francisco Bonilla Division of Immunology, Children’s Hospital, Boston, MA, USA
Dr. Deborah L Hardie Medical Research Council Centre for Immune Regulation, The University of Birmingham Medical School, Birmingham, England, UK
Dr. Per Brandtzaeg Laboratory of Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway
Dr. Linda Hendershot Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN, USA
Dr. Marianne Bruggemann Laboratory of Developmental Immunology, The Babraham Institute, Babraham Hall, Babraham, Cambridge, UK
Dr. Tasuku Honjo Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto, Japan
Dr. Peter Burrows Department of Microbiology, University of Alabama at Birmingham, 383 Wallace Tumor Institute, Birmingham, USA
Dr. Ellen Hsu Department of Physiology & Pharmacology, The State University of New York Health Science Center at Brooklyn, Brooklyn, NY, USA
Dr. Kathryn Calame Departments of Microbiology and Biochemistry & Molecular Biophysics, Columbia Unversity, Collge of Physicians and Surgeons, New York, NY, USA
Dr. John F. Kearney Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, 6th Avenue South, Birmingham, AL, USA
Dr. Michael C. Carroll Department of Pediatrics, Harvard Medical School, The Center for Blood Research, Boston MA, USA
Dr. Paul W. Kincade Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
Dr. Michel Cogné Laboratoire d’Immunologie, Faculte de Medecine, Limoges Cedex, France
Dr. Kazuo Kinoshita Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, Japan
Dr. Max D. Cooper Howard Hughes Medical Institute, The University of Alabama at Birmingham, 378 Wallace Tumor Institute, Birmingham AL, USA
Dr. Katherine L. Knight Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, IL, USA
Dr. Jason Cyster Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, U.S.A.
Dr. Michael Krangel Department of Immunology, Duke University Medical Center, Jones Bldg, Research Drive, Durham, NC, USA
Dr. Nadia Danilova Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
Dr. Michael E. Lamm Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA
Dr. Randall S. Davis Divisions of Developmental and Clinical Immunology and Hematology/Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Dr. Dennis Lanning Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, IL, USA
Dr. Douglas T. Fearon Department of Medicine, University of Cambridge School of Clinical Medicine_Addenbrookes Hospital, Cambridge, UK
Dr. Tucker W. LeBien University of Minnesota Cancer Center, Minneapolis, MN, USA
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Contributors
Dr. Gerard Lefranc Laboratoire d’Immunogenetique Moleculaire, Institut de Genetique Humaine, Universite Montpellier II, Montpellier Cedex 5, France
Dr. Michael Reth Department of Molecular Immunology, Faculty of Biology III, University of Freiburg and Max Planck Institute for Immunobiology, Freiburg, Germany
Dr. Marie-Paul Lefranc Laboratoire d’Immuno Genetique Moleculaire, LIGM, Universite Montpellier II, UPR CNRS Institut de Genetique Humaine, Montpellier Cedex 5, France
Dr. Roy Riblet Torrey Pines Institute for Molecular Studies, San Diego, CA, USA
Dr. Susanna Lewis Genetics and Genomic Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada Dr. Gary W. Litman Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, FL, USA Dr. A. Thomas Look Department of Pediatrics, Children’s Hospital, DanaFarber Cancer Institute, Boston, MA, USA Dr. Ian C. M. MacLennan Medical Research Council Centre for Immune Regulation, The University of Birmingham Medical School, Birmingham, UK Dr. Nancy Maizels Departments of Immunology and Biochemistry, University of Washington Medical School, Seattle, WA, USA Dr. Roy Mariuzza Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, MD, USA Dr. Jim Marks Department of Anesthesia, San Francisco General Hospital, San Francisco, CA, USA Dr. Fumihiko Matsuda Centre National de Genotypage, Evry Cedex, France Dr. Fritz Melchers Deptartmen of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland Dr. Herbert C. Morse III Laboratory of Immunopathology, National Institutes of Health, Bethesda, MD, USA Dr. H. Craig Morton Laboratory of Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway Dr. Masamichi Muramatsu Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, Japan Dr. Lars Nitschke Institute of Virology and Immunobiology, Wuerzburg, Germany Dr. Marjorie A. Oettinger Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Dr. Barbara A. Osborne Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA, USA
Dr. Matthew Scharff Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Dr. Mark S. Schlissel Department of Molecular and Cellular Biology, Division of Immunology, University of California-Berkeley, Berkeley, CA, USA Dr. JoAnn Sekiguchi Howard Hughes Medical Institute, The Children’s Hospital, The Center for Blood Research, Boston, MA, USA Dr. Ranjan Sen Department of Biology, Brandeis University, Waltham, MA, USA Dr. Mark Shlomchik Section of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Dr. Robero Sitia Department of Molecular Pathology and Medicine, Universita Vita-Salute San Raffaele, DIBIT-HSR Scientific Institute, Milan, Italy Dr. Janet M. Stavnezer Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA, USA Dr. Lisa A. Steiner Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA Dr. Freda Stevenson Molecular Immunolgy Group, Tenovus Laboratory, Southampton University Hospitals Trust, Southampton, UK Dr. Eric Sundberg Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, MD, USA Dr. Naoya Tsurushita Protein Design Labs, Inc., Fremont, CA, USA Dr. Maximiliano Vásquez Protein Design Labs, Inc., Fremont, CA, USA Dr. Ulrich H. von Andrian The Center for Blood Research and the Department of Pathology, Harvard Medical School, Boston, MA, USA Dr. Urich H. von Andrian Department of Pathology, The Center for Blood Research, Boston, MA, USA Dr. Gregory W. Warr Department of Biochemistry, and Center for Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC, USA Dr. Jurgen Wienands Department of Biochemistry & Molecular Immunology, Universität Bielefeld, Abteilung Biochemie I, Bielefeld, Germany Dr. Catherine Willett Phylonix Pharmaceuticals, Inc., USA
Dr. Andreas Radbruch Deutsches Rheumaforschungszentrum Berlin, 10117 Berlin, Germany
Dr. Gillean Wu Dean, Faculty of Pure and Applied Science, York University, Toronto, Ontario, Canada
Dr. Klaus Rajewsky Harvard Medical School, Center for Blood Research, 200, Longwood Avenue, Boston, MA, USA
Dr. Hans G. Zachau Adolf-Butenandt-Institut Molekularbiologie, Muenchen, Germany
Dr. Jeffrey V. Ravetch Laboratory of Molecualr Genetics and Immunology, The Rockefeller University, New York, NY, USA
Dr. Zhixin Zhang Howard Hughes Medical Institute, University of Alabama at Birmingham, Birmingham, AL, USA
Contents
Preface
5. The Mechanisms of V(D)J Recombination
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JOANN SEKIGUCHI, FREDERICK W. ALT, AND MARJORIE OETTINGER
1. Human Immunoglobulin Heavy Chain Locus
Antigen Receptor Gene Assembly 62 Mutational Analyses of Recombination Signal Sequences 64 “Beyond 12/23” Restriction of V(D)J Rearrangements 64 Influence of Coding Flanks 65 The Biochemistry of V(D)J Cleavage 65 RAG1/2-RSS Binding 66 RAAG1/2 Post-Cleavage Complex 67 A Role for HMG1 (or HMG2) in V(D)J Recombination 67 A Closer Look at RAAG1 and RAG2 68 Colding and Signal Joint Formation Requires the NHEJ Pathway 71
FUMIHIKO MATSUDA
Organization of the Human VH Locus 2 Analysis of Human VH Segments 7 Evolution of the Human VH Locus 10 Human CH Locus 12
2. Immunoglobulin Heavy Chain Genes of Mouse ROY RIBLET
Igh-V or VH Genes of the Ighb Haplotype Polymorphism in VH Genes 20 Evolution 24 Genomic Considerations 24
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6. Transcription of Immunoglobulin Genes KATHRYN CALAME AND RANJAN SEN
3. Immunoglobulin K Genes of Human and Mouse
Transcriptional Regulatory Elements in Immunoglobulin Heavy and Light Chain Genes 83 Proteins Binding in Ig Transcriptional Regulatory Elements 86 Areas of Current Research 89 Discoveries Resulting from the Study of Ig Gene Transcription 93
HANS G. ZACHAU
General Features of Human and Mouse K Genes Human Immunoglobulin K Genes 27 Mouse Immunoglobulin K Genes 30 Aspects of Evolution of the K Genes 33
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4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
7. Early B Cell Development to a Mature, Antigen-Sensitive Cell
MARIE-PAULE LEFRANC AND GÉRARD LEFRANC
FRITZ MELCHERS AND PAUL KINCADE
IGL Genes and IMGT-ONCOLOGY 37 The Human IGL Genes 40 The Mouse IGL Genes 50
Three Waves of Hematopoiesis During Embryonic Development 101
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Pluripotent Hematopoietic Stem Cells 103 Pathways of Hematopoietic Progenitor Cells Toward B Lymphocyte Lineage Commitment and Differentiation 106 Control of Lymphoid Cell Development by Transcription Factors 107 Plasticity if PAX-5–Deficient Pre-B Cells 109 The Surrogate Light Chain 110 Pre-B Cells and Their Differentiation to More Mature B Lineage Cells 112 Rearrangements at the L Chain Loci at the Transition from Large to Small Pre-B-II Cells 114 Immature B Cells 116 Selections of Immature B Cells 117
8. Allelic Exclusion, Isotypic Exclusion, and the Developmental Regulation of V(D)J Recombination
10. Development and Function of B Cell Subsets JOHN T. KEARNEY
Selection and Differential Survival Mechanisms—B Cell Receptor Signaling 156 Compartmentalization of B Cell Subsets 157 Other Factors Involved in Formation of B Cell Subsets 157
11. Structure and Function of B Cell Antigen Receptor Complexes MICHAEL RETH AND JURGEN WIENANDS
Structure of the BCR Complex 161 Coupling Between the BCR and SYK 162 Redox Regulation of BCR Signaling 162 ITAM- and Non-ITAM-Controlled Signaling Pathways to SLP-65 163 ITAM-Independent Signaling and Fine-Tuning 165
MICHAEL S. KRANGEL AND MARK S. SCHLISSEL
Rag Expression 127 The 12/23 Rule 128 Accessibility Hypothesis 128 Enhancer and Promoter Control of V(D)J Recombination 128 Trans-Acting Factors 130 Chromatin Dynamics and V(D)J Recombination 130 Ordered Rearrangement Within Ig and TCR Loci 132 Allelic Exclusion at Ig and TCR Loci 133 Ig Light Chain Isotypic Exclusion 136 Future Directions 136
9. The Development of Human B Lymphocytes PETER D. BURROWS, TUCKER LEBIEN, ZHIXIN ZHANG, RANDALL S. DAVIS, AND MAX D. COOPER
Stages of Human B Cell Differentiation 141 Sites of Human B Cell Development 143 Human Immunoglobulin Genes 143 The Role of Surrogate Light Chains in Human B Cell Development 144 Repertoire Diversification via Receptor Editing and VH Replacement 145 Regulation of Antibody Production by B Cell Receptors 147 Immunodeficiency Diseases 148 B Lineage Leukemia 149
12. Regulation of Antigen Receptor Signaling by the Co-Receptors, CD19 and CD22 LARS NITSCHKE AND DOUGLAS T. FEARON
CD19 171 Inhibitory Co-Receptors on B Cells
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13. The Dynamic Structure of Antibody Responses IAN C. MACLENNAN AND DEBORAH L. HARDIE
Three Routes to Antibody Production 187 Stages of Adaptive Antibody Responses 187 How and Where B Cells Encounter Antigen 188 Primary Cognate Interaction of B Cells with Primed T Cells 189 Exponential Growth of Activated B Cells 190 Proliferation, Hypermutation, and Selection in GC 192 Sustained Survival of Memory B Cell Clones and Plasma Cells 197
14. Dynamics of B Cell Migration to and within Secondary Lymphoid Organs JASON G. CYSTER AND ULRICH H. VON ANDRIAN
Lymphoid Organ Entry 203 Compartmentalization of Mature B Cells 209 B Cells at Sites of Inflammation 213 Homing of Antibody Secreting Cells (ACSs) 213
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15. Characteristics of Mucosal B Cells with Emphasis on the Human Secretory Immune System PER BRANDTZAEG, H. CRAIG MORTON, AND MICHAEL E. LAMM
Immune-Inductive Tissue Compartments 223 Characteristics of B Cells in Secretory Effector Tissues 226 B-Cell Stimulation in MALT Structures 229 Class Switch and Ig A Isotype Promotion 232 Mechanisms Directing Homing and Retention of Mucosal B Cells 234 What Is Actually Known About Human Mucosal B Cells? 238
16. The Cellular Basis of B Cell Memory KLAUS RAJEWSKY AND ANDREAS RADBRUCH
Generation of B Cell memory and Memory B Cells in T Cell-Dependent Antibody Responses 247 Memory Plasma Cells 252 Adaptive B Cell Memory 254
17. Immunoglobulin Assembly and Secretion
19. Regulation of Class Switch Recombination MICHEL COGNÉ AND BARBARA K. BIRSHTEIN
CSR Requires Specific Stimuli Occurring in a Defined Germinal Center (GC) Microenvironment 289 Proximal CIS Regulatory Elements for GT 291 Distant Regulatory Region for GT and CSR: The 3¢ IGH Enhancers 293 Mechanisms for 3¢ IGH Regulatory Region-Mediated Regulation of GT 295 Coordinated Regulation of Transcription, Recombination, and Replication 300
20. Molecular Mechanisms of Class Switch Recombination JANET STAVNEZER, KAZUO KINOSHITA, MASAMICHI MURAMATSU, AND TASUKU HONJO
Outline of Mechanisms for CSR 307 Isotype Specificity of CSR 312 AID, The Sole B Cell-Specific Factor Required for CSR 313 Cleavage of the S Region 314 Processing and Joining of DNA Ends After Cleavage 315 Comparison of CSR with SHM 319
LINDA M. HENDERSHOT AND ROBERTO SITIA
Mechanisms of IG Synthesis and Assembly 261 Multiple Layers of Quality Control Exist to Aid and Monitor the Assembly of Functional IGs 264 Transport of Assembled IG Molecules to the Golgi 267 Degradation of Misfolded and Unassembled IG Subunits 267 Differentiation to Plasma Cell 268
18. Fc and Complement Responses JEFFREY V. RAVETCH AND MICHAEL C. CARROLL
Consequences of FCgRIIB Deficiency 275 Consequences of Complement and Complement Receptor Deficiencies 276 Fc Receptors 2276 Complement Receptors 280 Co-Receptor Signaling Versus Antigen Localization to FDC 281 Frontiers: Complement Versus Fc Receptors 285
21. Molecular Mechanisms of Hypermutation NANCY MAIZELS AND MATTHEW D. SCHARFF
Characteristics of Somatic Hypermutation of Immunoglobulin Variable Regions 327 Activation and Targeting of Hypermutation by Transcription and CIS-Elements 329 Hypermutation Occurs Within a Limited Window of B Cell development 330 The AID Gene Is Critical for Hypermutation 331 Phase One of Hypermutation: C Æ U Deamination and Base Excision Repair 332 Mismatch Repair Factors in Phase Two of Hypermutation 332 DNA Breaks in Hypermutation 334 Competing Pathways of Repair: Error-Prone DNA Synthesis or Strand Transfer 335 Evolution and Hypermutation 335
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22. Selection During Antigen-Driven B Cell Immune Responses: The Basis for High Affinity Antibody MARK J. SHLOMCHIK
Overview of the B-Cell Immune Response 339 Affinity Maturation in the Early Stages of the B-Cell Immune Response 341 The CG Is a Second Major Site for Affinity Maturation 342 Affinity-Based Selection Continues After the GC Reaction Has Ended 344 An Integrated View of the Strategic Design of the B-Cell Immune Response: Future Directions 344
23. Chromosomal Translocations in B Cell Leukemias and Lymphomas A. THOMAS LOOK AND ADOLFO FERRANDO
Translocations Associated with a Block in Lymphoid Differentiation 349 Translocations Associated with Suppression of Apoptosis During Lymphoid Development 352 Translocations Associated with Increased Proliferation in Lymphoid Precursors 354 Activation Cell Cycle Regulation in Mantle Cell Lymphoma and Myeloma 355
24. Classification and Characteristics of Mouse B Cell–Lineage Lymphomas
26. Immunodeficiencies Caused by B Cell Defects FRANCISCO A. BONILLA AND RAIF S. GEHA
Clinical Features of the Agammaglobulinemias 403 Autosomal Recessive Hyper-IGM Syndrome 410 Murine Models of Human B-Cell Deficiency 411
27. Diverse Forms of Immunoglobulin Genes in Lower Vertebrates GARY W. LITMAN, MARTIN F. FLAJNIK, AND GREGORY W. WARR
Cartilaginous Fish: An Unusual Example of Gene Multiplicity 417 Bony Fish: IG Heavy Chain Genes Resemble IgM and IgD 419 Lobe-Finned Fish: A “Transitional” Arrangement of Recombining Elements 422 Fleshy-Finned Fish: An Ancient Origin for Isotype Diversity 422 Amphibians and Reptiles: The Possible Origins of Class Switching 422 Light Chain Genes: Diverse Structures and Organization in Lower Vertebrates 423 Transcriptional Control of IG Genes in Lower Vertebrates 425 A Unifying Hypothesis to Explain the Origins of the Adaptive Immune Receptor 427 Immune Molecules in Jawless Vertebrates 427 Protochordates: Different Contexts for Diversified B Regions 428
HERBERT C. MORSE III
Comparative Classification of Mouse and Human B Cell–Lineage Neoplasms 366 Characteristics of Mouse B Cell–Lineage Lymphomas 368 Pathogenesis 373
25. B Cells Producing Pathogenic Autoantibodies
28. Immunoglobulin Genes and Generation of Antibody Repertoires in Higher Vertebrates: A Key Role for GALT DENNIS LANNING, BARBARA A. OSBORNE, AND KATHERINE L. KNIGHT
Avians 433 Lagomorphs 436 Artiodactyls 440 Other Mammals 444
CONSTANTIN A. BONA AND FREDA K. STEVENSON
Subsets of Autoantibodies 382 Criteria to Define Pathogenic Autoantibodies 383 Genetics of Autoantibodies 384 Molecular and Immunochemical Characteristics of Human Pathogenic Autoantibodies 388 Human Pathogenic Autoantibodies with Murine Counterparts 392
29. The Zebrafish Immune System LISA A. STEINER, CATHERINE E. WILLETT, AND NADIA DANILOVA
Hematopoiesis 450 Adaptive Immunity in Zebrafish: Organs and Molecules 452
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Genetic Approaches 457 Major Histocompatibility Complex (MHC) Innate Immunity 460 Infection 464
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30. The Origin of V(D)J Diversification SUSANNA M. LEWIS, GILLIAN E. WU, AND ELLEN HSU
The Alien Seed 473 The Evolution of BCR and TCR Loci 481 Considerations on the UR-V Gene 483
Antibody Phage Display 516 Use of Phage Display to Bypass Hybridoma Technology 519 Use of Phage Display to Bypass Immunization 520 A Comparison of Different Phage Antibody Library Types and Applications 521 Strategies for Selection of Phage Antibodies 522 Increasing Antibody Affinity Using Phage Display 522 Alternative Antibody Display Technologies 524
33. Humanization of Monoclonal Antibodies NAOYA TSURUSHITA AND MAXIMILIANO VASQUEZ
31. Antibody Structure and Recognition of Antigen ERIC J. SUNDBERG AND ROY A. MARIUZZA
A Structural Framework for Molecular Recognition 491
Murine, Chimeric, and Humanized Antibodies 533 Computer-Guided Design of Humanized V Regions 534 Other Humanization Methods 537 Immunogenicity of Humanized Antibodies 540 Humanized Antibodies Approved for Clinical Use 540
32. Monoclonal Antibodies from Display Libraries
34. Human Monoclonal Antibodies from Translocus Mice
JIM MARKS
MARIANNE BRÜGGEMANN
Overview of Antibody Phage Display 513 Prokaryotic Expression of Antibody Fragments 514 Generation of Antibody Gene Repertoires Using the Polymerase Chain Reaction 515
Human IG Transloci 547 The Mouse Strains 552 Index 563
Preface
Studies of immunoglobulin (Ig) genes have been one of the major focal points in modern biology. The apparently unique property of Ig gene loci, and their related T cell receptor loci, to be somatically assembled from germline gene segments has long fascinated biologists. Moreover, studies of the mechanisms by which Ig genes are expressed, and how this relates to the development of B lymphocytes, have contributed much to our understanding of fundamental genetic and cellular processes, ranging from transcription, differential RNA processing, site- and region-specific recombination, general DNA repair, and cellular signaling mechanisms. Because of the unique insights and wealth of instructive materials that derived from these studies, 14 years ago we decided to cover the field and its advances in a book on Immunoglobulin Genes. Eight years ago, the second edition of Immunoglobulin Genes continued to track this progress, and now we have continued and expanded this coverage in the third edition of Immunoglobulin Genes, which we have entitled Molecular Biology of B Cells. The change in title reflects the increased scope of the current volume, which covers the elucidation of the intimate links between Ig genes and many of the fundamental processes involved in generating and affecting the humoral arm of the immune response. The first edition of Immunoglobulin Genes appeared in 1989. At that time, the advent of DNA cloning and molecular biology had allowed a relatively full elucidation of the dynamic mechanisms involved in the somatic assembly of Ig variable (V) region gene segments. We knew then, at least in general terms, about some of the basic aspects of the V(D)J recombination, IgH class switch recombination (CSR), and somatic hypermutation (SHM) processes that form the fundamental basis for the diverse humoral immune response. In particular, molecular genetic approaches had provided an enormous amount of information on the structure, organization, assembly, and expression of Ig genes in a variety of organisms and had also provided the tools to investigate the general mechanisms by which Ig gene assem-
bly was tied to the developmental programs of B and T lymphocytes. Moreover, we had begun to get a glimpse of how the expression of Ig receptors and other molecules on the surface of particular types of B lineage cells was linked to aspects of B cell development and function. The second edition of Immunoglobulin Genes appeared in 1995. Over the intervening six years, tremendous progress had been made on several fronts. Substantial organizational information had been obtained with respect to the IgH and Igk loci in humans and mice. In fact, the complete IgH V region locus had been isolated on overlapping cosmids and YACs. A huge breakthrough came from the identification of the developing lymphocyte-specific RAG gene products, which are the specific components of the site-specific VDJ recombinase required to assemble Ig and TCR variable region genes in B and T cells, respectively. The availability of the RAG products allowed dissection of the VDJ recombination mechanism in detail and also facilitated identification of the generally expressed nonhomologous DNA end-joining proteins, which are co-opted by the VDJ reaction to complete the joining phase of V(D)J recombination. Gene targeted mutational studies had also begun to be employed to test the function of Ig genes and some of their regulatory sequences as well as that of other molecules that function in developing and mature B lymphocytes. In the eight years that have passed since the publication of the second edition of Immunoglobulin Genes, we have witnessed remarkable progress on several fronts. First, as anticipated, the IgH and IgL loci have now been fully sequenced in humans and mice. In fact, the sequences of the entire genome of human, mouse, and many other organisms have now been obtained. Another formidable advance has been the elucidation of the basic mechanisms underlying CSR and SHM that are critical to generation of antigenspecific antibodies. The explosion of information on these processes was stimulated by the discovery of AID, an enzyme that is fundamental to both CSR and SHM, as well as to the gene conversion process that diversifies chicken Ig
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genes. Much like the impact that the discovery of RAG had on elucidating the VDJ recombination process, the discovery of AID has now allowed the generation of new insights into the mechanisms that work to effect SHM and CSR at the mechanistic level. In another five years, it is likely that our knowledge of these processes will advance to the level that we now understand VDJ recombination. As for VDJ recombination, we have learned that the RAGs likely evolved from elements of a transposase, a finding that along with other aspects Ig Gene organization has provided some notion about how the Ig gene assembly system may have evolved. Studies of AID and its relatives are beginning to provide similar insights into the evolution of the CSR and SHM processes. As anticipated, gene targeted mutational studies have continued to provide great insights into the function of Ig genes and the mechanisms that regulate their expression, and have also helped to elucidate the function of many other molecules involved in the differentiation of B cells and in their activation and effector functions. Exciting developments have also take place in the area of the cellular dynamics that regulate migration of B cells for participation in effector functions at specific locations of the body. Application of immunoglobulins to clinical fields, not only diagnosis but also therapy, and understanding of molecular basis of human B cell defects and malignancy has also witnessed remarkable advances. Still, there are major questions remaining to be solved at many levels and with respect to many processes. One that
has been particularly enigmatic is the molecular details of how accessibility regulation occurs at the level of the chromosome and chromatin structure. This clearly is a problem that is not restricted to B cells but bears on generally relevant control mechanisms. There are also many aspects of B cell physiology (such as the mucosal antibody response and the biology of memory B cells) that remain to be fully dissected by molecular approaches. However, the new technologies now available will hopefully allow substantial advances to be made in the study of all these processes before the next edition appears. The chapters of Molecular Biology of B Cells, as in previous editions, are written by authors who have very actively participated in the accumulation of knowledge in the area that they cover. Moreover, in this edition, we have tried something new; now most chapters are authored by two separate authorities on the subject covered. In this way, we have hoped to achieve the most balanced view of each individual field and, in some cases, to generate novel points of view from the cooperative efforts of authors with somewhat different viewpoints. As before, we continue to look forward to many more exciting developments in research on Ig genes and B lymphocyte development and function over the next five years. Tasuku Honjo Frederick Alt Michael Neuberger
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1 Human Immunoglobulin Heavy Chain Locus FUMIHIKO MATSUDA Centre National De Genotypage 2 Rue Gaston Cremieux, 91057 Evry Cedex France and Department of Genome Epidemiology, Kyoto University Graduate School of Medicine, Yoshida Sakyo-Ku, Kyoto 606 Japan
provides evidence to estimate the relative contribution of germline VH repertoire, VDJ recombination, somatic hypermutation, and subsequent selection of B lymphocytes to the Ig repertoire. Accumulating evidence indicates that the human VH locus is highly polymorphic. It is interesting to investigate the polymorphic variation of the number and repertoire of germline VH segments and CH genes and its association with disease susceptibility. It is known that some VH segments are overrepresented in the antibody repertoire, suggesting that the utilization of each VH segment may not be random. It is important to know whether the germline organization or structure of VH segments predicts such a preferential usage. Isolation of the total human VH segments has played important roles in the generation of human Ig in J segment–disrupted mice carrying human Ig mini loci (Taylor et al., 1994; Green et al., 1994; Mendez et al., 1997). Finally, from an evolutionary viewpoint, multigene families are considered to have evolved through repeated duplication and recombination of DNA. Diversification of newly generated VH members by such events contributes to the germline VH repertoire. Evolutionary studies of VH organization and structure and, in particular, comparison of VH loci between related species, will provide insight to the molecular mechanisms that govern the evolution of multigene families. Needless to say, studies on the complete organization of the CH locus are the basis for understanding the molecular mechanism for class switching and regulation of IgH expression (see the chapter by Honjo). Significant progress was made in the structural analysis of the human IgH locus between the first (1988) and second (1995) editions of this book by completion of the physical mapping of the human VH segments using YAC clones. Since then, with a rapid evolution of genome sequencing technology, the complete nucleotide sequence of the entire
The immunoglobulin (Ig) molecule is composed of heavy (H) and light (L) chains, both of which consist of variable (V) and constant (C) regions. The V region is responsible for antigen binding, whereas the CH region specifies the isotype of Ig. Genes encoding IgH V regions are split into VH, diversity (DH), and joining (JH) segments. One each of the three segments is generally assembled into a functional VH gene by a somatic genetic event called VDJ recombination. In Homo sapiens, VH region genes are mapped to chromosome 14 q32.33 (Croce et al., 1979; Kirsch et al., 1982). Recent completion of the nucleotide sequence of the 957kilobase (kb) DNA covering the entire human VH locus demonstrated that it consists of 123 copies of VH segments, 26 DH segments, and six JH segments. Conversely, the human CH locus comprises 11 CH genes, of which 2 are pseudogenes (Matsuda et al., 1998). The VH and CH loci are physically linked on the chromosome in the order of 5¢-VHDH-JH-CH-3¢. The distance between the 3¢-most VH locus segment (JH) and the 5¢-most CH gene (Cm) is approximately 8 kb in man (Ravetch et al., 1981). The 298-kb DNA of the entire human CH locus was also sequenced (Heilig et al., 2003; Nicodeme et al., submitted). Thus, the IgH locus, which combines the VH and CH loci, constitutes a huge multigene family, encompassing the 1.3-Mb DNA of the distal end of chromosome 14. The complete knowledge of the organization and structure of the IgH locus will provide clear answers to a number of questions essential to Ig repertoire formation and Ig expression. Obviously, the total number of VH segments determines the upper limit of the germline Ig repertoire, although somatic genetic events, including VDJ recombination, hypermutation, and gene conversion further tremendously amplify the expressed repertoire. Comparisons between the total germline and expressed VH sequences
Molecular Biology of B Cells
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Copyright 2004, Elsevier Science (USA). All rights reserved.
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Matsuda
human IgH locus is now available. This achievement provides an enormously beneficial reference to map expressed VH genes and their polymorphisms, as well as a detailed structure of the human CH locus.
ORGANIZATION OF THE HUMAN VH LOCUS Studies on the physical mapping of the human VH locus were initiated by cosmid cloning (Kodaira et al., 1986). The distribution of VH families on 23 cosmid clones has shown that members of different VH families are interspersed, in contrast to the finding that the same family members tend to cluster in the mouse VH locus (Kemp et al., 1981; Rechavi et al., 1982). Another important conclusion from early studies is the presence of abundant pseudogenes (about 40%), many of which are highly conserved, with only a few point mutations (Givol et al., 1981; Kodaira et al., 1986). The complete physical map was constructed for the 80-kb of DNA encompassing the 3¢-most V6-1 segment, DH cluster, and JH cluster (Matsuda et al., 1988; Buluwela et al., 1988; Buluwela and Rabbits, 1988; Sato et al., 1988; Schroeder et al., 1988). A more general overview of the whole human VH locus has been provided by studies using pulse field gel electrophoresis (PFG), which allowed examination of the VH content on a few hundred kb to 1,000 kb DNA fragments. The total size of the human VH locus was estimated to be about 2.5 to 3.0 Mb (Berman et al., 1988; Matsuda et al., 1988), including the D5-hybridizing fragments that later mapped to chromosome 15. PFG analysis using twodimensional electrophoresis has provided a more precise organization of the entire VH locus of about 1.2 Mb, on which 76 human VH segments were mapped (Walter et al., 1990). The same group further refined the mapping using the deletion profile of VH segments associated with VDJ recombination in human B cell lines (Walter et al., 1991a). Introduction of the yeast artificial chromosome (YAC) vector has been key to completing the physical mapping of the entire human VH locus (Figure 1.1). The first report using YAC cloning has identified and located five VH segments proximal to the DH and JH segments (Shin et al., 1991). These authors proposed to rename all the VH segments by the family number and the order from the 3¢ end of the VH locus. The nomenclature of VH segments was controversial not only because investigators named VH segments in their own way, but also because many expressed VH sequences containing somatic mutations could not be easily assigned as different VH segments. The newly proposed nomenclature defined VH segments only when they were mapped on the chromosome, which has been well accepted by the scientific community. The same group in Kyoto has completed the mapping of 64 VH segments in 0.8 Mb of the human VH locus
by analyzing more than seven overlapping YAC clones. The nucleotide sequences of all these VH segments were determined (Matsuda et al., 1993). The transcriptional orientation of 43 VH segments that are located either 5¢-most, middle, or 3¢-most part of the contig was determined. All had the same polarity as the JH segments, unlike in the human Vk locus, where the gross inversion of the distal duplicated copy of 360-kb DNA containing 59 Vk segments and relics is observed (Kawasaki et al., 2001, see Chapter 3). The physical mapping of the human VH locus was completed by identification of a YAC clone in human subtelomeric region–specific YAC libraries (Cook et al., 1994). A 200-kb clone that physically links to the upstream portion of the 0.8Mb contig was isolated and 17 novel VH segments were identified, suggesting that the total number of the VH segment is 81. The same region was cloned independently by the Kyoto group, confirming the telomeric end of the physical map by the Cambridge group. The last and biggest effort to complete the physical map was made by the Kyoto group. The complete nucleotide sequence of the 957,090-bp DNA upstream of the human JH cluster was determined (Matsuda et al., 1998). The 5¢-most part of the locus contains a diverged human telomere repeat and subtelomeric region, confirming the proximity of the VH locus to chromosome 14q telomere. A total of 123 VH segments and pseudogenes was identified in the 883-kb DNA between 73 and 956 kb upstream of the JH cluster. The highly interspersed organization of the VH segments belonging to seven different families was confirmed. Sixteen of the 17 distal VH segments were identified at the position proposed. However, the VH sequence corresponding to the V777 segment was not identified at the suggested position even though the physical maps of the corresponding portions are exactly identical. V3-82P, the 5¢-most VH segment, is located 1,480 bp downstream of the 5¢ terminus of the locus. The distances between neighboring VH segments are quite variable, with the largest being 41.4 kb (between V1-2 and V41.1P) and the smallest as little as 418 bp (between V3-67.2P and V4-67.1P). However, clustering of VH segments, as shown in the human Vl locus, was not evident (Frippiat et al., 1995; Kawasaki et al., 1997). The transcriptional polarities of all the VH segments are the same as that of the JH segments. Southern hybridization detects many DNA fragments in YACs and cosmids that weakly hybridize with VH probes, although such hybridization is not detectable against human genomic DNA, thus suggesting the presence of additional VH-related sequences including those of novel VH families. Nucleotide sequencing newly identified 43 such sequences. However, all these VH segments were classified as a member of seven known VH families, excluding the existence of novel VH families in humans. Interestingly, they all carry defects in their structure and are categorized as pseudogenes.
1. Human Immunoglobulin Heavy Chain Locus
3
FIGURE 1.1 Organization of the entire human IgH locus. Five thick horizontal lines show the 1.3-MB DNA with the 3¢ end at the bottom right corner. VH segments are indicated by vertical lines with their names (newly identified VH segments are shown with asterisk). VH segments containing truncations are shown by shorter vertical lines. DH segments and CH genes are indicated by diamonds and open boxes, respectively. Thirteen locus-specific repeats are indicated below by boxes of different pattern. Enhancers, predicted matrix attachment region, and nonimmunoglobulin genes are also shown with their names. Modified from Matsuda et al. (1998).
The Total Number of VH Segments One of the most important goals in the study of the human VH locus is to determine the total number of functional VH segments that can participate in functional heavychain formation. Some discrepancy was noted regarding the classification of VH segments into functional and pseudo-
genes, in part due to the incomplete nucleotide sequence of some VH segments. Given the complete nucleotide sequencing of the human VH locus, 123 VH segments were classified into four different categories based on the following criteria (Matsuda et al., 1998). The 79 VH segments without open reading frames (ORF) (due to various defects including frame shift and truncation) were classified as pseudogenes.
4
Matsuda
The other 44 VH segments with a complete ORF were further subdivided into “functional,” “transcribed,” or “ORF” group as follows:
that ancient truncation events were followed by gene duplication.
• The “functional” VH segments have an intact exonintron structure, a complete ORF, and no fatal defects in recombination signal sequences (RSS). In addition, their expression was confirmed by identification of the corresponding full-length VH mRNAs. • The “transcribed” VH segments correspond to those whose sequence identity with partial VH mRNA sequences have been identified. • The “ORF” segments consist of the VH segments with a complete ORF, yet not demonstrated to be transcribed (Table 1.1). Obviously, the direct proof of the functional VH segment is to identify its sequence in the IgH amino acid sequence.
Polymorphism of the Human VH Segments
Among 44 VH segments that have a complete ORF, 39 VH segments are translated and classified as functional. V428 is >97% identical to partial VH mRNA sequences in the database. However, its translation product remains to be identified and, hence, it was classified as “transcribed.” The remaining four genes, namely, V3-16, V3-35, V3-38, and V7-81, were categorized as “ORF” due to the absence of their transcripts in the database. Indeed, V3-38 has a truncation at the 5¢ untranslated region that results in the complete loss of its 5¢ regulatory region. Moreover, these four VH segments carry a diverged RSS heptamer sequence. They might not be employed for VDJ rearrangement. The 79 VH segments classified as pseudogenes were subdivided into 29 VH segments with point mutations and 50 with truncation(s) (Table 1). Indeed, none of these 79 VH segments corresponded to any VH mRNAs. Interestingly, 12 VH3 pseudogenes have the 5¢ truncation at the same position in their introns. Similarly, 13 VH4 pseudogenes contain the common 5¢ truncation in the second exon, suggesting
TABLE 1.1
RFLP and DNA sequencing have shown a number of polymorphic VH alleles. Among a variety of polymorphisms in the locus, insertion/deletion of VH segments and single nucleotide polymorphisms (SNPs) within the coding region are more likely to have functional significance. One obvious possibility is the expansion of repertoire. Polymorphic VH may affect the affinity of the antibody for its ligand, as even mutations in framework residues of the Ig have been shown to influence the binding affinity (Foote and Winter, 1992). Furthermore, expression of particular allelic variants could influence the efficiency of H-L chain pairing or interaction with B cell super-antigens. Variation of the germline VH segment copy number may associate with the preferential utilization and expression level of specific VH segments (Sasso et al., 1996). To date, three insertion/deletion polymorphisms have been mapped along the human VH locus. An insertional V74.1 segment is present between V2-5 and V4-4 in 65% and 72% of alleles among the Caucasian and Japanese populations, respectively. Another frequent insertion polymorphism of 50-kb DNA containing five functional VH segments was identified in the region between V3-31 and V3-30 by “HAPPY mapping” (Walter et al., 1993); this polymorphism is present in 73% of the Caucasian population. Interestingly, the insertion is located between a tandem homology pair of three VH segments, namely, V3-33/V3-32P/V4-31 and V330/V3-29P/V2-28, suggesting the highly recombinogenic nature of the region. A large insertion polymorphism of 80kb DNA, containing at least one each of VH2- and VH3family segments, was localized in the region between V2-70 and V1-67P by 2D-PFGE analysis (Walter et al., 1990). One
Summary of the human VH segments VH family
Chromosome
Classification
1
2
3
4
5
6
7
Total
14q32.33
Functional Transcribed ORF
9 0 0
3 0 0
19 0 3
6 1 0
1 0 0
1 0 0
0 0 1
39 1 4
Pseudogene Point mutator Truncation
3 2
1 0
21 22
2 23
0 1
0 0
2 2
29 50
14
4
65
32
2
1
5
123
15q11
Total
6
0
1
1
0
0
0
8
16p11
4
1
11
0
0
0
0
16
The number of VH segments on chromosome 14 is calculated by the results from Matsuda et al. (1998). VH segments with polymorphic insertion are not included. Information on VH segments on chrmonsome 15 and 16 is taken from Nagaoka et al. (1994) and Tomlinson et al. (1994).
1. Human Immunoglobulin Heavy Chain Locus
of the most polymorphic VH segment is V1-69, having 13 known alleles including duplication and deletion (Sasso et al., 1993); this segment is located very close to the region of the large polymorphic deletion. In addition, several VH segments mapped to chromosome 14 have not been located in the current map, suggesting the possibility of other deleterious polymorphisms. It is important to test whether VH polymorphisms are associated with disease susceptibility. However, this is a controversial area; some reports suggested the association of VH polymorphisms with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis (Yang et al., 1990; Walter et al., 1991b), whereas others failed to find a clear association (Hashimoto et al., 1993; Shin et al., 1993a). This might be due to the usage of only a limited number of VH segments or polymorphic markers in the analysis. To address this question, it would be essential to perform a large-scale genetic analysis of the entire VH locus. An SNP genotyping program is under way to determine the functional VH segments and the pseudogenes among a large number of DNA samples of multiple ethnic backgrounds (F. Matsuda unpublished). Preliminary results show that the content and frequency of SNPs differ largely between individual VH segments. The V1-69 segment has as many as eighteen alleles in ninety-six Japanese DNAs, a rather homogenous population, with different copy numbers among individuals, whereas no SNPs are detected in another VH segment. Other VH segments that have copy-number variation in the Caucasian population are V1-2, V3-23, V2-26, and V2-70. The systematic screening of VH locus haplotypes, generated by the combination of identified VH alleles against a large cohort of autoimmune and immune deficiency patients, will provide some ideas on possible associations.
Organization of the Human DH Segments The 70-kb region of the 3¢-most part of the human VH locus is occupied by DH and JH gene segments (Figure 1.1). The human JH cluster contains three pseudo JH segments interspersed among six functional JH segments (Ravetch et al., 1981). These are clustered approximately 8 kb upstream of Cm, the 5¢-most constant region gene. A human counterpart to the murine DQ52 segment is located about 100 bp upstream of the JH1 segment. Initially, a family of DH segments (D1-D4 or DLR1-DLR4) was identified, using as a probe an aberrantly rearranged DH-JH segment in a CLL cell clone (Siebenlist et al., 1981). Physical mapping studies showed that these are ordered at regular 9-kb intervals along the chromosome, suggesting the generation of the human DH cluster by gene duplication. However, the fact that these four segments corresponded to a smaller part of DH sequences in functional VDJ rearrangement raised a possibility of novel DH families in the genome. Later, a number of additional
5
human DH segments were identified, including ones homologous to the murine DFL16 segments as well as a number of those that are markedly dissimilar in size and sequence (Schroeder et al., 1987; Zong et al., 1988; Ichihara et al., 1988a, b; Buluwela et al., 1988; Sonntag et al., 1989; Shin et al., 1993b). Five novel DH families (DM, DXP, DA, DK, and DN) were identified in the order of 5¢-DM-D(LR)DXP-DA-DK-DN-3¢ by the nucleotide sequencing of a 15-kb DNA fragment covering the D(LR)1 segment and flanking regions (Ichihara et al., 1988b). Southern blot analysis strongly suggested the existence of a set of six DH segments in each copy of duplicated 9-kb DNA. An additional DH family (DIR) with unusual structure was identified in the 5¢ adjacent portion of DM family segments. The possible involvement of the DIR family in D-D rearrangement was pointed out because of its irregular spacer length (23 bp) of RSS. The definitive answer to the content of DH segments was given by the nucleotide sequencing analyses of the entire human DH locus (Corbett et al., 1997). A total of 26 DH segments was identified in four tandemly arrayed copies of the 9-kb DNA (Figure 1.1). These consist of five DM and DXP segments and four each of D(LR), DA, DK, and DN family gene segments. However, the number of DH segments shows allelic variation. One example is the polymorphic deletion of the 9-kb DNA containing the D(LR)1 segment, which occurs at a high frequency among the Japanese population (48% of alleles are D(LR)1 negative) (Zong et al., 1988). An extensive analysis of DH segment usage in rearranged heavychain sequences classified the 27 DH segments—including the unique DQ52 segment—into 25 functional and two pseudogenes (DM2 and DN3) (Corbett et al., 1997). The authors demonstrated the highly biased utilization of different DH segments and reading frames. In contrast, no evidence was obtained for the usage of DIR segments, inverted DH segments, or DD recombination in functional VDJ rearrangements.
VH and DH Segments on Chromosomes 15 and 16 Although the VH locus is located at the telomere end of chromosome 14q32, several VH and DH clusters remained unmapped for some time. The first evidence that a DH segment is located on chromosome 15 was obtained by in situ hybridization (Chung et al., 1984). Subsequently, studies using in situ hybridization, as well as human/rodent somatic hybrid cells (Cherif and Berger, 1990; Matsuda et al., 1990; Nagaoka et al., 1994; Tomlinson et al., 1994), identified two VH orphon loci on chromosome 15q11 and chromosome 16p11. Studies of cosmid and YAC clones derived from these orphon loci revealed that approximately 40% of VH segments in both loci (three out of seven VH on chromosome
6
Matsuda
16 and one out of three VH on chromosome 15) are apparently functional, without any structural defects, in the coding region as well as in RSS (Nagaoka et al., 1994) (Figure 1.2). A totally different approach, based on PCR and using somatic cell hybrid DNAs as templates, specifically amplified 24 VH segments, including 10 apparently functional ones, on chromosomes 15 and 16 (Tomlinson et al., 1994). Among them, 16 VH segments (including the above 7 VH segments) were mapped on chromosome 16, of which 14 make seven pairs of closely related VH sequences. The authors pointed out the possibility of intrachromosomal duplication of the DNA containing the seven VH segments. Taken together, the total number of VH segments on chromosome 16 would be sixteen, although the critical test for the duplication depends on their mapping along the chromosome. Florescence in situ hybridization mapped two independent contigs containing human DH segments (D5-a and D5b) to chromosome 15q11–12 (Nagaoka et al., 1994). Each consists of five DH segments in the order 5¢-DM5-D(LR)5DXP5-DA5-DK5–3¢, whereas the DN segment, the 3¢-most DH segment in the DH clusters on chromosome 14 is absent (Matsuda et al., 1990). Nucleotide sequence homology of the corresponding DH segments is much higher between D5a and D5-b clusters than between the D5 and any of the
D1–D4 clusters. One of the DH clusters (D5-b) is flanked by three VH segments. Interestingly, these three VH segments are located 3¢ to the D5-b cluster, and one of them (V13C) is apparently functional, having a complete ORF. The polarity of one of them (V3) had the same transcriptional orientation relative to DH (Matsuda et al., 1990). Quantitative hybridization estimated the copy number of D5 clusters to be at least four (Nagaoka et al., 1994). Chromosomespecific PCR amplification identified eight VH segments on chromosome 15 (Tomlinson et al., 1994). Nucleotide sequencing of both of these translocated VH loci is under way as a part of the human genome project. The future completion of the sequencing will provide us with definitive information regarding the number and organization of orphon VH and DH segments. Moreover, the evolutionary origin and mechanisms of translocation will be elucidated through comparative structural analysis between these loci and those on chromosome 14.
Nonimmunoglobulin Genes in the Human VH Locus Computer-assisted homology searches using the 957-kb DNA identified eight DNA sequences that are highly
D5-a
D5-b
M XP K LR A
M XP K LR A
V3 V54 V13C
Chr.15 82% 83%
95%
2-26 1-24P 3-22P 3-21 3-25P 3-23
77%
3-16P 1-14P 1-12P 3-15 3-13 3-11 1-17P
Chr.14 3-20
1-18 3-19P
95%
93%
96% 95%
93%
93%
95%
Chr.16 (VH-F) F2-26
0
F3-16P F3-15
50
100
F1-14P F3-13 F3-11 F1-12P 150
200(kb)
FIGURE 1.2 Comparison of VH segments on chromosomes 15 and 16 with their counterparts on chromosome 14. Corresponding VH segments are indicated with the percentage of homologies of coding and intron sequences. Neighboring VH segments of V1-18 were compared with V3 or V13C on the D5-b region (shown by dashed lines). Modified from Nagaoka et al. (1994).
7
1. Human Immunoglobulin Heavy Chain Locus
homologous to known DNA sequences in the databases. The 7883-bp cDNA of the KIAA0125 gene (Nagase et al., 1995) showed 99.8% identity to the DNA sequence between the V6-1 segment and the D gene cluster (Figure 1.1). This gene is encoded by a single exon and its relative transcriptional orientation is opposite to the VH segments. KIAA0125 has several interesting features that are often found in imprinted genes, including an extremely short putative protein coding region (77 amino acids) and very long 5¢- and 3¢-untranslated regions (1,289 and 6,087 nucleotides, respectively) and the presence of tandem repeats of 68 and 48 bp units in the 3¢-untranslated region (Neumann et al., 1995). Interestingly, its expression is limited to lymphoid organs such as spleen, thymus, and peripheral blood leukocyte (Nagase et al., 1995). The other seven DNA sequences are homologous to the human ribosomal protein S8, the metalloprotease-like, disintegrin-like, cystein-rich protein (MDC) family of Macaca, the human leukemia virus receptor 1 (GLVR1) (2 copies), and the human golgin-245 (three copies). All are processed pseudogenes.
a
ANALYSIS OF HUMAN VH SEGMENTS VH Subgroups and Families Human VH regions were divided into three subgroups based on amino acid sequences (reviewed in Kabat et al., 1991). These protein subgroups have been further subdivided into seven distinct VH families defined by the nucleotide sequence homology; VH segments that show 80% or greater identity are considered to be in the same family whereas VH segments that have less than 70% identity to one another form different VH families (Kodaira et al., 1986; Lee et al., 1987; Shen et al., 1987; Berman et al., 1988). Such criteria have been supported by construction of the phylogenetic tree of 114 VH segments (Figure 1.3) (Matsuda et al., 1998). It clearly shows three VH subgroups, namely VHI, VHII, and VHIII, subdivided into the VH1/VH5, VH2/VH4/VH6, and VH3 families, respectively. It is interesting to note that the VH4 (Lee et al., 1987), VH5 (Shen et al.,
b Truncated VH4
VH4 VH6
Human/Mouse Segregation
V3-52P/V4-51.2P
V4-44.1P
V3-22.2P/V4-22.1P
VH2
V3-50P/V4-49.1P
73 VH1
V3-32P/V4-31.1P V3-29P/V4-28.1P
132
13
VH3 VH7
35
44
100
V3-63P/V4-62.1P V3-79P/V4-78.1P V3-54P/V4-53.1P
VH5 54
75(Myr)
V3-33.2P/V4-33.1P 39
V3-30.2P/V4-30.1P 10
Truncated VH3
FIGURE 1.3 (a) A phylogenetic tree of the human VH segments based on their nucleotide sequence alignment. Three distinct sets of the VH segments, which correspond to VHI, VHII, and VHIII subgroups, are separated with boxes and indicated by Roman numerals. (b) Estimation of divergence time between 10 homologous units containing a pair of the VH3 and VH4 segments. The divergence time is indicated in million years ago (Myr) and the human/mouse divergence is shown by a vertical line. Modified from Matsuda et al. (1998).
8
Matsuda
1987), and VH6 (Berman et al., 1988) families have been identified by the comparison of nucleotide sequences of VH segments. The VH4 family members are most strongly conserved, suggesting that VH4 may have evolved most recently (Lee et al., 1987; Haino et al., 1994). However, frequent recombination between VH segments makes it difficult to estimate the precise time of divergence among VH segments. The VH5 and VH6 families contain only two and one members, respectively. Subgroup I contains a unique set of VH segments that share about 80% overall homology with the VH1 family but much less similarity to VH1 at a clustered region between framework 2 (FR2) and FR3. This group was also identified from nucleotide sequence homology and has been proposed to be classified as VH7 family (Schroeder et al., 1990). According to the above definition of the VH family, VH7 should be a subfamily of VH1 or a family captured in transition from VH1 to independence (Kirkham and Schroeder, 1994). However, Southern blot and sequencing analysis revealed that the VH7 family is a small but discrete VH family consisting of five to eight members that are dispersed within the VH locus (van Dijk et al., 1993), indicating that the classification of VH7 is practically useful. Of interest, a set of 12 VH3 pseudogenes that have the 5¢truncation at the same position constitutes an independent cluster of the VH3 family in the phylogenetic tree (Figure 1.3). Another group of 13 VH4 pseudogenes, sharing the common 5¢-truncation in the second exon, again branched off from the common ancestor. Since they are scattered across the locus, the initial truncation probably took place in an ancestral VH segment, followed by interspersion of duplicated copies throughout the locus. The V4-44.1P segment, which shares <40.6% amino acid similarity to the other human VH segments, constitutes an independent branch in the tree (Figure 1.3). Interestingly, a similar level of amino acid similarity was obtained from those of a variety of vertebrates including mouse (38.8%), rat (30.0%), rabbit (38.6%), dog (34.4%), caiman (36.4%), Xenopus (33.7%), teleost fish (36.7%), and horned shark
TABLE 1.2
(28.6%). This fact suggests that V4-44.1P might be a putative ancestral VH segment or a very old pseudogene having an accumulation of the point mutations. Several VH family-specific conserved regions occur in human germline VH segments (Kabat et al., 1991; Tomlinson et al., 1992; Matsuda et al., 1993; Haino et al., 1994). Family-specific sequences were found in the codons 9–30 in FR1 and the codons 60–85 of FR3. It is important to note that the codons 60–65 in the 3¢ portion of complementarity-determining region 2 (CDR2) were conserved in a family-specific way. Generally conserved universally were codons 1–8, FR2 (codons 38–47), and codons 86–92, in which the embedded heptamer recombination signal is located. A more extensive structural comparison of VH subregions is found elsewhere (Tomlinson et al., 1992; Kirkham and Schroeder, 1994).
5¢ Regulatory Regions The 5¢ flanking region of the VH segments contains two cis-acting elements, namely the octamer motif that regulates tissue-specific expression of IgH genes and the TATA box essential for the general transcription machinery. Extensive comparison of 500-bp of 5¢-flanking sequences of 79 VH segments without 5¢ truncation revealed striking familyspecific conservation (Haino et al., 1994; Matsuda et al., 1998) (Table 1.2). Locations of the octamer motif and TATA box are conserved within the same family but are different between different families. Forty out of 44 VH segments having a complete ORF contain an octamer sequence identical to the consensus (ATGCAAAT). The V3-20, V353, and V6-1 segments carry slightly less conserved octamer sequence but are known to be translated. The V3-38 segment in the ORF group has completely lost octamer (and TATA) due to 5¢-truncation. Of note, the octamer sequence is less conserved in pseudogenes and as many as 15 of 33 pseudogenes without 5¢-truncation have diverged octamer motif.
Summary of the 5¢ regulatory sequences and RSS of the human VH segments 5¢ regulatory region
RSS
VH family
Heptamcer
(bp)*
Octamer
(bp)*
TATA
(bp)*
7mer
(bp)
9mer
VH1 VH2 VH3 VH4 VH5 VH6 VH7
CTCATGA — — — — — TTCATGA
2 — — — — — 2
ATGCAAAT ATGCAAAT ATGCAAAT ATGCAAAT ATGCAAAT AGGCAAAT ATGCAAAT
19 26 18 39 18 19 8
TAAATAT TT(G/C)AAAA ATGAAAA TTAAATT ACTTAAA TTTAAAT GGAATAT
81 41 101 59 79 78 79
CACAGTG CACAGAG CACAGTG CACAGTG CACAGTG CACAGTG CACAGTG
23 23 23 23 23 23 23
TCAGAAACC ACAA(A/G)AACC ACACAAACC ACA(C/A)AAACC CTAAAACCC ACACAAACC TCAGAAACC
* Most common distance between the motifs is shown.
1. Human Immunoglobulin Heavy Chain Locus
In contrast, the sequence of the TATA box is well conserved within the same VH family, but very different between different families (Table 1.2). In addition, like the octamer motif, pseudogenes have a less conserved TATA motif. A heptamer sequence (CTCATGA), which is reported to be essential for full VH promoter activity in mouse lymphoid cells (Ballard and Bothwell, 1986; Eaton and Calame, 1987; Siu et al., 1987), is found in the human VH1 and VH7 families and, as in mice, similarly located (2- to 22-bp upstream of octamer). However, no heptamer element is found or similarly placed in the other VH families. Hence, this provided no further supportive evidence for the hypothesis that the heptamer element is involved in the activation of the Hchain promoters by the oct protein before the activation of the L-chain promoters that do not contain the heptamer motif (Kemler et al., 1989). Another interesting finding is that the 5¢-flanking regions of three VH3 segments, namely V3-9, V3-20, and V3-43 have a common 65-bp deletion in the region at 251- to 315bp upstream of the initiation codon (Haino et al., 1994). Since all are found in the full-length VH mRNAs, the deletion would not drastically reduce the promoter activity. No other conserved nucleotide sequences or potential candidates for a novel cis-acting element of VH transcription regulation are identified, in spite of an extensive investigation of nucleotide sequence alignment. However, future studies to correlate VH promoter activity and nucleotide sequence variation in the 5¢ regulatory region may identify such elements.
Recombination Signal Sequence The RSS of VH segments, which is located immediately 3¢ to the coding region, is composed of highly conserved heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by a 23-bp spacer. In vitro analysis of the RSS clearly showed that the first three nucleotides (CAC) in the heptamer and the fifth and sixth nucleotides (AA) in the nonamer are critical for the efficient V(D)J recombination (Ramsden et al., 1996; Couno et al., 1996). All 40 VH segments classified as either “functional” or “transcribed” maintain the first four and the last nucleotides (CACANNG) in the heptamer, and 35 of them have intact heptamers (Matsuda et al., 1998). The other five VH segments have either AA, GA, or GC at their fifth and sixth positions in the heptamer sequence. Conservation of the nonamer sequence is weaker and relatively family specific (Table 1.2). Again, the critical two nucleotides are well conserved in the more than 40 VH segments, except V2-26 and V2-70, which carry GA instead of AA. The nonamer sequence having G nucleotide at its fifth position is shown to be still active in V-J recombination in the human Vl genes (Kawasaki et al., 1997). The VH1 and VH7 families have a
9
family-specific nonamer TCAGAAACC. In the ORF group, the heptamer signal of the V3-16 (TCCTGTG) and V3-38 (TACACAG) segments are highly diverged without conservation of the first three nucleotides, suggesting their incapability for functional VDJ rearrange-ment. Likewise, V3-35 and V7-81 contain diverged heptamers (CACTGAG and CACCATG, respectively) but with the first three nucleotides intact. However, the effect of these mutations on the efficiency of VDJ recombination is not yet known. Obviously, they all maintain 23-bp spacer length. In contrast, RSS of VH pseudogenes are much less diverged. The 64 pseudogenes that have not lost RSS by 3¢truncation contain 26 pseudogenes with at least one mutation in the five critical positions. In addition, one, two, six, and seven pseudogenes have an irregular spacer length of 17, 20, 22, or 24 bp, respectively, although V segments with 22- and 24-bp spacers are acceptable for the V(D)J rearrangement in the human Vl and TCRb loci (Kawasaki et al., 1997; Rowen et al., 1996). However, roughly 35% or 28 of 79 of the pseudogenes retain the flawless RSS with a 23-bp spacer. Assuming that the VDJ recombination possibility is the same for any of the 70 VH segments (39 functional, 1 transcribed, 2 ORF, and 28 pseudogenes), the probability of productive VH to DH-JH rearrangement per allele is 1/3 (frame) ¥ {40 (functional/transcribed VH segments)/70} = 0.19 or 19%.
Primary Repertoire of the Human VH Region The number of germline VH, DH, and JH segments is largely different between different species. This corresponds to the genetic mechanism through which VH region diversity is created. One extreme example is the chicken VH locus, in which a multiple number of VH pseudogenes function as a donor of somatic gene conversion to a single functional VH segment in VDJ rearrangement (Raynaud et al., 1987). In humans, VH diversity is obtained in a “classical” way; the number of germline VH, DH, and JH segments generates the genetic basis of the VH region repertoire. The immune response of Ig-transgenic mice (xenomice) gives important hints to the minimum number of functional VH segments necessary to provide a full antibody repertoire. The xenomouse II strains that carry 35 and 18 (respectively) functional human VH and Vk segments develop a human adultlike antibody repertoire with high-affinity human antibodies against diverse antigens produced by a sufficient number of mature B-lymphocytes (Mendez et al., 1997). In contrast, in the xenomouse I strains bearing five functional VH and three functional Vk segments, maturation of Blymphocytes is severely affected and only a modest immune response is observed. This strongly suggests the importance of the primary V-region repertoire in the highly diverse human antibody response. The combinatorial diversity of
10
Matsuda
the human VH genes can be estimated as 40 (functional/transcribed VH segments) ¥ 25 (functional DH segments) ¥ 6 (functional JH segments) = 6,000. This is certainly an approximate value since the number of functional VH segments varies between alleles due to deleterious polymorphisms.
VH Segment Usage and Repertoire Formation The biased usage of particular VH, DH, and JH segments during early phases of ontogeny was first reported in mouse (Yancopoulos et al., 1984; Reth et al., 1986). In humans, VH5 and VH6 families are selectively expressed at 7 weeks of gestation, when B-lineage development is initiated (Cuisinier et al., 1989). A rapid expansion of the VH repertoire, including biased usage of specific VH segments, takes place between 8 and 15 weeks of gestation. The utilization of VH segments may be influenced by two groups of factors: (a) those affecting the recombination frequency and (b) those affecting selection of B cells expressing particular VH segments. Group (a) includes the distance between DH (or JH) and VH segments, variation in the recombination signal sequence, and locations that favor the recombinase accessibility. Group (b) includes self-antigens and bacterial super-antigens. The initial observation of the preferential usage of JHproximal VH segments in mouse led to the hypothesis that the proximity of VH segments to JH favors biased expression of VH segments in early stages of ontogeny. According to the studies investigating the utilization of VH segments in early development, those VH segments often used preferentially in early stages of ontogeny were V6-1, V1-2, V2-5, V3-13, V3-15, V3-23, V3-30, V5-51, V3-53, V4-59, and V1-69 segments (Schroeder et al., 1987; Schroeder and Wang, 1990; Cuisinier et al., 1993). In particular, the V3-30 segment is utilized at the highest rate in these studies. The V1-69 segment is also used frequently in peripheral B cells (Schwartz and Stoller, 1994), B-cell leukemia, and autoantibodies (Zouali, 1992). On the physical map, however, they are scattered across the region between 70-kb (V6-1) and 900-kb (V1-69) upstream of the JH cluster. Furthermore, none of the JH-proximal functional VH segments (including V1-2, V1-3, V2-5, V3-11) were found repeatedly in either examination. The results indicate that VH segments preferentially used in early stages of ontogeny do not necessarily cluster in the JH-proximal region. Surprisingly, three overexpressed VH segments in early ontogeny (V3-23, V3-30, and V1-69) have polymorphic variations of germline copy number. Moreover, Sasso et al. (1996) reported that the expression of V1-69 is proportional to its germline copy number. It is interesting and feasible to test the correlation between polymorphisms of specific VH segments and their utilization.
EVOLUTION OF THE HUMAN VH LOCI Evolution of the VH Locus on Chromosome 14 The evolution of multigene families, including Ig, has been driven by dynamic reorganization of the gene locus including duplication, deletion, and translocation. Comparative analysis of VH loci among different vertebrates from shark to man revealed dramatic differences of H-chain gene organization among species, yet all of them carry multiple VH segments, thus indicating that duplication of VH segments must have started quite a long time ago. It is also important to realize that reorganization of the VH locus is still ongoing on a variety of scales, as evidenced by dramatic difference in the VH locus organization between mouse and man. Recent translocation of VH and DH segments to chromosome 15 and 16 is more evidence of the dynamic reshuffling of the VH locus. Homology matrix analysis of the 957-kb sequence against itself showed that 67% of the entire VH locus is occupied by the thirteen DNA sequences of variable length (4 kb to 24 kb) that appear at least twice across the locus (Figure 1.1). These homologous units contain the DNA fragments previously shown to cross-hybridize with fourteen nonrepetitive intergenic probes using Southern blot analysis (Matsumura et al., 1994). One of such sequence, which appears 11 times across the locus, contains a pair consisting of a VH3 segment and a VH4 segment having the 5¢ truncation. Molecular evolutionary analysis by comparison of the highly conserved spacer sequence between different VH3 and VH4 units showed that the reorganization took place at least eight times between 132 and 10 million years ago (Matsuda et al., 1998). Of these, six took place after the mammalian radiation at 75 million years ago (Figure 1.3b). A similar calculation between DNA regions containing the truncated VH segments, namely, V367.3P/V3-67.2P and V3-5.2P/V3-5.1P, deduced the divergence time to be 61 million years ago, again after the divergence between mouse and human (Matsuda et al., 1998). These results strongly suggest that recent DNA reorganizations play a key role in the generation of the germline VH-region repertoire in human.
Evolution of Orphon VH and DH Loci It is striking that orphon VH and DH loci contain a remarkably high percentage (about 40%) of apparently functional VH segments. Several possible explanations for such conservation might be: • recent translocation; • functional constraint through their usage by transchromosomal rearrangement, as shown in the
1. Human Immunoglobulin Heavy Chain Locus
human g and d T cell receptor loci (Tycko et al., 1991); or • correction mechanisms, such as gene conversion (discussed below). Putative origins for the orphon VH segments on chromosome 15 and 16 were found in the 0.43- to 0.25-Mb JH proximal VH region on chromosome 14 (Nagaoka et al., 1994) (Figure 1.2). The orphon VH locus on chromosome 16 (designated as VH-F) is structurally similar to the region between the V3-11 segment and the V2-26 segment on chromosome 14. In addition, nucleotide sequence homologies of seven corresponding VH segments between the two loci total more than 93% (Figure 1.2). Most remarkable homology was found between two truncated pseudogenes VF1-12P and V1-12P, in which the homology extends into the region 3¢ to the truncation site. The time of the translocation of the VH-F locus from chromosome 14 was estimated to be, at the earliest, 20 million years ago, using the synonymous site substitution in the coding region between corresponding VH segments. In contrast, no obvious counterparts of the D5 clusters could be identified on chromosome 14. However, the V54 segment in the D5-b locus showed a significant homology of 94.7% to the V1-18 segment, which is located in the putative origin of VH-F locus, although the other two segments are less homologous to the corresponding VH segments (Figure 1.2). The segregation time of V54 and V1-18 was estimated to be approximately 13 million years ago. These findings suggest that a DNA fragment of greater than 100 kb might have been translocated simultaneously to chromosome 15 and 16 approximately 20 million years ago.
Pseudogenes and Gene Conversion The completion of the nucleotide sequence of the entire human VH locus revealed that as much as 65% (79 out of 123) of the VH segments are pseudogenes. The presence of abundant VH pseudogenes and orphon VH segments raises the question of whether they have any functional significance. Since 40% of orphon VH segments are apparently functional, they can theoretically recombine with DH-JH rearrangements on chromosome 14 through interchromosomal recombination. Recombination between a VH–DH rearrangement on chromosome 15 and a JH segment on chromosome 14 is also possible since a VH–DH fusion product was isolated from a human B-cell line (Shin et al., 1993b) and from B lymphocytes of transgenic mice carrying an IgH mini locus (Tuaillon et al., 1994). In addition, germline transcripts of orphon VH have been identified in human fetal liver (Cuisinier et al., 1993), suggesting that orphon VH loci might be targets of recombinase. Unfortunately, however, no direct evidence for the expression or recombination of translocated VH segments has been reported to date. More-
11
over, an extensive analysis of the human D segment usage ruled out the possible involvement of D5 segments on chromosome 15 in functional VDJ rearrangement (Corbett et al., 1997). Conserved pseudogenes have been already shown to serve as sequence donors for gene conversion in other species. Somatic gene conversion (or double unequal crossing-over) has been shown to take place to amplify the V region repertoire in chicken (Raynoud et al., 1987; Tompson and Neiman, 1987) and rabbit (Becker and Knight, 1990) but not in mouse and man. One attempt to identify evolutionary gene conversion is based on the theory of molecular evolution (Haino et al., 1994). In the course of evolution, the introns and the synonymous positions of the coding region evolve at high and remarkably similar rates in different genes, and base substitutions are accumulated at approximately constant rates with respect to geological time (Miyata et al., 1980; Hayashida and Miyata, 1983). Hence, the recipients of gene conversion would be found by comparing the substitution rates in the intron and the synonymous position of pairs of VH segments; clear differences in substitution rates of the two portions in a given pair of VH segments suggests some recombination events. One of the recently duplicated VH segment pairs, V3-62P and V3-60P, which show 94% nucleotide homology and share the same mutation in RSS (Kodaira et al., 1986), w ere chosen for the analysis. These two VH segments displayed a significant difference in the substitution rates in the intron (Kci = 0.1637 ± 0.0461) and the synonymous position of the coding region (Kcs = 0.0821 ± 0.0339), thus indicating possible segmental change in the intron in either V3-60P or V3-62P (Figure 1.4). Sequences of these two VH segments were compared with those of the other VH3 members, to look for the putative donor of the segmental transfer. As a result, V3-43 and V3-62P were found to have significantly smaller Kci (0.0820 ± 0.0280) than Kcs (0.4165 ± 0.0761). Such unusual homology of the 5¢ half region between V3-43 and V3-62P is most likely explained by the unidirectional segmental transfer of the V3-43 sequence to V3-62P. This example supports the hypothesis that gene conversion contributes to the maintenance of the pseudogene structure. The same method was applied to VH segments belonging to the VH4 family, which is most conserved (>90%) and richest in functional VH segments among the seven human VH families (Lee et al., 1987). Rather frequent unidirectional correction was observed between VH4 segments, thus demonstrating that the VH4 family members evolved by recent duplication, followed by gene conversion (Figure 1.4). It is to be noted that V4-55P served as a donor of two functional VH segments, V4-4b and V4-28. This might indicate that the high percentage of pseudogenes should also contribute to the generation of the germline VH repertoire.
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FIGURE 1.4 Schematic demonstration of gene conversion. The values of Kci and Kcs between VH segments are calculated for introns and synonymous positions of codons -4/92, respectively. Vertical arrows indicate direction of sequence transfer. Modified from Haino et al. (1994).
HUMAN CH LOCUS The human CH gene family is mapped to the q32 band of chromosome 14 (Kirsch et al., 1982) and consists of nine functional genes and two pseudogenes. Between mouse and human, the characteristic difference in the organization of the CH gene cluster is the presence of the duplication of 70kb DNA, consisting of two Cg genes and one each Ce and Ca genes in human (Figure 1.1) (Flanagan and Rabbitts, 1982). In addition, a pseudo Cg gene has been genetically mapped between the duplication unit (Bech-Hansen et al., 1983). The 5¢ Ce or Ce2 gene is a pseudogene with a 5¢-
truncation, resulting in the absence CH1 and CH2 exons. The other pseudogene Ce3 is processed and translocated to chromosome 9 (Battey et al., 1982). The complete nucleotide sequence of the entire CH locus permitted the precise organization of the human CH locus as follows: 5¢-JH-(8 kb)-Cm(5 kb)-Cd-(65 kb)-Cg3-(26 kb)-Cg1-(19 kb)-Ce2-(13 kb)-Ca1-(34 kb)-yCg-(20 kb)-Cg2-(18 kb)-Cg4-(23 kb)Ce1-(10 kb)-Ca2–3¢ (Ravetch et al., 1981; Flanagan and Rabbitts, 1982; Word et al., 1989; Heilig et al., 2003; Nicodeme et al., submitted), which is in agreement to that of previous studies based on hybridization (Bottaro et al., 1989b; Hofker et al., 1989).
1. Human Immunoglobulin Heavy Chain Locus
Three heavy chain transcription enhancers are known in the human CH locus, all of which are located at a place similar to those of mouse (Figure 1.1). The 5¢-lost enhancer, or Em enhancer, is located in the intron between JH and Cm (Rabbitts et al., 1983). Two nearly identical copies of enhancer arrays homologous to the mouse Ca 3¢-enhancer (3¢aE) were identified at the 3¢ flanking region of each of the two human Ca genes, namely at the 3¢ end of the CgCg-Ce-Ca duplication (Mills et al., 1997). A novel regulatory motif cluster with a potential B lymphocyte-specific enhancer function (Ed-g3) was recently identified (Mundt et al., 2001) in the 55-kb DNA between the Cd and Cg3 genes, where the existence of a strong candidate region for matrix attachment was predicted by computer programs (Nicodeme et al., submitted). This region has an exceptionally low G + C nucleotide content (average 42%) in the G + C predominant human CH locus (average 58%). Three non-immunoglobulin DNA sequences were identified in the CH locus (Figure 1.1). The 1.7-kb mRNA sequence of AK056731 showed 99.7% homology to a DNA sequence between yCg and Cg2 genes. This single exon
13
gene, having a 545-bp ORF, is expressed in placenta, but the function of the protein is unknown. A processed pseudogene of ELK2 is located upstream of the yCg gene (Harindranath et al., 1997). Another processed pseudogene of ATP6V1G1, a vacuolar ATPase, was identified in the region between the Cd and Cg3 genes.
Structure of CH Genes All the human CH genes have been isolated and sequenced completely. References for complete CH gene sequences with detailed information are available from Ig databases (for example, IGMT database; http:// imgt.cines.fr). The human CH genes for secretory forms are composed of three (d, g, and a) or four (m and e) exons, each encoding a functional and structural unit of the H chain, namely a domain (Edelman et al., 1969) (Figure 1.5). Cd has an additional exon that encodes a C-terminal tail for the secretory-form IgD 2-kb downstream of its CH3 exon. Exons corresponding to hinge regions are located between the CH1 and CH2 exons in the Cd and Cg genes, and their number
FIGURE 1.5 Exon/intron structure of human CH genes and pseudogenes. Coding exons, sterile (I) exons, hinge exons, and membrane exons are shown by open box, hatched box, vertical line, and striped box, respectively. Switch regions are indicated with vertical stripes. The exon of the Cd gene for soluble form (CH-S) is indicated. Note that I exons of Cg2, Cg4, and yCe2 and membrane exons of Cg4 and yCe2 are predicted by homology search.
14
Matsuda
and length vary between different CH genes and subclasses. The hinge region of Ca genes is exceptionally encoded by the CH2 exon, and there is no obvious hinge region in the Cm and Ce genes. In addition, one (a) or two (others) separate exons encode the hydrophobic transmembrane and short intracytoplasmic segments that are used for a membrane-form Ig. The size of each CH exon is similar to that of the CL exon, suggesting that the CH gene evolved through the duplication of a primordial single exon gene, like the CL gene. Such exon–intron organization of the CH gene is consistent with the domain hypothesis that states that the Hchain protein consists of a tandem array of three or four functional units (Edelman et al., 1969). The total length of each CH gene is therefore variable, ranging from 4 to 9 kb (sterile exons are not taken account). All functional CH genes except Cd have the switch (S) region at the 5¢ flanking region of the CH1 exon. S regions consist of tandem repeats of pentameric nucleotides and are responsible for class switch recombination. The presence of germline transcripts prior to class switching arising from untranslated exons (I exons) was reported previously for most of the CH subclasses (Sideras et al., 1989; Nilsson et al., 1991; Kuzin et al., 2000; Mage et al., 1989; Bachl et al., 1996). Expression of such transcripts is driven by a promoter located upstream of the S region. Missing information for the I exons of the Cg2, Cg4, and yCe genes was recently obtained by nucleotide sequence alignment of the 5¢ flanking region between different Cg and Ce genes (Nicodeme et al., submitted). Most of the CH genes carry a single I exon, except two Cg genes; Cg1 has three I exons as does its duplicated copy, Cg4. No putative I exons were found in the Cd gene, which is transcribed together with Cm as a single transcript. The absence of the pseudo Cg gene product, despite a complete set of coding exons without defects, is explained by the deletion of S region and I exons.
TABLE 1.3
Expression of the membrane exons is controlled by differential splicing. Transcripts of the membrane exons are spliced to the 3¢-most domain exons by removing the last few residues of the secreted Ig tail. Membrane segments, except those of the Ca genes, are encoded by two exons. The hydrophobic transmembrane segment of 26 residues is relatively conserved among all the H chains, suggesting the possibility that membrane-form Ig is anchored by a common membrane protein (Yamawaki-Kataoka et al., 1982). Since the intracytoplasmic segments of the membrane-form Ig are too short (27 residues for Cg and Ce chains, 13 residues for Ca, and 2 residues for Cm and Cd) to catalyze any enzymatic activity such as phosphorylation, transduction of the triggering signal of the antigen–antibody interactions may require involvement of at least one other protein. This hypothesis has been verified by subsequent identification of Iga and b proteins (see Chapter 11).
Polymorphisms of the Human CH Locus The human CH locus is highly polymorphic, with different alleles carrying deletion and duplication of CH genes. Eleven types of deletions and eight duplications involving one or more CH genes have been identified to date (summarized in Table 1.3). One of the most common polymorphism is the Cg4 gene duplication, which is present in 44% of Caucasian chromosomes (Brusco et al., 1995a). Large differences in the frequency of the CH haplotype were observed between different ethnic groups in an inter-racial genetic study (Rabbani et al., 1996). Of interest, seven of these polymorphisms appear as both deletion and duplication, suggesting that unequal crossing-over between highly homologous regions played a major role for the CH locus polymorphisms. Looping-out excision is also conceivable as another genetic mechanism (Bottaro et al., 1989a). It is
Summary of the human CH locus polymorphisms Reference
Polymorphism Cg1 Cg1-Ca1 Cg1-Cg2 Cg1-Cg4 yCe2-yCg yCe2-Ce1 Ca1-Ce1 yCg yCg-Ca2 Cg2 Cg2-Cg4 Cg4
Approximate size (in
Deletion
Duplication
— 50 110 130 70 120 120 — 110 — 35 —
Smith et al. (1989) Rabbani et al. (1995) Smith et al. (1989) Lefranc et al. (1982) Lefranc et al. (1983) Migone et al. (1984) Migone et al. (1984) N.I. Bottaro et al. (1989a); Hendriks et al. (1989) Bottaro et al. (1989a); Hendriks et al. (1989) Olsson et al. (1991) Bottaro et al (1990)
N.I. N.I. N.I. Rabbani et al. (1996) N.I. Bottaro et al. (1991) Bottaro et al. (1991) Rabbani et al. (1996) Brusco et al. (1995a) Bech-Hansen and Cox (1986) Brusco et al. (1995a) Brusco et al. (1995a)
* The size of deletion/duplication for those comprising multiple CH genes was estimated from the physical map. N.I.; not identified to date.
1. Human Immunoglobulin Heavy Chain Locus
rather surprising that individuals with deletions of multiple CH genes have not shown any severe clinical symptoms, thus suggesting that Cg and Ca subclass genes are capable of substituting each other and that the Ce genes might not be obligatory but might facilitate efficient protection from parasite infection. Ig allotype typing is usually performed with serological methods based on hemagglutination inhibition. Allotypes of Ig are mostly explained by specific amino acid substitutions in CH regions. In humans, Ig allotypes have been identified for five human CH genes, the Cg1, Cg2, Cg3, Ca2, and Ce1 genes, and are designated as G1m, G2m, G3m, A2m, and Em, respectively. Molecular typing of Ig allotypes has been done for different CH genes. SNPs specific to G2m and G3m allotypes (Brusco et al., 1995b; Dard et al., 2001) were identified by nucleotide sequencing and were confirmed by population-based tests.
Acknowledgments We thank all our colleagues for their contribution to accomplish this study, and Dr. David H. Gelfand (Roch Molecular Systems, Inc.) for the critical reading of the manuscript. Most of the work was done in the Center for Molecular Biology and Genetics and the Department of Medical Chemistry, Kyoto University Graduate school of Medicine (on the VH locus) and Centre National de Genotypage (on the CH locus). The work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture in Japan and from the Science and Technology Agency of Japan. The CNG is supported by the Ministere de la Recherche et des Nouvelles Technologies.
References Bachl, J., Turck, C. W., and Wabl, M. (1996). Eur J Immunol 26, 870–874. Ballard, D. W., and Bothwell, A. (1986) Proc Natl Acad Sci U S A 83, 9626–9630. Battey, J., Max, E. E., McBride, W. O., Swan, D., and Leder, P. (1982). Proc Natl Acad Sci U S A 79, 5956–5960. Bech-Hansen, N. T., and Cox, D. W. (1986). Am J Hum Genet 38, 67– 74. Bech-Hansen, N. T., Linsley, P. S., and Cox, D. W. (1983). Proc Natl Acad Sci U S A 80, 6952–6956. Becker, R. S., and Knight, K. L. (1990). Cell 63, 987–997. Berman, J. E., Mellis, S. J., Pollock, R., Smith, C. L., Suh, H., Heinke, B., Kowal, C., Surti, U., Chess, L., Cantor, C. R., and Alt, F. W. (1988). EMBO J 7, 727–738. Bottaro, A., Cariota, U., DeMarchi, M., and Carbonara, A. O. (1991). Am J Hum Genet 48, 745–756. Bottaro, A., Cariota, U. G., de Lange, G. G., DeMarchi, M., Gallina, R., Oliviero, S., Vlug, A., and Carbonara, A. O. (1990). Hum Genet 86, 191–197. Bottaro, A., de Marchi, M., de Lange, G. G., and Carbonara, A. O. (1989a). Exp Clin Immunogenetics 6, 55–59. Bottaro, A., de Marchi, M., Migone, N., and Carbonara, A. O. (1989b). Genomics 4, 505–508. Brusco, A., Cinque, F., Saviozzi, S., Boccazzi, C., DeMarchi, M., and Carbonara, A. O. (1995a). Hum Genet 100, 84–89. Brusco, A., de Lange, G. G., Boccazzi, C., and Carbonara, A. O. (1995b). Immunogenetics 42, 414–417.
15
Buluwela, L., and Rabbitts, T. H. (1988). Eur J Immunol 18, 1843–1845. Buluwela, L., Albertson, D. G., Sherrington, P., Rabbitts, P. H., Spurr, N., and Rabbitts, T. H. (1988). EMBO J 7, 2003–2010. Cherif, D., and Berger, R. (1990). Genes Chromosomes Cancer 2, 103–108. Chung, J. H., Siebenlist, U., Morton, C. C., and Leder, P. (1984). Fed Proc 43, 1486. Cook, G., Tomlinson, I. M., Walter, G., Riethman, H., Carter, N. P., Buluwela, L., Winter, G., and Rabbitts, T. H. (1994). Nat Genet 7, 162–168. Corbett, S. J., Tomlinson, I. M., Sonnhammer, E. L. L., Buck, D., and Winter, G. (1997). J Mol Biol 270, 587–597. Couno, C. A., Mundy, C. L., and Oettinger, M. A. (1996). Mol Cell Biol 16, 5683–5690. Croce, C. M., Sander, M., Martins, J., Cicurel, L., D’Ancona, G. G., Dolby, T. W., and Koprowski, H. (1979). Proc Natl Acad Sci U S A 76, 3416–3419. Cuisinier, A. M., Guigou, V., Boubli, L., Fougereau, M., and Tonnelle, C. (1989) Scand J Immunol 30, 493–497. Cuisinier, A. M., Gauthier, L., Boubli, L., Fougereau, M., and Tonnelle, C. (1993). Eur J Immunol 23, 110–118. Dard, P., Lefranc, M. P., Osipova, L., and Sanchez-Mazas, A. (2001). Eur J Hum Genet 9, 765–772. Eaton, S., and Calame, K. (1987). Proc Natl Acad Sci U S A 84, 7634– 7638. Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottliep, P. D., Rutishauser, U., and Waxdal, M. J. (1969). Proc Natl Acad Sci U S A 63, 78–85. Flanagan, J. G., and Rabbitts, T. H. (1982). Nature 300, 709–713. Foote, J., and Winter, G. (1992). J Mol Biol. 224, 487–499. Frippiat, J. P., Williams, S. C., Tomlinson, I. M., Cook, G. P., Cherif, D., Le Paslier, D., Collins, J. E., Dunham, I., Winter, G., and Lefranc, M. P. (1995). Hum Mol Genet 4, 983–991. Givol, D., Zakut, R., Effron, K., Rechavi, G., Ram, D., and Cohen, J. B. (1981). Nature 292, 426–430. Green, L. L., Hardy, M. C., Maynard-Currie, C. E., Tsuda, H., Louie, D. M., Mendez, M. J., Abderrahim, H., Noguchi, M., Smith, D. H., Zeng, Y., David, N. E., Sasai, H., Garza, D., Brenner, D. G., Hales, J. F., McGuiness, R. P., Capon, D. J., Klapholz, S., and Jakobovits, A. (1994) Nat Genet 7, 13–21. Haino, M., Hayashida, H., Miyata, T., Shin, E. K., Matsuda, F., Nagaoka, H., Matsumura, R., Taka-ishi, S., Fukita, Y., Fujikura, J., and Honjo, T. (1994). J Biol Chem 269, 2619–2626. Harindranath, N., Mills, F. C., Mitchell, M., Meindl, A., and Max, E. E. (1997). Gene 221, 215–224. Hashimoto, L. L., Walter, M. A., Cox, D. W., and Ebers, G. C. (1993). J Neuroimmunol 44, 77–83. Hayashida, H., and Miyata, T. (1983). Proc Natl Acad Sci U S A 80, 2671–2675. Heilig, R., Eckenberg, R., Petit, J. L., Fonknechten, N., DaSilva, C., Cattolico, L., Levy, M., Barbe, V., DeBerardinis, V., Ureta-Vidal, A., Pelletier, E., Vico, V., Anthouard, V., Rowen, L., Madan, A., Qin, Sun, S. H., Du, H., Pepin, K., Artiguenave, F., Robert, C., Cruaud, C., Brüls, T., Friedlander, L., Samson, G., Brottier, P., Cure, S., Ségurens, B., Samain, S., Abbasi, N., Aiach, N., Boscus, D., Dickhoff, R., Dors, M., Dubois, I., Friedman, C., Gouyvenoux, M., James, R., Madan, A., Mairey–Estrada, B., Mangenot, S., Martins, N., Ménard, M., Oztas, S., Ratcliffe, A., Shaffer, T., Vacherie, B., Bellemere, C., Belser, C., Besnard-Gonnet, M., Bartol–Mavel, D., Boutard, M., Briez-Silla, S., Combette, S., Dufosse-Laurent, V., Ferron, C., Lechaplais, C., Louesse, C., Muselet, D., Magelenat, G., Pateau, E., Petit, E., SirvainTrukniewicz, P., Trybou, A., Vega-Czarny, N., Bataille, E., Bluet, E., Bordelais, I., Dubois, M., Dumont, C., Guérin, T., Haffray, S., Hammadi, R., Muanga, J., Laure, L., Pellouin, V., Robert, D., Wunderle, E., Anière, F., Gauguet, G., Roy, A., Sainte-Marthe, L., Verdier, J., Verdier-Discalla, C., Hillier, L., Fulton, L., McPherson, J., Matsuda, F.,
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Trask, B. J., Wilson, R., Scarpelli, C., Gyapay, G., Wincker, P., Saurin, W., Quétier, F., Waterston, R., Hood, L., and Weissenbach, J. (2003) Nature 421, 601–607. Hendriks, R. W., van Tol, M. J., de Lange, G. G., and Schuurman, R. K. (1989). Scand J Immunol 29, 535–541. Hofker, M. H., Walter, M. A., and Cox, D. W. (1989) Proc Natl Acad Sci U S A 86, 5567–5571. Ichihara, Y., Abe, M., Yasui, H., Matsuoka, H., and Kurosawa, Y. (1988a). Eur J Immunol 18, 649–652. Ichihara, Y., Matsuoka, H., and Kurosawa, Y. (1988b). EMBO J 7, 4141–4150. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of proteins of immunological interest, 5th ed. (Washington, DC: NIH). Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Schmeits, J. L., Wang, J., and Shimizu, N. (1997) Genome Res 7, 250–261. Kawasaki, N., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Asakawa, T., Klobeck, H.-G., Combriato, G., Zachau, H. G., and Shimizu, N. (2001) Eur J Immunol 31, 1017–1028. Kemler, I., Schreiber, E., Müller, M. M., Matthias, P., and Schaffner, W. (1989) EMBO J 8, 2001–2008. Kemp, D. J., Tyler, B., Bernard, O., Gough, N., Gerondakis, S., Adams, J. M., and Cory, S. (1981). J Mol Appl Genet 1, 245–261. Kirkham, P. M., and Schroeder, H. W. Jr. (1994). Semin Immunol 6, 347–360. Kirsch, I. R., Morton, C. C., Nakahara, K., and Leder, P. (1982). Science 216, 301–303. Kodaira, M., Kinashi, T., Umemura, I., Matsuda, F., Noma, T., Ono, Y., and Honjo, T. (1986). J Mol Biol 190, 529–541. Kuzin, I. I., Ugine, G. D., Wu, D., Young, F., Chen, J., and Bottaro, A. (2000). J Immunol 164, 1451–1457. Lee, K. H., Matsuda, F., Kinashi, T., Kodaira, M., and Honjo, T. (1987). J Mol Biol 195, 761–768. Lefranc, M. P., Lefranc, G., and Rabbitts, T. H. (1982). Nature 300, 760–762. Lefranc, M. P., Lefranc, G., de Lange, G. G., Out, T. A., van den Broek, P. J., van Nieuwkoop, J., Radl, J., Helal, A. N., Chaabani, H., van Loghem, E., and Rabbitts, T. H. (1983). Mol Biol Med 1, 207–217. Mage, R. G., Newman, B. A., Harindranath, N., Bernstein, K. E., Becker, R. S., and Knight, K. L. (1989). Mol Immunol 26, 1007–1010. Matsuda, F., Lee, K. H., Nakai, S., Sato, T., Kodaira, M., Zong, S. Q., Ohno, H., Fukuhara, S., and Honjo, T. (1988). EMBO J 7, 1047–1051. Matsuda, F., Shin, E. K., Hirabayashi, Y., Nagaoka, H., Yoshida, M. C., Zong, S. Q., and Honjo, T. (1990). EMBO J 9, 2501–2506. Matsuda, F., Shin, E. K., Nagaoka, H., Matsumura, R., Haino, M., Fukita, Y., Taka-ishi, S., Imai, T., Riley, J. H., Anand, R., Soeda, E., and Honjo, T. (1993). Nat Genet 3, 88–94. Matsuda, F., Ishii, K., Bouvagnet, P., Kuma, K., Hayashida, H., Miyata, T., and Honjo, T. (1998). J Exp Med 188, 2151–2162. Matsumura, R., Matsuda, F., Nagaoka, H., Shin, E. K., Fukita, Y., Haino, M., Fujikura, J., and Honjo, T. (1994). J Immunol 152, 660–666. Mendez, M. J., Green, L. L., Corvalan, J. R. F., Jia, X.-C., Maynard-Currie, C. E., Yang, X.-D., Gallo, M. L., Louie, D. M., Lee, D. V., Erickson, K. L., Luna, J., Roy, C. M.-N., Abderrahim, H., Kirschenbaum, F., Noguchi, M., Smith, D. H., Fukushima, A., Hales, J. F., Finer, M. H., Davis, C. G., Zsebo, K. M., and Jakobovits, A. (1997). Nat Genet 15, 146–156. Migone, N., Oliviero, S., de Lange, G., Delacroix, D. L., Boschis, D., Altruda, F., Silengo, L., DeMarchi, M., and Carbonara, A. O. (1984). Proc Natl Acad Sci U S A 81, 5811–5815. Mills, F. C., Harindranath, N., Mitchell, M., and Max, E. E. (1997). J Exp Med 186, 845–858. Miyata, T., Yasunaga, T., and Nishida, T. (1980). Proc Natl Acad Sci U S A 77, 7328–7332.
Mundt, C. A., Nicholson, I. C., Zou, X., Popov, A. V., Ayling, C., and Brüggemann, M. (2001). J Immunol 166, 3315–3323. Nagaoka, H., Ozawa, K., Matsuda, F., Hayashida, H., Matsumura, R., Haino, M., Shin, E. K., Fukita, Y., Imai, T., Anand, R., Yokoyama, K., Eki, T., Soeda, E., and Honjo, T. (1994). Genomics. 22, 189–197. Nagase, T., Seki, A., Tanaka, K., Ishikawa, K., and Nomura, N. (1995). DNA Res 2, 167–174. Nicodeme, S., Gartioux, C., Heilig, R., Petie, J.-L., Sepulveda, A., Birshtein, B., Weissenbach, J., Lathrop, M., and Matsuda, F. Submitted. Nilsson, L., Islam, K. B., Olafsson, O., Zalcberg, I., Samakovlis, C., Hammarstrom, L., Smith, C. I., and Sideras, P. (1991). Int Immunol 3, 1107–1115. Neumann, B., Kubicka, P., and Barlow, D. P. (1995). Nat Genet 9, 12–13. Olsson, P. G., Hofker, M. H., Walter, M. A., Smith, S., Hammarstrom, L., Smith, C. I. E., and Cox, D. W. (1991). J Immunol 147, 2540–2546. Rabbani, H., Kondo, N., Smith, C. I., and Hammarstrom, L. (1995). Clin Immunol Immunopathol 76, 214–218. Rabbani, H., Pan, Q., Kondo, N., Smith, C. I., and Hammarstrom, L. (1996). Immunogenetics 45, 136–141. Rabbitts, T. H., Forster, A., Baer, R., and Hamlyn, P. H. (1983). Nature 306, 806–9. Ramsden, D. A., McBlane, J. F., van Gent, D. C., and Gellert, M. (1996). EMBO J 15, 3197–3206. Ravetch, J. V., Siebenlist, U., Korsmeyer, S., Waldmann, T., and Leder, P. (1981). Cell 27, 583–591. Raynaud, C.-A., Anques, V., Grimal, H., and Weill, J. C. (1987). Cell 48, 379–388. Rechavi, G., Bientz, B., Ram, D., Ben-Neriah, Y., Cohen, J. B., Zakut, R., and Givol., D. (1982). Proc Natl Acad Sci U S A 79, 4405–4409. Reth, M., Jackson, N., and Alt, F. W. (1986). EMBO J 5, 2131–2138. Rowen, L., Koop, B. F., and Hool, L. (1996). Science 272, 1755–1762. Sanz, I., Dang, H., Takei, M., Talal, N., and Capra, J. D. (1989). J Immunol 142, 883–887. Sasso, E. H., van Dijk, K. W., Bull, A. P., and Milner, E. C. B. (1993). Proc Natl Acad Sci U S A 89, 10430–10434. Sasso, E. H., Johnson, T., and Kipps, T. J. (1996). J Clin Invest 97, 2074–2080. Sato, T., Matsuda, F., Lee, K. H., Shin, E. K., and Honjo, T. (1988). Biochem Biophys Res Commun 154, 265–271. Schroeder, H. W. Jr., Hillson, J. L., and Perlmutter, R. M. (1987). Science 238, 791–793. Schroeder, H. W. Jr., Walter, M. A., Hofker, M. H., Ebens, A., Willem van Dijk, K., Liao, L. C., Cox, D. W., Milner, E. C. B., and Perlmutter, R. M. (1988). Proc Natl Acad Sci U S A 85, 8196–8200. Schroeder, H. W. Jr., Hillson, J. L., and Perlmutter, R. M. (1990). Int Immunol 20, 41–50. Schroeder, H. W. Jr., and Wang, J. Y. (1990). Proc Natl Acad Sci U S A 87, 6146–6150. Schwartz, R. S., and Stoller, B. D. (1994). Immunol Today 15, 27–32. Shen, A., Humphries, C., Tucker, P., and Blattner, F. (1987). Proc Natl Acad Sci U S A 84, 8563–8567. Shin, E. K., Matsuda, F., Nagaoka, H., Fukita, Y., Imai, T., Yokoyama, K., Soeda, E., and Honjo, T. (1991). EMBO J 10, 3641–3645. Shin, E. K., Matsuda, F., Ozaki, S., Kumagai, S, Olerup, O., Strom, H., Melchars, I., and Honjo, T. (1993a). Immunogenetics 38, 304–306. Shin, E. K., Matsuda, F., Fujikura, J., Akamizu, T., Sugawa, H., Mori, T., and Honjo, T. (1993b). Eur J Immunol 23, 2365–2367. Sideras, P., Mizuta, T. R., Kanamori, H., Suzuki, N., Okamoto, M., Kuze, K., Ohno, H., Doi, S., Fukuhara, S., Hassan, M. S., Hammarstrom, L., Smith, C. E., Shimizu, A., and Honjo, T. (1989). Int Immunol 1, 631–642. Siebenlist, U., Ravetch, J. V., Korsmeyer, S., Waldmann, T., and Leder, P. (1981). Nature 294, 631–635.
1. Human Immunoglobulin Heavy Chain Locus Siu, G., Springer, E. A., Huang, H. V., Hood, L. E., and Crews, S. T. (1987). J Immunol 138, 4466–4471. Smith, C. I., Hammarstrom, L., Henter, J. I., and de Lange, G. G. (1989). J Immunol 142, 4514–4519. Sonntag, D., Weingartner, B., and Grutzmann, R. (1989). Nucleic Acids Res 17, 1267. Taylor, L. D., Carmack, C. E., Huszar, D., Higgins, K. M., Mashayekh, R., Sequar, G., Schramm, S. R., Kuo, C. C., O’Donnell, S. L., Kay, R. M., Woodhouse, C. S., and Lonberg, N. (1994). Int Immunol 6, 579–591. Tomlinson, I. M., Walter, G., Marks, J. D., Llewelyn, M. B., and Winter, G. (1992). J Mol Biol 227, 776–798. Tomlinson, I. M., Cook, G. P., Carter, N. P., Elaswarapu, R., Smith, S., Walter, G., Buluwela., L., Rabbitts, T. H., and Winter, G. (1994) Hum Mol Genet 3, 856–860. Tompson, C. B., and Neiman, P. E. (1987). Cell 48, 369–378. Tuaillon, N., Miller, A. B., Longberg, N., Tucker, P. W., and Capra, J. D. (1994). J Immunol 152, 2912–2920. Tycko, B., Smith, S. D., and Sklar, J. (1991). J Exp Med 174, 867–873. Van Dijk, K. W., Mortari, F., Kirkham, P. H., Schroeder, H. W., and Milner, E. C. B. (1993). Eur J Immunol 23, 832–839.
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Walter, G., Tomlinson, I. M., Cook, G. P., Winter, G., Rabbitts, T. H., and Dear, P. H. (1993). Nucleic Acids Res 21, 4524–4529. Walter, M. A., Surti, U., Hofker, M. H., and Cox, D. W. (1990). EMBO J 9, 3303–3313. Walter, M. A., Dosch, H. M., and Cox, D. W. (1991a). J Exp Med 174, 335–349. Walter, M. A., Gibson, W. T., Ebers, G. C., and Cox, D. W. (1991b). J Clin Invest 87, 1266–1273. Word, C. J., White, M. B., Kuziel, W. A., Shen, A. L., Blattner, F. R., and Tucker, P. W. (1989). Int Immunol 1, 296–309. Yamawaki-Kataoka, Y., Nakai, S., Miyata, T., and Honjo, T. (1982). Proc Natl Acad Sci U S A 79, 2623–2627. Yancopoulos, G. D., Desiderio, S. V., Pasking, M., Kearney, J. F., Baltimore, D., and Alt, F. W. (1984). Nature 311, 727–733. Yang, P.-H., Olsen, N. J., Siminovitch, K. A., Olee, T., Kozin, F., Carson, D. A., and Chen, P. P. (1990). Proc Natl Acad Sci U S A 88, 7907– 7911. Zong, S. Q., Nakai, S., Matsuda, F., Lee, K. H., and Honjo, T. (1988). Immunol Lett 17, 329–334. Zouali, M. (1992). Immunol Rev 128, 73–99.
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2 Immunoglobulin Heavy Chain Genes of Mouse ROY RIBLET Torrey Pines Institute for Molecular Studies San Diego, California, USA
using a large number of landmarks derived from prior assembly of a C57BL yeast artificial chromosome (YAC) contig (Chevillard et al., 2002). The sequence-ready tiling path was selected by a team at Washington University, led by John MacPherson. BAC sequencing was performed by the Genome Therapeutics Corporation sequencing group directed by Douglas Smith. This summary of our findings was written in advance of primary publication.
The immunoglobulin heavy chain locus, Igh in mouse, is an unusual genetic locus that must undergo molecular recombination to yield an active expressible gene for its antibody heavy chain product. Before this genetic rearrangement occurs, the locus is comprised of an array of clusters of gene segments of four types, Variable (Vh), Diversity (Dh), Joining (Jh), and Constant (Ch) gene segments. A rearranged active heavy chain V gene is constructed by fusing together a V, D, and J segment (Sakano et al., 1980). In mouse, this array of gene segments is near the telomere of chromosome 12 and comprises about 3 Mb (million basepairs) of DNA. DNA sequence variation occurs between mouse strains across the entire locus, so that it is helpful to analyze a single allelic state of the array, such as is found in an inbred mouse strain. This allelic form of the entire length is termed a haplotype. Prior to the initiation of the mouse genome project, many years of characterization of the mouse Igh locus focused primarily on the Igha haplotype of BALB/c due to the extensive collection of mineral oil–induced plasmacytomas and their monoclonal antibody products that were available in this strain (Potter, 1977). This work was well reviewed in the last edition of this book (Honjo and Matsuda, 1995) and is covered in a comparative manner here. The DNA sequence of the C57BL/6 mouse genome is nearly completed, and this includes the Igh locus. Mouse Igh was expected to be difficult to analyze since it is two to three times longer than human IGH (Chevillard et al., 2002) and is known to contain an unusually high level of Line1 repetitive elements (Herring et al., 1998). Because Igh was accepted as a locus of high biological interest, it was sequenced from a bacterial artificial chromosome (BAC) contig rather than assembled from shotgun reads. A deeply redundant BAC contig was assembled in my laboratory
Molecular Biology of B Cells
Igh-V OR VH GENES OF THE Ighb HAPLOTYPE A search for Vh gene segments in the current assembly of the Ighb sequence identified 170 full length coding sequences (plus additional truncated gene fragments). An additional 20 to 30 sequences are expected in the unfinished 5¢ end of the locus. Of the 170 sequences, 69 have obvious defects in the coding sequence that preclude their expression as functional Vh genes. A search of GenBank, including the EST database, indicates that many of the 101 apparently functional sequences are present, at least as recovered mRNAs. Many others have not been observed and may have defects in their promoter or RSS sequences that render them unable to be expressed. The 101 potentially functional Vh coding sequences were aligned and are displayed as a neighbor joining tree in Figure 2.1. The gene relationships radiate from a central trifurcation that reflects the three Vh gene and protein subgroups originally noted by Kabat (1991). The subgroups further divide into the 15 Vh gene families described by Brodeur and other groups (summarized in Mainville et al., 1996) for the Vh sequences of BALB/c and the Igha haplotype. Strain surveys by Southern blot hybridization indicated
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Copyright 2004, Elsevier Science (USA). All rights reserved.
20
Riblet
FIGURE 2.1 Neighbor-joining tree of the C57BL/6 Vh gene segments. The 101 candidate functional Vh genes group into three major clades (the subgroups of Kabat) and 15 Vh gene families. The Vh gene coding sequences corresponding to the mature heavy chain peptide were extracted from the mouse genome assembly. Apparent pseudogenes containing termination codons were omitted. Vh sequences were aligned and a neighbor-joining tree calculated using ClustalX (Thompson et al., 1997); the tree was plotted with DrawGram in the PHYLIP package (Felsenstein, 1993).
similar gene family organization and content in C57BL/6 and many different Igh haplotypes in lab strains and wild mice (Tutter and Riblet, 1988; Tutter and Riblet, 1989b). The complexity or content of the Vh gene families in the Ighb haplotype of C57BL/6 is listed in Table 2.1. Evident pseudogenes are tabulated separately from sequences that appear functional. The numbers of genes in each Vh gene
family in the assembled C57BL/6 sequence are in general agreement with, but tend higher than, the restriction fragment counts from a C57BL YAC contig of Ighb (Chevillard et al., 2002), and the estimates from BALB/c made by cloning individual genes and counting bands on blots (Brodeur and Riblet, 1984). Most families are small, with one to six members; three of the four Group I families are
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2. Immunoglobulin Heavy Chain Genes of Mouse
TABLE 2.1 Mouse Vh repertoire in the Ighb haplotype Mouse Vh family VhQ52 Vh36–60 Vh3609P Vh12 VhJ558 VhGam3–8 VhSm7 VhX24 Vh7183 VhJ606 VhS107 Vh10 Vh11 Vh15 Vh3609N Totals
Intact genes
Pseudogenes
Total
8 6 8 1 43 4 4 1 10 5 3 2 2 1 3
3 2 4 2 31 0 0 1 16 1 2 4 1 1 1
11 8 12* 3 74* 4 4 2 26 6 5 6 3 2 4
101
69
170
* Gene numbers in these two families represent sequencing from 1.2 Mb. An additional 0.2 to 0.3 Mb remains to be sequenced.
somewhat larger with 8 to more than 12 members. The 7183 family contains 26 sequences, and the J558 family is largest, with 74 sequences currently identified. When finished, the 5¢ portion of the locus should contain an additional 10 to 20 J558 and 3609P sequences. Overall, 40% of the Vh sequences have obvious defects in the coding sequence that preclude their expression, and most families contain such pseudogenes. A majority of sequences in the Vh7183, Vh10, and Vh12 families is defective. A physical map of the Vh gene array is shown in Figure 2.2. It begins at D12Mit263, arbitrarily taken as a boundary between the Dh and Vh gene segment regions, and ends more than two million base pairs later at the 5¢ end of the current assembly. Comparison to the YAC contig and other data indicates that 200 to 300 kb remain to be sequenced. The placement of Vh families in the locus is in agreement with the deletion map of Brodeur (Mainville et al., 1996). The first megabase of the Vh array contains all of 13 of the families. Each family is localized in a subregion, interspersed with several other families. The 5¢ 1100 kb (plus 200 to 300 kb) of the locus contains exclusively VhJ558 and Vh3609P genes. Most of the Vh region is densely occupied by Vh genes, with roughly 10 kb spacing, but genes are more sparse in the distal, 5¢ 600 kb, where spacing averages 20 kb.
POLYMORPHISM IN VH GENES The array of Vh, Dh, and Jh gene segments in Igh defines the germ line, or inherited, antibody heavy chain repertoire, the spectrum of antibody structures that the B cell popula-
tion will make initially and throughout life, although its diversity will be much enhanced by somatic mutation, N region addition, and other junctional mechanisms. This is a basic measure of the universe of bindable antigens. The inherited Vh gene array, the starting library of antibody specificities, can vary between inbred mouse strains, and can affect specific antibody responses. Understanding the extent of the variation between mouse strains, and then between species, will teach us about the acceptable limits for the inherited repertoire and what antibody diversity an animal needs to start with in order to build a successful humoral immune system. We can begin to compare the repertoires of two mouse strains, C57BL/6 and BALB/c. Extensive random cDNA and focused genomic cloning and sequencing efforts have characterized all members of several Vh gene families in BALB/c, and we can compare these to the genomic C57BL/6 repertoire. Figure 2.3 shows a tree of BALB and C57BL/6 Vh genes of the Vh10, VhS107, and Vh7183 families. Both strain sequences were previously known for Vh10 (Whitcomb et al., 1999) and VhS107 (Perlmutter et al., 1985). The Igha haplotype sequences (from strain 129) for Vh7183 were recently completed (Williams et al., 2001). Figure 2.3 shows only the subset of Vh genes in each strain that are clear alleles, and it tabulates the nucleotide divergence between alleles. These differences range from zero (identity throughout the mature protein coding sequence) to 6%. Perhaps more significantly, not shown in this figure are those members of each gene family that do not have alleles in both strains. These have resulted from gene duplications and deletions that occurred independently in the history of the two haplotypes. In the Vh10 family, one additional functional member in Igha was previously known (Whitcomb et al., 1999), and we see a total of five members in C57BL/6, although the three not shown are all pseudogenes. In VhS107, an additional pseudogene occurs in C57BL/6. In Vh7183, 9 allelic pairs are shown in Figure 2.3, but there are also 11 BALB/c and 17 C57BL/6 Vh7183 genes that clearly have no allelic relationship. With the development of such extensive discordance (not sequence divergence as such) over a relatively short evolutionary span (1 to 3 million years), it is apparent that, at least in mice, the inherited library can vary quite extensively with respect to sequence. Whether this is reflected in comparable variation in functional binding specificities cannot yet be addressed.
Dh Figure 2.4 displays the physical map of the D-J-C region of Ighb. Dh sequences in BALB/c have been extensively analyzed (Kurosawa and Tonegawa, 1982; Wood and Tonegawa, 1983; Feeney and Riblet, 1993). One Dh segment, DhQ52, is only 700 bp from the first Jh segment. Additional Dh segments are scattered in a region of at least
74 psi
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FIGURE 2.2 Physical map of the C57BL/6 Vh gene cluster. The positions of 170 full-length Vh gene segments of the Ighb haplotype are shown to scale. The genes are named according to their family, “b” haplotype, and position in the array starting at the 3¢ end. For example, Vh7183.b1Psi and Vh7183.b2 are the b alleles of E4Psi and Vh81X, respectively. Apparent pseudogenes are shown in gray. The scale is in kb, and 2.1 Mb of the Vh cluster is shown. An estimated 200 to 300 kb at the 5¢ end of the Vh array remain to be sequenced. 0p si
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2. Immunoglobulin Heavy Chain Genes of Mouse
apart, but DhFL16b and Vh7183.b1Psi (the C57BL/6 allele of E4Psi) are separated by 90 kb.
Jh The four Jh gene segments were isolated as 1,340 bp Jh locus PCR products from mouse strains of ten Igh haplotypes and sequenced by Solin and Kaartinen (1992). The J locus from C57BL/10 reported by Solin is identical to the genomic sequence of C57BL/6 and differs from BALB/c at eight nucleotides. The Jh coding segments are identical, except for one nucleotide difference resulting in an amino acid replacement in Jh1 between BALB and the C57BL strains.
Ch
FIGURE 2.3 Tree of alleles of 3 Vh families. For the Vh10, VhS107, and Vh7183 gene families the alleles from the Igha and Ighb haplotypes were aligned and a neighbor-joining tree calculated. For each allele pair, the percentage nonidentity was calculated. Vh gene segments from the Ighb haplotype are shown in black, the Igha haplotype in gray.
80 kb between DhQ52 and the first Vh gene, E4Psi. These include two DFL16 segments, nine DSP2 segments, a DST4 segment, and an undetermined number of defective D-like pseudogenes. These 13 listed Dh segments of the Igha haplotype are found in productive VDJ rearrangements (Feeney and Riblet, 1993). Comparable detailed analysis of Dh usage in the Ighb haplotype is lacking. On the basis of genomic sequence, nine Dh segments have an intact RSS on each side. These include evident homologs for 3¢ DhQ52 and DhST4 segments and the 5¢ DhFL16.1; these enclose a series of six DhSP2 segments. Additionally, eight homologs of the BALB/c D1Psi pseudogene alternate with the DhSP2 and DhFL16 segments. These apparent pseudo-Dh sequences lack one or both consensus RSS segments. A dotplot of this region reveals a pattern of 5 kb duplications yielding the alternating Dh–DhPsi pattern. The spacing between the separate clusters of Jh and Dh, and Dh and Vh gene segments is interestingly different. Jh1 and DhQ52 are only 700 bp
The heavy chain constant region gene segments (Ch) encode the C-terminal major portion of the heavy chain protein. The eight classes or isotypes of constant regions and the respective isotypes of serum immunoglobulin in mouse are m and IgM, d and IgD, g1 and IgG1, g2a and IgG2a, g2b and IgG2b, g3 and IgG3, e and IgE, and a and IgA. The physical map of the 200 kb long Ch gene region in BALB/c was determined by Shimizu et al. (1982). The sequencebased map of Ighb shown here is in agreement with BALB/c, with minor variations in intergenic spacing. The significant difference in the Ch genes of the two haplotypes is the replacement of IgG2a in BALB/c by IgG2c in C57BL/6, as previously described (Fukui et al., 1984; Morgado et al., 1989). This is apparently the result of the duplication of an ancestral g2 gene to create g2a and g2c isotypes, subsequent sequence divergence, and finally the loss of alternate genes in the two haplotypes. This explanation is confirmed by the presence of both isotypes in wild Asian and European mouse haplotypes (Fukui et al., 1984; JouvinMarche et al., 1989).
3¢ Regulatory Region (Enhancer) Downstream (3¢) of the Ca gene segment is the 3¢ regulatory region (3¢RR), which has enhancer activity for the transcription of Igh. This region is important in the regulation of isotype switching as well (Manis et al., 2003; Pinaud et al., 2001). It is characterized by a complex series of direct and inverted repeats that produce nested palindromes (Chauveau and Cogne, 1996). Recently, the complete sequence of this region was obtained from a 125 kb BAC from 129, an Igha haplotype mouse strain (Zhou et al., 2002a). This work also defined the positions of the nearest genes flanking Igh on the centromeric side: Crip and Mta1. The genomic sequence of C57BL/6 matches the 129 sequence with small variations in nucleotide sequence and intergenic distances.
24
Ce
D1 2M it4 1
Ca
D1 2M it1 9
D1 2M it1 8
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Cg2b
Cg1
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01 b
200 Dh Dh02b Dh03b 04 b Dh 05 Dh b 06 b Dh Dh07b 08 b Dh Dh09b 10 b Dh 11 Dh b 12 b Dh Dh13b 14 b Dh 1 Dh 5b 16 b
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3
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it2 0
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FIGURE 2.4 Physical map of the Ch, Jh, and Dh clusters. 300 kb of the Ighb locus is diagrammed, starting at the 3¢ end of Igh, which is centromeric on chromosome 12. The eight Ch gene segments and the small cluster of four Jh segments occupy 200 kb. The Dh segments occupy the next 100 kb, ending at the simple sequence marker D12Mit263. The Dh segments are identified as Dh01b–Dh17b. Dh01b is the Ighb allele of the BALB DhQ52, Dh02b the allele of DhST4, and Dh16b the allele of DhFL16.1. Dh04b, Dh06b, Dh08b, Dh10b, Dh12b, and Dh14b are DhSP2 segments, and the alternating Dh03b, Dh05b, Dh07b, Dh09b, Dh11b, Dh13b, Dh15b, and Dh17b are Dh1Psi pseudogenes.
EVOLUTION Comparison of the human IGH and mouse Igh loci reveals some basic similarities: The constant region classes or isotypes, IgM, IgD, IgG, IgE, and IgA, are present in both and probably in all mammals. Each species may have different numbers of duplicated members of some isotypes, particularly IgG and IgA genes. Similarly, the Jh and Dh gene segments vary in number; the human content is higher than the mouse in both cases. In contrast, the mouse Vh gene array is larger than the human; the mouse has about 200 Vh gene segments spaced over more than 2 Mb, compared with about 100 Vh genes in 1 Mb in man. Analysis of the V sequences in both species reveals the same trifurcation into three subgroups, noted by Kabat (1991) as shown for mouse in Figure 2.1. This evolutionary trifurcation is ancient, existing in most mammals (Tutter and Riblet, 1989a). In each species, there is division of subgroups into gene families, seven in man and fifteen in mouse as shown. Similarities of certain families exist across species (Tutter and Riblet 1989a), but with no evident correspondence of individual Vh genes between mouse and man. Evolutionary analysis of individual Vh gene sequence and specificity will require comparison of different mouse haplotypes and comparisons of mouse and rat. Such studies are in progress.
GENOMIC CONSIDERATIONS The genomic sequence facilitates consideration of the Igh locus as a whole, as a functional entity, and enables us to ask what distinguishes it from surrounding genes, what
boundaries mark the edges of the locus-specific regulation of Igh? What characteristics of the locus relate to the mechanisms that activate and regulate the intricate series of genomic alterations involved in VDJ recombination, class switch recombination, somatic mutation, and allelic exclusion during the development of a B cell?
3¢ Border The genes that flank Igh on its 3¢ side, Crip and Mta1, are not B cell–specific; rather, they are expressed in many cell types and are regulated independently of Igh. The boundary of transcriptional regulation between the Igh region (the Igh structural genes and 3¢RR) and these flanking loci coincides with a remarkable developmentally regulated origin of DNA replication (Zhou et al., 2002a). In non-B cells and in mature B cells, a replication fork initiates at this origin at the beginning of S phase in the cell cycle and travels 5¢ though the 3¢ RR, Ch, Jh, and Dh regions and into the Vh genes. This progression occurs over nearly the entire span of the S phase and covers a distance of over 400 kb. Late in S phase, the remaining portion of Igh, more than 2 Mb containing most Vh gene segments, is replicated by forks moving in both directions. In a contrasting pattern in pro- and pre-B cells, where Igh becomes activated to undergo VDJ recombination, the entire locus is replicated early in S by forks moving in both directions. The significance of this unusual replication pattern is not clear, but its two transitions, first as the locus is activated for rearrangement, and second as the locus completes rearrangement and is ready for stable high-efficiency transcription, are correlated with other changes in chromatin structure and accessibility.
25
2. Immunoglobulin Heavy Chain Genes of Mouse
5¢ Border The 5¢ end of Igh is distal (telomeric) in both mouse and man. In man, IGH is immediately adjacent to the chromosome 14 telomere (Cook et al., 1994). In mouse, this is not the case; although Igh is far distal on chromosome 12 there are several million base pairs of genome before the telomere. The genes flanking the 5¢ end of Igh are Zfp386, a Kruppel-like zinc finger protein identified in EST sequencing, and Vipr2. In man, homologous sequences are located on 7q36. 1 to 2 Mb farther distal in mouse are the genes Sp4 and Dnahc11 (iv, situs inversus). In man, these are on 7p15–21. This mouse genomic information is based partially on genomic sequencing and clone assembly, and it extends previous published and unpublished genetic mapping studies (Brueckner et al., 1989; de Meeus et al., 1992).
Nuclear Location and Chromatin Structure The C57BL/6 and 129/Sv BAC contigs that were assembled for the genomic sequencing of Igh have provided the reagents for other studies of the locus as a whole. Fluorescent in situ hybridization (FISH) studies using BAC probes showed that the Igh locus undergoes a cyclical change in location in the nucleus in differentiating B cells (Kosak et al., 2002; Zhou et al., 2002b). In non-B cells and hematopoietic progenitors, the Igh locus is positioned at the nuclear periphery, associated with the nuclear lamina. In pro- and pre-B cells, Igh repositions towards the nuclear center, and in B- and plasma cells it moves back to the edge. In addition, the locus undergoes a compaction when it leaves the periphery. The ends of this 3 Mb locus are brought closer together, presumably to facilitate VDJ recombination (Kosak et al., 2002). Increased knowledge of the sequence and structure of Igh has also facilitated detailed studies of transcriptional regulation and chromatin changes across the locus during B cell development (Chowdhury and Sen, 2001; Johnson et al., 2003). These have shown a strong correlation of histone acetylation with accessibility and activation of the locus for rearrangement. It is straightforward to hypothesize that these alterations in chromatin structure and locus accessibility are mechanistically correlated with developmental alterations in replication patterns, nuclear location, and compactness of Igh. However, which, if any, of these parameters is the initiator or first link in the intricate chain of developmental events, and how these different steps are linked together, are important questions yet to be addressed.
CONCLUSION Several decades of structural studies of Igh have culminated in the nearly complete DNA sequence of this 3 Mb
locus. This has yielded a complete definition of all the gene segments in the mouse locus that can now be manipulated to answer questions about the immune repertoire and can be compared to human and other species. It has also led to novel findings of global changes in replication patterns, nuclear location, and chromatin structure that offer new avenues to study antibody gene actions and B cell development.
References Brodeur, P. H., and Riblet, R. (1984). The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. Eur J Immunol 14, 922–930. Brueckner, M., D’Eustachio, P., and Horwich, A. L. (1989). Linkage mapping of a mouse gene, iv, that controls left-right asymmetry of the heart and viscera. Proc Natl Acad Sci U S A 86, 5035–5038. Chauveau, C., and Cogne, M. (1996). Palindromic structure of the IgH 3¢locus control region. Nat Genet 14, 15–16. Chevillard, C., Ozaki, J., Herring, C. D., and Riblet, R. (2002). A threemegabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus. J Immunol 168, 5659–5666. Chowdhury, D., and Sen, R. (2001). Stepwise activation of the immunoglobulin mu heavy chain gene locus. EMBO J 20, 6394–6403. Cook, G. P., Tomlinson, I. M., Walter, G., Riethman, H., Carter, N. P., Buluwela, L., Winter, G., and Rabbitts, T. H. (1994). A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q. Nat Genet 7, 162–168. de Meeus, A., Alonso, S., Demaille, J., and Bouvagnet, P. (1992). A detailed linkage map of subtelomeric murine chromosome 12 region including the situs inversus mutation locus IV. Mamm Genome 3, 637–643. Feeney, A. J., and Riblet, R. (1993). Dst4: A new, and probably the last, functional Dh gene in the BALB/c mouse. Immunogenetics 37, 217–221. Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package). Fukui, K., Hamaguchi, Y., Shimizu, A., Nakai, S., Moriwaki, K., Wang, C. H., and Honjo, T. (1984). Duplicated immunoglobulin gamma 2a genes in wild mice. J Mol Cell Immunol 1, 321–330. Herring, C. D., Chevillard, C., Johnston, S. L., Wettstein, P. J., and Riblet, R. (1998). Vector-hexamer PCR isolation of all insert ends from a YAC contig of the mouse Igh locus. Genome Res 8, 673–681. Honjo, T., and Matsuda, F. (1995). Immunoglobulin heavy chain loci of mouse and human. In Immunoglobulin genes, T. Honjo and F. W. Alt, eds. (London, Academic Press), pp. 145–171. Johnson, K., Angelin-Duclos, C., Park, S., and Calame, K. L. (2003). Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development. Mol Cell Biol 23, 2438–2450. Jouvin-Marche, E., Morgado, M. G., Leguern, C., Voegtle, D., Bonhomme, F., and Cazenave, P. A. (1989). The mouse Igh-1a and Igh-1b H chain constant regions are derived from two distinct isotypic genes. Immunogenetics 29, 92–97. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991). Sequences of proteins of immunological interest. U.S. Dept of Health and Human Services, Washington, D.C. Kosak, S. T., Skok, J. A., Medina, K. L., Riblet, R., Le Beau, M. M., Fisher, A. G., and Singh, H. (2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296, 158–162. Kurosawa, Y., and Tonegawa, S. (1982). Organization, structure, and assembly of immunoglobulin heavy chain diversity segments. J Exp Med 155, 201–218.
26 Mainville, C., Sheehan, K., Klaman, L. D., Giorgetti, C. A., Press, J. L., and Brodeur, P. H. (1996). Deletional mapping of fifteen mouse Vh gene families reveals a common organization for three Igh haplotypes. J Immunol 156, 1038–1046. Manis, J. P., Michaelson, J. S., Birshtein, B. K., and Alt, F. W. (2003). Elucidation of a downstream boundary of the 3¢ IgH regulatory region. Mol Immunol 39, 753–760. Morgado, M. G., Cam, P., Gris-Liebe, C., Cazenave, P. A., and Jouvin-Marche, E. (1989). Further evidence that BALB/c and C57BL/6 gamma 2a genes originate from two distinct isotypes. EMBO J 8, 3245–3251. Perlmutter, R. M., Berson, B., Griffin, J. A., and Hood, L. (1985). Diversity in the germline antibody repertoire. Molecular evolution of the T15 VH gene family. J Exp Med 162, 1998–2016. Pinaud, E., Khamlichi, A. A., Le Morvan, C., Drouet, M., Nalesso, V., Le Bert, M., and Cogne, M. (2001). Localization of the 3¢ IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15, 187–199. Potter, M. (1977). Antigen-binding myeloma proteins of mice. Adv Immunol 25, 141–211. Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. (1980). Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286, 676–683. Shimizu, A., Takahashi, N., Yaoita, Y., and Honjo, T. (1982). Organization of the constant region gene family of the mouse immunoglobulin heavy chain. Cell 28, 499–506. Solin, M. L., and Kaartinen, M. (1992). Allelic polymorphism of mouse Igh-J locus, which encodes immunoglobulin heavy chain joining (JH) segments. Immunogenetics 36, 306–313. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997). The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Riblet Tutter, A., and Riblet, R. (1988). Duplications and deletions of Vh genes in inbred strains of mice. Immunogenetics 28, 125–135. Tutter, A., and Riblet, R. (1989a). Conservation of an immunoglobulin variable-region gene family indicates a specific, noncoding function. Proc Natl Acad Sci U S A 86, 7460–7464. Tutter, A., and Riblet, R. (1989b). Evolution of the immunoglobulin heavy chain variable region (Igh-V) locus in the genus Mus. Immunogenetics 30, 315–329. Whitcomb, E. A., Haines, B. B., Parmelee, A. P., Pearlman, A. M., and Brodeur, P. H. (1999). Germline structure and differential utilization of Igha and Ighb VH10 genes. J Immunol 162, 1541–1550. Williams, G. S., Martinez, A., Montalbano, A., Tang, A., Mauhar, A., Ogwaro, K. M., Merz, D., Chevillard, C., Riblet, R., and Feeney, A. J. (2001). Unequal v(h) gene rearrangement frequency within the large v(h)7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location. J Immunol 167, 257–263. Wood, C., and Tonegawa, S. (1983). Diversity and joining segments of mouse immunoglobulin heavy chain genes are closely linked and in the same orientation: implications for the joining mechanism. Proc Natl Acad Sci U S A 80, 3030–3034. Zhou, J., Ashouian, N., Delepine, M., Matsuda, F., Chevillard, C., Riblet, R., Schildkraut, C. L., and Birshtein, B. K. (2002a). The origin of a developmentally regulated Igh replicon is located near the border of regulatory domains for Igh replication and expression. Proc Natl Acad Sci U S A 99, 13693–13698. Zhou, J., Ermakova, O. V., Riblet, R., Birshtein, B. K., and Schildkraut, C. L. (2002b). Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells. Mol Cell Biol 22, 4876–4889.
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3 Immunoglobulin k Genes of Human and Mouse HANS G. ZACHAU Adolf Butenandt Institut, Molekularbiologie, Universität München, Germany
The immunoglobulin k gene chapter in the first edition of this book covered all that was known at the time about the genes of humans and mouse (Zachau, 1989), and the chapter in the second edition concentrated on the human k genes (Zachau, 1995). Although an enormous amount of data on the structure, function, and evolution of the k genes has accumulated in the meantime, the present chapter again deals with the genes of both species. This is possible only since several aspects of the k genes are covered in other chapters of this book. With respect to the early work on k genes, the reader is referred to the previous reviews. In the present chapter, some basic facts on the k genes are recounted, but the emphasis is on the recent work. If a topic is dealt with in several publications, only the latest one is quoted here.
that show a lower or higher degree of sequence variation, respectively. The recombination signal sequences at the 3¢ side of the Vk genes consist of conserved 7mer and 9mer sequences at a distance of 12 bp from each other. The early research on antibodies and antibody genes of mouse, human, and other species is recounted in the book by Kindt and Capra (1984). The molecular genetics of immunoglobulins is presented not only in textbooks, but also in reviews (e.g., Max, 1999).
2. HUMAN IMMUNOGLOBULIN k GENES The work on human k genes has been reviewed by Zachau (1989, 1995, 1996, 2000), by Lefranc and Lefranc (2001), and in the database of Lefranc (2002). The results of our group are summarized on the Internet (Zachau, 2001).
1. GENERAL FEATURES OF HUMAN AND MOUSE k GENES
2.1 Elucidation of the Human k Locus
The human and the mouse k loci contain extended Vk gene regions and one Jk–Ck gene region. A typical Vk gene consists of upstream regulatory sequences, a leader sequence, the region coding for the k protein, and, at the 3¢ side, the recombination signal sequences. The upstream regulatory elements comprise, in addition to a TATA box, more or less conserved 10mer and 15mer sequences (Falkner and Zachau, 1984; Schäble and Zachau, 1993; Bemark et al., 1998). The leader sequence is interrupted by an intron, which results in an L and L¢ sequence, the latter being contiguous with the sequence coding for the k protein. Comparison of all known k protein sequences in the database of Kabat (2002; Johnson and Wu, 2001) led to the definition of three framework and three complementarity-determining regions
Molecular Biology of B Cells
A prominent feature of the human k locus is the duplication of most of its Vk gene region. The first pairs of very similar but not identical Vk genes were detected by Bentley and Rabbitts (1983) and by Pech et al. (1985). Numerous cosmid and phage l clones were mapped in our laboratory by restriction nuclease cleavage and assembled in large contigs (review Zachau, 1995). When yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) became available, we also used those (BrensingKüppers et al., 1997; Kawasaki et al., 2001). A so-called Ck proximal (p) contig of 600 kb comprises, in addition to the Jk–Ck gene region, 40 Vk genes; a distal (d) contig of 440 kb contains 36 Vk genes. The p and d copies of the k locus are arranged in opposite 5¢–3¢ polarities. These
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Copyright 2004, Elsevier Science (USA). All rights reserved.
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Zachau
are inverted repeats with a still uncloned region of 800 kb in between, which does not seem to contain any Vk genes (Weichhold et al., 1993a). The structure is largely symmetrical starting from a center in the uncloned region. The data on the region of the k locus between the first Vk gene 23 kb upstream of Jk1, which we called B3, and the k deleting element (kde) 24 kb downstream of Ck (Klobeck and Zachau, 1986) were reviewed (Zachau, 1995). No nonVk gene sequences were detected within the k locus, but a transcribed region was found 46 kb downstream of Ck; this was termed BENE (Lautner-Rieske et al., 1995) because of its homology to the membrane protein MAL (de Marco et al., 2001 and earlier literature). A Vk orphon sequence (2.5) was found 1.5 Mb downstream of Ck (Huber et al., 1994),
and another 0.5 Mb further downstream the CD8a locus was localized (Weichhold et al., 1993b).
2.2 Vk Genes, Pseudogenes, Relics, and Repetitive Elements Within the Human k Locus In our laboratory in Munich, we sequenced only the Vk genes and regions of special interest, but more recently we gave our clones to N. Shimizu’s group in Tokyo, who sequenced with them the whole locus (Kawasaki et al., 2001). The results of the mapping and sequencing work on the k locus are summarized in Figure 3.1.
FIGURE 3.1 Comprehensive map of the human immunoglobulin k locus, taken from Kawasaki et al. (2001). (A) Locations of the clones used in sequencing. Sequenced and unsequenced regions are depicted as red and green lines, respectively. (B) Locations of the genes. Vk (red), Jk1-5 (sky blue), and Ck (blue) genes with the same transcriptional polarity are indicated as small vertical lines on the same side of the horizontal line. Lines with full height, 2/3 height, and 1/3 height represent Vk genes with ORFs, pseudogenes with >200 bp, and relics with <200 bp in length, respectively. Relics consisting only of exon I are not included. The names of the Vk genes with ORFs are shown. Thick horizontal lines within the k locus represent duplicated regions; thin horizontal lines indicate regions, which exist in either the proximal or the distal unit. Wedge-shaped shadows indicate deletion events that happened after the inverted duplication. The 6-kb regions between the two wedges show sequence homology but do not seem to be inverted duplication counterparts; rather they correspond to adjacent biock duplicates generated prior to the inverted duplication. (C) Six categories of interspersed repeats are indicated; Alu (green), MIR (blue), LINE L1 and L2 (red), LTR (yellow), DNA transposons (sky blue), and others (purple). (D) The GC content was plotted with a window size of 4,000 nt and with a sliding size of 2,000 nt. (E) Sequence identity without indels between the proximal unit and the distal unit was plotted with a window size of 10,000 nt and with a sliding size of 500 nt. Thirteen homology blocks (A–M) and the average sequence identities (red dashed lines) are indicated. The scale at the bottom of the figure shows the proximal unit; it does not take into account the gaps in the distal unit. See color insert.
3. Immunoglobulin k Genes of Human and Mouse
Seventy-six Vk genes and pseudogenes were identified in the k locus by restriction mapping and sequencing. Eight solitary Vk genes and 34 gene pairs occur with 95 to 100% sequence identity between the p and d copy genes. Thirtytwo of the 76 Vk genes are potentially functional, and 25 are pseudogenes. Minor defects were found in 16 genes, and three genes have potentially functional alleles and alleles with minor defects. A minor defect was defined as a one or two 1-bp alteration in a gene; for example, the occurrence of a stop codon and/or a deviation from the canonical sequences of a regulatory element, a splice site, or a recognition sequence. The genes with minor defects are taken as a separate class of genes, since functional alleles may exist in the human population for more than the three aforementioned genes. This is not to be expected for pseudogenes, which usually carry several defects each. The Vk genes and pseudogenes, including all alleles known to us at the time, and the conserved sequence elements, were compared in a review by Schäble and Zachau (1993). The pseudogenes, the unique sequences, and the repetitive sequences of the k locus were dealt with by Schäble et al. (1994). The sequencing of the whole locus revealed (in addition to the 76 Vk genes) 55 truncated pseudogenes and relics located in the stretches between the Vk genes (Kawasaki et al., 2001). In this publication, the total number of genes and relics is given as 132, since the orphon Z0 (see 2.5) is included in the number; this is not done in this review. The truncated pseudogenes (>200 bp) and the relics (<200 bp) had not been detected by the hybridization techniques employed in our mapping work. The structural features of all sequence elements of the locus and the presence or absence of their rearrangement, transcription, and translation products are compiled in Table 1 of Kawasaki et al. (2001). The classification of k proteins into four subgroups, as used in the database of Kabat (2002), was fully confirmed when the gene sequences became known (Schäble and Zachau, 1993). For subgroups V–VII, no proteins have been found; they are defined on the basis of the nucleotide sequences only. The same is true for the truncated pseudogenes and relics, which have been grouped into five further subgroups VIII–XII. In a phylogenetic tree of the genes and relics, all subgroups can be distinguished (Kawasaki et al., 2001). The average GC content of the k locus is 40.7%, indicating that it belongs to an AT-rich L isochore (Bernardi, 2000). More than 34.9% of the locus consists of interspersed repeats. These were assigned to seven different classes (Kawasaki et al., 2001) and are depicted as a colorful bar in Figure 3.1. The nomenclature of Vk genes used in our publications (O1–O18, A1–A30, L1–L25, B1–B3) evolved as the elucidation of the locus proceeded cluster by cluster. However, in retrospect it became clear that at least some of the clus-
29
ters have a structural, probably evolution-derived significance, since their gene regions share certain sequence features. A systematic nomenclature of the 76 Vk genes and pseudogenes was proposed by Lefranc (2001). All 131 Vk genes, pseudogenes, and relics were designated by a systematic nomenclature stating the gene family and the location within the locus; sequences in homologous positions in the two copies of the locus are distinguished by the suffixes “p” and “d” (Kawasaki et al., 2001); various nomenclatures are compared of this publication. Our old nomenclature is used by many groups today. However, now that the k locus has been sequenced, anybody starting new systematic work on the k genes or gene products also may employ one of the systematic nomenclatures and correlate it to the historic one.
2.3 Rearranged and Expressed Human Vk Genes How many germline Vk genes are actually rearranged, transcribed, and translated? This question was studied by sequencing 70 clones from a cDNA library prepared from spleen mRNA, as well as by comparing numerous rearranged genomic Vk genes, cDNAs, and k proteins from the literature (Klein et al., 1993). Not all potentially functional Vk genes were found to be rearranged and expressed, whereas the alleles of some genes with minor defects are rearranged and expressed. The assignment of the products to the germline genes was summarized in a figure (Klein et al., 1993; Figure 2 in Zachau, 1995) and in a recent survey (Table 1 in Kawasaki et al., 2001). Due to somatic mutations and processes at the V–J junction, several rearrangement and expression products cannot be clearly assigned to one or the other gene of a homologous gene pair. Such products must be attributed to the gene pairs, and both genes of the pair were assumed to be active. In this way, the observed rearrangement products are derived from 42, the cDNAs from 35, and the proteins from 33 germline genes. Considering the aforementioned uncertainties of some assignments, it is reasonable to suppose that about half the 76 germline Vk genes contribute to the light chain repertoire. Possible reasons for the absence of functional products from certain potentially functional genes have been discussed (Klein and Zachau, 1995; Kawasaki et al., 2001). Tomlinson et al. (1995) defined 40 germline Vk genes as “functional” on the basis of structural considerations of the main chain conformations, or canonical structures, they encode. The authors state that all or most of those genes are rearranged. The expression of the genes was not studied. The systematic search for rearrangement and expression products also permitted two conclusions: In mapping the k locus by hybridization, we had not missed any Vk genes, and the Vk genes outside the locus (the orphons [2.5]) are not rearranged or expressed.
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The alignment of the sequences of the germline genes and their products, which were, of course, derived from many different individuals, indicated that the extent of allelic polymorphism in the k locus is low (2.4). The alignments were also useful in defining the preferred sites of somatic mutation. The 5¢–3¢ polarity of the Vk genes within the locus determines the type of Vk–Jk rearrangement. The two Jk proximal Vk genes and all Vk genes of the d copy, whose polarities are opposite to that of Jk–Ck, are arranged by an inversion mechanism (3.2), whereas the other genes rearrange by deletion of the DNA between the Vk and Jk genes (Weichhold et al. 1990). In Figure 3.1 the polarities are indicated by the gene bars pointing either up or down. The work on the germline and expressed Vk genes in apparently healthy individuals was also the basis of studies on the k repertoires in different developmental stages (e.g., Girschick and Lipsky, 2001), in particular disease states (e.g., Pyon et al., 2001), or in population studies (e.g., Padyukov et al., 2001). In a search for allelic variations in the k locus, an additional Vk gene was found in 12 of 57 individuals (Juul et al., 1998). This gene, termed La, is rearranged and transcribed, but it was not yet mapped; it was found to be only 94% similar to the closest known gene of the k locus. Transgenic mice with human immunoglobulin genes produced human immunoglobulins (e.g., Gallo et al., 2000). A Ck sequence altered in one codon was found to give rise to amyloid deposits in a patient (Solomon et al., 1998). Many aspects of immunoglobulin gene expression were reviewed in a symposium (Casali and Silberstein, 1995).
2.4 Polymorphisms in the Human k Locus Since the polymorphisms were extensively discussed earlier (Zachau, 1995; 1996), only three facts should be mentioned here. 1. The extent of allelic variation in the k locus seems to be fairly low (2.3). Therefore, no problems were encountered when we used DNA samples from different human individuals in our structural work 2. In the elucidation of the duplicated parts of the locus, the duplication-differentiating probes played an important role (Pargent et al., 1991) 3. The only startling polymorphism detected is one in which the whole d copy, with its 36 Vk genes, is missing. This so-called haplotype 11 was found in an apparently healthy individual, who is homozygous for it (Pargent et al., 1991). The d-copy gene A2 codes for the most common light chain in the Haemophilus influenzae response. Vaccination of individual 11 with the appropriate carbohydrate vaccine gave rise to antibodies whose light chains were derived, of course, from p-copy genes but carried more mutations than the usual A2-derived light chains (Scott et al., 1992). Haplotype 11 is rather rare (Schäble et al., 1993). Indirect evidence indicates that it is due to a deletion
rather than to the persistence of an evolutionary early nonduplicated structure (Weichhold et al., 1993a).
2.5 Vk Genes Outside the Human k Locus The Vk genes dispersed to positions outside the k locus were first found by Lötscher et al. (1986). They are called orphons in analogy to the histone and ribosomal RNA genes located outside the respective loci. All Vk orphons contain introns and, therefore, certainly have been dispersed at the DNA level and not by mRNA retrotranscription. A group of VkI orphons, the so-called Z family, has closely similar sequences. To date, 24 Vk orphons have been cloned and sequenced. About a dozen further orphons, some of them belonging to the Z family, were detected but not further studied; they may represent new loci or may be alleles of known orphons. Two Vk orphons have no defects in their sequences, but they are included in this group because of their location outside of the locus. The other orphons are pseudogenes, also according to their sequence characteristics. Vk orphons were localized on chromosome 1, 2, 22, and others (review Zachau, 1995). The 12 Vk orphons on the long arm of chromosome 2 (2cen-q13) are of particular interest. These were probably translocated by a pericentric inversion from 2cen-p13, where also the k locus is located. Such inversions seem to be ongoing processes, since 0.1% of the human population carries inverted chromosomes 2 with the k locus on the long arm and the group of orphons on the short arm (LautnerRieske et al., 1993). An orphon on the short arm of chromosome 2, called Z0, was found at a distance of about 140 kb beyond the last Vk gene of the distal copy of the locus (Brensing-Küppers et al., 1997). Z0 may have been the first orphon that left the k locus and became the parent of the other Z-family orphons (Kawasaki et al., 2001). The sequence of ZO is 99.3% and 97.7% identical with the sequences of the Z-family orphons on chromosomes 1 and 22, respectively, but at best 92.6% to the Vk genes within the k locus. The cluster of five orphons on chromosome 22 has no direct counterpart within the k locus either, suggesting that its precursor, as the one of the Z orphons, left an early k locus by a nonduplicative transposition (Kawasaki et al., 2001). A practical note: In searching through hybridization or polymerase chain reactions (PCR) for the germline origin of a rearranged Vk gene or a cDNA, one has a good chance of finding not only genes in the locus, but also orphon sequences.
3. MOUSE IMMUNOGLOBULIN k GENES The earliest work on immunoglobulin genes was on the k genes of the mouse. This research took place in 1974–1975
3. Immunoglobulin k Genes of Human and Mouse
in the laboratory of S. Tonegawa in Basel. In our laboratory, the work on mouse immunoglobulin k genes started in the late 1970s with studies on the chromatin structure of germline and rearranged k genes, on Vk–Jk rearrangements, on nonfunctional Vk–Jk joining, on somatic hypermutation, and on the expression of k genes (review Zachau, 1989). Human k genes were included in some of those experiments for comparison and, from the early 1980s on, we concentrated on the elucidation of the structure of the human k locus (2.1); this research took about 12 years. Only in 1992 did we return to the mouse k genes.
3.1 Elucidation of the Mouse k Locus The strategy of work on the mouse k locus was similar to that used on the human locus: identification of germline Vk genes by hybridization with subgroup-specific probes, cloning of the gene regions, contructing contigs from overlapping clones, and, finally, chromosomal walking to link the contigs. But the work proceeded faster than that on the human k locus, since YAC- and BAC-cloning and longrange PCR had been introduced into genome work in the meantime. Numerous Vk germline and rearranged genes, as well as cDNAs, had been characterized in various mouse strains (review Kofler et al., 1992), but only one contig of two Vk genes was known at the time (Lawler et al., 1992). We prepared a cosmid library of C57BL/6J mouse DNA and screened it with 18 probes, which were more or less specific for the different Vk gene families. The contigs and the still unlinked cosmid clones covered 1.6 Mb, with 85 strong and 11 weak Vk hybridization signals (Zocher et al., 1995). First experiments with Vk gene containing YACs were reported by George et al. (1995). In a systematic study, 43 YACs with C57BL/6J and C3H mouse DNA were analyzed and the first evidence for about 140 Vk genes was obtained (Kirschbaum et al., 1996). A size of 3.5 Mb of the k locus was estimated on the basis of YAC contigs (Schupp et al., 1997). To obtain a detailed restriction map of the locus and exactly localize the Vk genes, cosmid sublibraries were prepared from the YACs. Since the YACs tended to undergo recombinations and deletions, BAC clones were included into our analysis. All maps were eventually established on the level of cosmid clones and subclones thereof. Contigs were designated Z1 at the Ck end to Z9 at the 5¢ end of the locus, and this is how the maps and the gene localizations were reported in the literature up to 1999. The results on the 3¢ and the central parts of the locus were published by Kirschbaum et al. (1998; 1999) and those on the 5¢ part were published by Röschenthaler et al. (1999; 2000). Special attention was paid to the Vk genes and gene families (Thiebe et al., 1999), as well as to the relics and orphons (Schäble et al., 1999). A panel of BAC clones allowed Röschenthaler et al. (2000) to close the gaps in the 5¢ part of the k locus and to show that one of the previously defined contigs (Z7) is
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located near, but not within, the locus. The three pseudogenes of this contig are therefore orphons (3.3). Conversely, three additional Vk genes were found on the BAC clones. The picture of the k locus, as it stood in 1999–2000, at the time of my retirement when our laboratory closed, is that there is a 5¢ contig of 1.88 Mb with 82 Vk genes and a 3¢ contig of 1.04 Mb with 51 Vk genes. In between are two contigs of 65 and 105 kb with 2 and 5 genes, respectively. The detailed restriction maps of the k locus on the Internet (Zachau, 2001) represent this structure. Three gaps of 10 to 40 kb each, comprising together about 90 kb, in the central part of the locus were not bridged in our work because of internal duplications and difficulties with rearranged YACs. A new sublibrary, which would have been required to close the gaps, was not established, since the missing data should become available soon from the mouse genome project. The size of the locus is taken to be about 3.2 Mb, in fair agreement between results of pulsed field gel electrophoresis (PFGE) experiments and the addition of the sizes of the numerous clones. An a-tubulin genelike sequence and an S-adenosyl methionin decarboxylase genelike sequence were detected in the 5¢ and 3¢ contigs, respectively (Röschenthaler et al., 1999; Kirschbaum et al., 1999). The regulatory sequences upstream and downstream of Ck were studied in detail (e.g., Liu et al., 2002). A gene coding for ribose 5-phosphate isomerase was localized 40 to 50 kb downstream of Ck (Apel et al., 1995).
3.2 Vk Genes and Pseudogenes Within the Mouse k Locus One hundred and forty Vk genes and pseudogenes were localized within the k locus, cloned, and sequenced. There are indications that two to five additional Vk genes or pseudogenes, which we were not able to identify, exist in the locus. Less than a third of the Vk genes are oriented in the same 5¢–3¢ polarity as Jk–Ck; the rest are in the opposite polarity. The map positions of the genes and their 5¢ to 3¢ polarities can be seen in the above-mentioned publications and in our final form on the Internet (Zachau, 2001). A so-called signal joint remains in the locus when the rearrangement occurs by inversion. This joint was first cloned from the DNA of a mouse myeloma and sequenced by Steinmetz et al. (1980). Seventy-five of the 140 genes are functional: that is, transcription and/or translation products are known; 21 potentially functional genes have no structural defects, but no expression products have been found yet; 44 Vk genes are pseudogenes (Thiebe et al., 1999; Röschenthaler et al., 2000). In the mouse k locus, we defined no “genes with minor defects” in the human k locus. The characteristics of the genes, their accession numbers, and the expression products are compiled in tables (Kirschbaum et al., 1998; Schäble et al., 1999). A combination of the tables is
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included, together with additional information, in our Internet site (Zachau, 2001). Instead of presenting here detailed maps and tables, an illustration of the mouse k locus, as it was envisaged in 1999, is shown (Figure 3.2). The Vk genes and pseudogenes were assigned to 18 families. The heterogeneous Vk9- and Vk10-gene families of previous classifications were combined into one family, and Vkdv was established as a new one-member gene family, leaving the total number of families unchanged. In general, the members of a family yield clear cross-hybridization signals under stringent conditions and have at least 80% homology in their exon II sequences. As can be seen in Figure 3.2, some clustering of gene families and also of the 5¢ to 3¢ polarities occurs, but notable cases of interspersion also occur. Since the characteristics of the gene families have been described in detail (Thiebe et al., 1999), only a few families should be mentioned here. The Vk4/5 family, which was called “young, dynamic, successful,” is remarkable for its size: 27 functional and potentially functional genes plus 6 pseudogenes were cloned. Since the gaps in our map are in the Vk4/5-gene region, the missing genes also may belong to this family. On the other side, there are the four onemember gene families: Vk22, Vk38C, VkRF, and Vkdv. The
Vk8, Vk19/28, and Vk22 gene families are similar and may be considered a clan. As with the human Vk genes, our nomenclature of the mouse Vk genes developed cluster by cluster, as the work proceeded. An abbreviated designation of the gene family was always included in the name, and once a contig was linked to Jk–Ck, its genes were numbered consecutively: the 22 Jk–Ck proximal genes were named Vk21-1 to Vk8-22. The genes of the yet-unlinked contigs are named by a combination of two letters and the designation of the gene family. For example, ar4 is a Vk4/5 gene in the contig Z2. The attempt to develop a systematic nomenclature was published by Martinez-Jean et al. (2001), but a final nomenclature must await the sequencing of the whole locus. A huge amount of work exists in the literature on mouse Vk genes, and a few recent publications should be mentioned. The germline diversity of the Vk9 and Vk10 gene families was studied by Ulrich et al. (1997) and Fitzsimmons et al. (2002), respectively. The Vk1 and Vk22 genes were the objects of Whitcomb and Brodeur (1998). Vk genes in autoantibodies and in antibacterial antibodies were investigated by Ye et al. (1996) and Emara et al. (1995), respectively. Rapid cloning and PCR techniques applied to Vk genes were frequently studied (e.g., Wang et al., 2000).
FIGURE 3.2 A researcher’s dream of the mouse immunoglobulin k locus, taken from Thiebe et al. (1999); the data of Röschenthaler et al. (2000) are not incorporated. The Jk–Ck and Vk genes are depicted as mice. Different gene families have different colors. Mice in full color designate potentially functional Vk genes, mice sketched only in outline are pseudogenes. Relics are not included. The 5¢, 3¢ direction of the Vk genes is indicated by the direction of the mice. See color insert.
3. Immunoglobulin k Genes of Human and Mouse
3.3 Mouse Vk Relics and Orphons Vk relics are genes with substantial deletions. Those in the human k locus were systematically studied only after the whole locus was sequenced (2.2), whereas in the mouse locus they were detected during the mapping work as weak hybridization signals and immediately sequenced. This was done because such signals sometimes turn out to be caused by cross-hybridization with functional genes of another family. The 18 relics found in the mouse k locus and their characteristics, accession numbers, and locations were compiled by Schäble et al. (1999). A fair number of additional relics also will probably be found in the mouse k locus once its sequence becomes known. Two mouse Vk orphons were localized to chromosomes 16 and 19 (Schupp et al., 1997). They were members of orphon clusters (Schäble et al., 1999). A map of the cluster on chromosome 16, which contains a Vk2, a Vk9, and a Vk20 orphon, was established at a time when it was not yet known that the genes were orphons (Figure 3.2 in Zocher et al., 1995). A Vk2 orphon was found near a Vk20 orphon on chromosome 19. The Vk2 orphon on chromosome 16 contains an internal inversion, but the two Vk2 orphon sequences still are more similar to each other than to the sequences of the Vk2 genes of the k locus. In the two Vk20 orphons, the leader and the exon II segments are separated by several kb of intracisternal A particle sequences. The Vk20 orphon sequences are 99% identical but less similar to those of the Vk20 genes of the locus. Apparently, first a Vk2 and a Vk20 orphon left the locus and both of them were later duplicated. When in the BAC analysis of the 5¢ part of the k locus the contig Z7 could not be linked to the other contigs (3.1), it was shown by fluorescence in situ hybridization (FISH) to be located near the k locus on chromosome 6 but not within the locus (Röschenthaler et al., 2000). Its cytogenetic distance from the locus exceeds 20 Mb. The single Vk1 and the two Vk9 pseudogenes of the contig are therefore classified as orphons.
4. ASPECTS OF EVOLUTION OF THE k GENES When in evolution was the human k locus duplicated? The locus is not yet duplicated in chimpanzees (see below), and the human and chimpanzee clades are thought to have diverged 5 to 7 million years ago. On the basis of 1% sequence divergence between the gene regions of the p and the d copies of the locus, we postulated that the duplication might have taken place 1 to 2 million years ago (Schäble and Zachau, 1993). However, the consideration of the total sequence of the locus led to the conclusion that the duplication had occurred at an earlier time, possibly shortly after the separation of the human clades and the chimpanzee
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clades. Thirteen blocks of homology between the sequences of the p and d copies were detected (A–M in Figure 3.1E), which may have arisen by inversion-mediated recombination. If one takes as the basis for the age of the duplication the sequence difference in the most highly diverged homology blocks, one arrives at the earlier date (Kawasaki et al., 2001). Several evolutionary events must have taken place before the duplication of the human k locus and others afterwards (Zachau, 1995; 2000). The amplification and interdigitation of the Vk genes of different subgroups, and most changes that converted functional genes to pseudogenes, occurred before the duplication. The insertion of an Alu element into the d and not into the p copy of the locus (Lautner-Rieske et al., 1992), the small deletions in one but not the other copy, and at least some of the gene-conversion–like events (Huber et al., 1993) took place after the duplication. The k loci of several nonhuman primate species were studied by Ermert et al. (1995). The Ck gene sequences of human and chimpanzee were found to be 99.6% identical, and the sequences of their Ck proximal Vk genes are remarkably similar. The restriction nuclease digestion patterns of the k loci of different primate species also are quite comparable. However, on the basis of the hybridization patterns obtained with 11 duplication-differentiating probes (2.4), it became clear that the chimpanzee and gorilla have only the part of the k locus in their genomes that corresponds to the p copy of humans. Since cosmid clones from the orphon regions of the human chromosomes 1 and 22 (2.5) hybridized in situ to the homologous chromosome bands in all great apes, the translocation may have happened early in primate evolution (Arnold et al., 1995). The pericentric inversion seems to have occurred after the gorilla and before the chimpanzee clades diverged from the human evolutionary tree. The role of recent duplications and duplicative transpositions in the evolution of the genomes of closely related primates was discussed by Eichler (2001). The mouse k locus is three times the size of the human locus and contains about twice as many Vk genes. Whereas the human locus has one large duplication, the mouse locus contains several smaller duplications. In the human locus, the genes of different families are intermingled, whereas in the mouse some gene families occur clustered. Serial amplifications of gene regions in the rodent clade were postulated as the reason for this clustering (Kirschbaum et al., 1999). Distinct homologies exist between the human gene families I–VII and several mouse gene families, generally ranging from 74 to 84% (for details see Thiebe et al., 1999). Apparently, the formation of gene families predated the divergence of the human and the mouse clades. Only the “young” Vk 4/5 gene family of mouse, with its 70% or less homology to the human genes, probably arose after the point of divergence (Thiebe et al., 1999).
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The evolution of the Vk gene loci was described as a continuous process of duplications and deletions or “birth and death” of the genes (Sitnikova and Nei, 1998). Whereas in humans the k and l proteins are found in comparable percentages, 95% k chains and only 5% l chains exist in the mouse. The ratios are related to the sizes of the light chain loci in the two species (Almagro et al., 1998). A general discussion of V gene evolution is presented by Rothenfluh et al. (1995).
5. CONCLUDING REMARKS The structures of the immunoglobulin k loci of human and mouse were elucidated by classical molecular biology techniques, in parallel to functional and mechanistic studies. Some colleagues involved in the large-scale genome projects called this the “cottage industry approach” (Zachau, 2000). However, most questions concerning the assembly processes and the maturation and functioning of the k genes could be answered in principle on the basis of data obtained through the classical approach, as is obvious from various chapters of this book. Because the human and mouse genomes have been largely sequenced now, the k loci of the two species will be looked at in detail and be annotated soon. Thus, the 800-kb Vk-gene free gap between the p and the d copies of the human k locus and the three small gaps in the mouse k locus will be closed. Also, the sequences adjacent to the k loci and to the orphons may be interesting and contribute to our insight into the processes of evolution and genome dynamics.
Acknowledgments I thank the members of our group for their contributions. The work of our laboratory was supported by Bundesministerium für Forschung und Technologie and by Fonds der Chemischen Industrie.
References Almagro, J. C., Hernández, I., Ramirez, M. C., and Vargas-Madrazo, E. (1998). Structural differences between the repertoires of mouse and human germline genes and their evolutionary implications. Immunogenetics 47, 355–363. Apel, T. W., Scherer, A., Adachi, T., Auch, D., Ayane, M., and Reth, M. (1995). The ribose 5-phosphate isomerase-encoding gene is located immediately downstream from that encoding murine immunoglobulin k. Gene 156, 191–197. Arnold, N., Wienberg, J., Ermert, K., and Zachau, H. G. (1995). Comparative mapping of DNA probes derived from the Vk immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization. Genomics 26, 147–150. Bemark, M., Liberg, D., and Leanderson, T. (1998). Conserved sequence elements in k promoters from mice and humans: Implications for transcriptional regulation and repertoire expression. Immunogenetics 47, 183–195.
Bentley, D. L., and Rabbitts, T. H. (1983). Evolution of immunoglobulin V genes: Evidence indicating that recently duplicated human Vk sequences have diverged by gene conversion. Cell 32, 181–189. Bernardi, G. (2000). Isochore and the evolutionary genomics of vertebrates. Gene 241, 3–17. Brensing-Küppers, J., Zocher, I., Thiebe, R., and Zachau, H. G. (1997). The human immunoglobulin k locus on yeast artificial chromosomes (YACs). Gene 191, 173–181. Casali, P., and Silberstein, L. E., eds. (1995). Immunoglobulin gene expression in development and disease. Ann N Y Acad Sci, 764 (New York: The New York Academy of Sciences). Eichler, E. E. (2001). Recent duplication, domain accretion and the dynamic mutation of the human genome. Trends Genet 17, 661–669. Emara, M. G., Tout, N. L., Kaushik, A., and Lam, J. S. (1995). Diverse VH and Vk genes encode antibodies to pseudomonas aeruginosa LPS. J Immunol 155, 3912–3921. Ermert, K., Mitlöhner, H., Schempp, W., and Zachau, H. G. (1995). The immunoglobulin k locus of primates. Genomics 25, 623–629. Falkner, F. G., and Zachau, H. G. (1984). Correct transcription of an immunoglobulin k gene requires an upstream fragment containing conserved sequence elements. Nature 310, 71–74. Fitzsimmons, S. P., Clark, K. J., and Shapiro, M. A. (2002). Evolutionary relationships and polymorphisms among Vk 10 genes from inbred and wild-derived inbred mice. Immunogenetics 54, 9–19. Gallo, M. L., Ivanov, V. E., Jakobovits, A., and Davis, C. G. (2000). The human immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans. Eur J Immunol 30, 534–540. George, J. B., Li, S., and Garrard, W. T. (1995). Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin k locus. Proc Natl Acad Sci U S A 92, 12421–12425. Girschick, H. J., and Lipsky, P. E. (2001). The kappa gene repertoire of human neonatal B cells. Mol Immunol 38, 1113–1127. Huber, C., Schäble, K. F., Huber, E., Klein, R., Meindl, A., Thiebe, R., Lamm, R., and Zachau, H. G. (1993). The Vk genes of the L regions and the repertoire of Vk gene sequences in the human germ line. Eur J Immunol 23, 2868–2875. Huber, C., Thiebe, R., and Zachau, H. G. (1994). A potentially functional Vk gene at a distance of 1,5 Mb from the immunoglobulin k locus. Genomics 22, 213–215. Johnson, G., and Wu, T. T. (2001). Kabat Database and its applications: Future directions. Nucleic Acids Res 29, 205–206. Juul, L., Hougs, L., and Barington, T. (1998). A new apparently functional IGVK gene (VkLa) present in some individuals only. Immunogenetics 48, 40–46. Kabat Database (2002). Sequences of proteins of immunological interest, continuously updated by G. Johnson and T. T. Wu. http://immuno.bme.nwu.edu Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Asakawa, S., Sasaki, T., Klobeck, H.-G., Combriato, G., Zachau, H. G., and Shimizu, N. (2001). Evolutionary dynamics of the human immunoglobulin k locus and the germline repertoire of the Vk genes. Eur J Immunol 31, 1017–1028. Kindt, Th. J., and Capra, J. D. (1984). The antibody enigma (New York: Plenum Press). Kirschbaum, T., Jaenichen, R., and Zachau, H. G. (1996). The mouse immunoglobulin k locus contains about 140 variable gene segments. Eur J Immunol 26, 1613–1620. Kirschbaum, T., Pourrajabi, S., Zocher, I., Schwendinger, J., Heim, V., Röschenthaler, F., Kirschbaum, V., and Zachau, H. G. (1998). The 3¢ part of the immunoglobulin k locus of the mouse. Eur J Immunol 28, 1458–1466. Kirschbaum, T., Röschenthaler, F., Bensch, A., Hölscher, B., LautnerRieske, A., Ohnrich, M., Pourrajabi, S., Schwendinger, J., Zocher, I.,
3. Immunoglobulin k Genes of Human and Mouse and Zachau, H. G. (1999). The central part of the mouse immunoglobulin k locus. Eur J Immunol 29, 2057–2064 Klein, R., Jaenichen, R., and Zachau, H. G. (1993). Expressed human immunoglobulin k genes and their hypermutation. Eur J Immunol 23, 3248–3262. Klein, R., and Zachau, H. G. (1993). Comparison of human germ-line Vk gene sequences to sequence data from the literature. Eur J Immunol 23, 3263–3271. Klein, R., and Zachau, H. G. (1995). Expression and hypermutation of human immunoglobulin k genes. Ann N Y Acad Sci 764, 74–83 (see Casali and Silberstein, eds.). Klobeck, H.-G., and Zachau, H. G. (1986). The human Ck gene segment and the kappa deleting element are closely linked. Nucleic Acids Res 14, 4591–4603. Kofler, R., Geley, S., Kofler, H., and Helmberg, A. (1992). Mouse variableregion gene families: Complexity, polymorphism and use in nonautoimmune responses. Immunol Rev 128, 5–21. Lautner-Rieske, A., Huber, C., Meindl, A., Pargent, W., Schäble, K. F., Thiebe, R., Zocher, I., and Zachau, H. G. (1992). The human immunoglobulin k locus. Characterization of the duplicated A regions. Eur J Immunol 22, 1023–1029. Lautner-Rieske, A., Hameister, H., Barbi, G., and Zachau, H. G. (1993). Mapping immunoglobulin gene related DNA probes to the central region of normal and pericentrically inverted human chromosome 2. Genomics 16, 497–502. Lautner-Rieske, A., Thiebe, R., and Zachau, H. G. (1995). Searching for non-Vk transcripts from the human immunoglobulin k locus. Gene 159, 199–202. Lawler, A. M., Umar, A., and Gearhart, P. J. (1992). Linkage of two pseudogenes from the Vk1 and Vk9 murine immunoglobulin families. Mol Immunol 29, 295–301. Lefranc, M.-P. (2001). Nomenclature of the human immunoglobulin kappa (IGK) genes. Exp Clin Immunogenet 18, 161–174. Lefranc, M.-P., and Lefranc, G. (2001). The immunoglobulin facts book (London, UK: Academic Press). Lefranc, M.-P. (2002). ImMunoGeneTics database, continuously updated. http://imgt.cnusc.fr:8104 Liu, Z.-M., George-Raizen, J. B., Li, S., Meyers, K. C., Chang, M. Y., and Garrard, W. T. (2002). Chromatin structural analyses of the mouse Igk gene locus reveal new hypersensitive sites specifying a transcriptional silencer and enhancer. J Biol Chem 277, 32640–32649. Lötscher, E., Grzeschik, K.-H., Bauer, H. G., Pohlenz, H.-D., Straubinger, B., and Zachau, H. G. (1986). Dispersed human immunoglobulin k light chain genes. Nature 320, 456–458. de Marco, M. D. C., Kremer, L., Albar, J. P., Martinez-Menárguez, J. A., Ballesta, J., Garcia-López, M. A., Marazuela, M., Puertollano, R., and Alonso, M. A. (2001). BENE, a novel raft-associated protein of the MAL proteolipid family, interacts with caveolin-1 in human endothelial-like ECV304 cells. J Biol Chem 276, 23009–23017. Martinez-Jean, C., Folch, G., and Lefranc, M.-P. (2001). Nomenclature and overview of the mouse (mus musculus and mus sp.) immunoglobulin kappa (IGK) genes. Exp Clin Immunogenet 18, 255–279. Max, E. E. (1999). Immunoglobulins: Molecular Genetics. In Fundamental immunology, 4th ed., W. E. Paul, ed. (Philadelphia: LippincottRaven), pp. 111–182. Padyukov, L., Hahn-Zoric, M., Blomqvist, S. R., Ulanova, M., Welch, S. G., Feeney, A. J., Lau, Y. L., and Hanson, L. A. (2001). Distribution of human kappa locus IGKV2-29 and IGKV2D-29 alleles in Swedish Caucasians and Hong Kong Chinese. Immunogenetics 53, 22–30. Pargent, W., Schäble, K. F., and Zachau, H. G. (1991). Polymorphisms and haplotypes in the human immunoglobulin k locus. Eur J Immunol 21, 1829–1835. Pech, M., Smola, H., Pohlenz, H.-D., Straubinger, B., Gerl, R., and Zachau, H. G. (1985). A large section of the gene locus encoding human
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immunoglobulin variable regions of the k type is duplicated. J Mol Biol 183, 291–299. Pyon, H. S., Ha-Lee, Y. M., Song, G. G., and Sohn, J. (2001). Analysis of the Igk light chain gene variable regions expressed in the rheumatoid synovial B cells. Scand J Immunol 53, 503–509. Röschenthaler, F., Kirschbaum, T., Heim, V., Kirschbaum, V., Schäble, K. F., Schwendinger, J., Zocher, I., and Zachau, H. G. (1999). The 5¢ part of the mouse immunoglobulin k locus. Eur J Immunol 29, 2065–2071. Röschenthaler, F., Hameister, H., and Zachau, H. G. (2000). The 5¢ part of the immunoglobulin k locus as a continuously cloned structure. Eur J Immunol 30, 3349–3354. Rothenfluh, H. S., Blanden, R. V., and Steele, E. J. (1995). Evolution of V genes: DNA sequence structure of functional germline genes and pseudogenes. Immunogenetics 42, 159–171. Schäble, K. F., and Zachau, H. G. (1993). The variable genes of the human immunoglobulin k locus. A review. Biol Chem Hoppe-Seyler 374, 1001–1022. Schäble, K. F., Thiebe, R., Flügel, A., Meindl, A., and Zachau, H. G. (1994). The human immunoglobulin k locus: pseudogenes, unique and repetitive sequences. Biol Chem Hoppe-Seyler 375, 189–199. Schäble, K. F., Thiebe, R., Bensch, A., Brensing-Küppers, J., Heim, V., Kirschbaum, T., Lamm, R., Ohnrich, M., Pourrajabi, S., Röschenthaler, F., Schwendinger, J., Wichelhaus, D., Zocher, I., and Zachau, H. G. (1999). Characteristics of the immunoglobulin Vk genes, Pseudogenes, relics and orphons in the mouse genome. Eur J Immunol 29, 2082– 2086. Schäble, G., Rappold, G. A., Pargent, W., and Zachau, H. G. (1993). The immunoglobulin k locus. Polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals. Hum Genet 91, 261–267; Erratum 92, 105. Schupp, I. W., Schlake, T., Kirschbaum, T., Zachau, H. G., and Boehm, T. (1997). A yeast artificial chromosome contig spanning the mouse immunoglobulin kappa light chain locus. Immunogenetics 45, 180–187. Scott, M. G., Zachau, H. G., and Nahm, M. H. (1992). The human antibody V region repertoire to the type b capsular polysaccharide of Haemophilus influenzae. Int Rev Immunol 9, 45–55. Sitnikova, T., and Nei, M. (1998). Evolution of immunoglobulin kappa variable region genes in vertebrates. Mol Biol Evol 15, 50–60. Solomon, A., Weiss, D. T., Murphy, C. L., Hrncic, R., Wall, J. S., and Schell, M. (1998). Light chain-associated amyloid deposits comprised of a novel k constant domain. Proc Natl Acad Sci U S A 95, 9547–9551. Steinmetz, M., Altenburger, W., and Zachau, H. G. (1980). A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments. Nucleic Acids Res 8, 1709–1720. Thiebe, R., Schäble, K. F., Bensch, A., Brensing-Küppers, J., Heim, V., Kirschbaum, T., Mitlöhner, H., Ohnrich, M., Pourrajabi, S., Röschenthaler, F., Schwendinger, J., Wichelhaus, D., Zocher, I., and Zachau, H. G. (1999). The variable genes and gene families of the mouse immunoglobulin k locus. Eur J Immunol 29, 2072–2081. Tomlinson, I. M., Cox, J. P. L., Gherardi, E., Lesk, A. M., and Chothia, C. (1995). The structural repertoire of the human Vk domain. EMBO J 14, 4628–4638. Ulrich, H. D., Moore, F. L., and Schultz, P. G. (1997). Germline diversity within the mouse Igk-V9 gene family. Immunogenetics 47, 91–95. Wang, Z., Raifu, M., Howard, M., Smith, L., Hansen, D., Goldsby, R., and Ratner, D. (2000). Universal PCR amplification of mouse immunoglobulin gene variable regions: The design of degenerate primers and an assessment of the effect of DNA polymerase 3¢ to 5¢ exonuclease activity. J Immunol Methods 233, 167–177. Weichhold, G. M., Klobeck, H.-G., Ohnheiser, R., Combriato, G., and Zachau, H. G. (1990). Megabase inversions in the human genome as physiological events. Nature 347, 90–92. Weichhold, G. M., Ohnheiser, R., and Zachau, H. G. (1993a). The human immunoglobulin k locus consists of two copies that are organized in opposite polarity. Genomics 16, 503–511.
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Weichhold, G. M., Huber, C., Parnes, J. R., and Zachau, H. G. (1993b). The CD8a locus is located on the telomere side of the immunoglobulin k locus at a distance of 2 Mb. Genomics 16, 512–514. Whitcomb, E. A., and Brodeur, P. H. (1998). Rearrangement and selection in the developing Vk repertoire of the mouse: An analysis of the usage of two Vk gene segments. J Immunol 160, 4904–4913. Ye, X. J., Marion, T. N., Terato, K., Aelion, J. A., Cremer, M. A., Tillman, D. M., Krug, M. S., Jackson, B., and Yo, T. J. (1996). Variable-region gene family usage for type II collagen autoantibodies in arthritis-susceptible DBA/1 mice. Clin Immunol Immunopathol 78, 263–275. Zachau, H. G. (1989). Immunoglobulin light-chain genes of the k type in man and mouse. In Immunoglobulin genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds. (London, UK: Academic Press), pp. 91–109. Zachau, H. G. (1995). The human immunoglobulin k genes. In Immunoglobulin genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds. (London, UK: Academic Press), pp. 173–191. Zachau, H. G. (1996). The human immunoglobulin k genes. Immunologist 4, 49–54. Zachau, H. G. (2000). The immunoglobulin k gene families of human and mouse: A cottage industry approach. Biol Chem 381, 951–954. Zachau, H. G. (2001). http://www.med.uni-muenchen.de/biochemie/ zachau/kappa.htm, homepage updated in 2001, containing the mouse k gene data; human k genes in the section . . . zachau/human_kappa.htm
Zocher, I., Röschenthaler, F., Kirschbaum, T., Schäble, K. F., Hörlein, R., Fleischmann, B., Kofler, R., Geley, S., Hameister, H., and Zachau, H. G. (1995). Clustered and interspersed gene families in the mouse immunoglobulin k locus. Eur J Immunol 25, 3326–3331.
Note Added in Proof This review was concluded at the time of submission in early October 2002. Before receiving the proofs a year later, several relevant publications appeared; for reasons of space only a few of them can be quoted here: Li, S., and Garrard, W. T. (2003). The kinetics of V-J joining throughout 3,5 megabases of the mouse Ig kappa locus fit a constrained diffusion model of nuclear organization. FEBS Lett 536, 125–129. Krangel, M. S. (2003). Gene segment selection in V(D)J recombination: accessibility and beyond. Nature Immunol 4, 624–630. Ohlin, M., and Zouali, M. (2003). The human antibody repertoire to infectious agents: implications for disease pathogenesis. Molec Immunol 40, 1–11. Brekke, K. M., and Garrard, W. T. (personal communication, 2003) assembled the nucleotide sequence of the mouse Igk locus using data from the recent genome sequencing effort. Their emphasis was on analyzing regulatory elements. They also counted 140 Vk genes, of which 95 were called potentially functional.
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4 Immunoglobulin Lambda (IGL) Genes of Human and Mouse MARIE-PAULE LEFRANC AND GÉRARD LEFRANC Université Montpellier II, CNRS, Montpellier, France
This chapter provides a description of the human and mouse germline immunoglobulin lambda (IGL) genes that are used to create the antibody lambda chain repertoire in human and mouse. In order to ensure accuracy, consistency and coherence in the immunoglobulin repertoire description between chain types and species, IMGT, the international ImMunoGeneTics information system® (http://imgt.cines.fr) (Lefranc, 2001a, 2003a), created in 1989 at the Université Montpellier II, CNRS, Montpellier, France, has developed a formal specification of the terms to be used in the domain of immunogenetics and immunoinformatics. This has been the basis of IMGT-ONTOLOGY (Giudicelli and Lefranc, 1999), the first ontology in the domain.
• Gene type—Three types of genes are involved in immunoglobulin lambda synthesis, the variable (V) and joining (J) genes, which encode the antigen binding sites, and the constant (C) genes. • Configuration—The configuration defines the status of the genes: “germline” or “rearranged” for the IGL V and J genes. Note that the C genes do not rearrange directly and therefore their configuration is not defined. • Chain type—The chain type identifies the nature of the peptidic chain potentially encoded by the immunoglobulin IGL genes. The chain type instances are defined by the C gene sequence characteristics. They are refered as Ig-Light-Lambda in IMGT/LIGM-DB (Lefranc, 2000c). • Functionality—The definition of functionality is based on the sequence analysis (Lefranc, 1998). As examples, the instances functional (F) (for germline IGLV and IGLJ, and for IGLC genes), and productive (for rearranged IGL V-J sequences) mean that the coding regions have an open reading frame without a stop codon, and that there is no described defect in the splicing sites, recombination signals, and/or regulatory elements. According to the gravity of the identified defects, the functionality can be defined as open reading frame (ORF) or pseudogene (P) (for germline IGLV and IGLJ, and for IGLC genes), or unproductive (for rearranged IGL V-J sequences).
IGL GENES AND IMGT-ONTOLOGY The human and mouse IGL gene description in this chapter follows the IDENTIFICATION, CLASSIFICATION, and DESCRIPTION concepts of IMGTONTOLOGY (Giudicelli and Lefranc, 1999). The first part of the chapter summarizes the rules of the IMGT Scientific chart (Lefranc et al., 1999a), based on these concepts, for the IGL genes. The second and third part of the chapter provide, for the first time, a description of the genes of a given immunoglobulin locus—the IGL locus—in two different species, human and mouse, with the same IMGT standardized rules for nomenclature and numbering.
IGL Genes and the IDENTIFICATION Concept
IGL Genes and the CLASSIFICATION Concept
The IDENTIFICATION concept allows scientists to identify immunoglobulin lambda sequences according to fundamental biological and immunogenetics characteristics (Giudicelli and Lefranc, 1999).
The CLASSIFICATION concept (Figure 4.1) organizes that immunogenetics knowledge useful to name and classify the immunoglobulin IGL genes (Giudicelli and Lefranc, 1999).
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Copyright 2004, Elsevier Science (USA). All rights reserved.
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the sequence level (Lefranc et al., 1998; Lefranc, 1998). Their sequences are compared to the reference sequence designated as *01 (see IMGT Scientific chart at http://imgt.cines.fr for IMGT description of mutations and IMGT allele nomenclature for sequence polymorphisms). Nomenclature of the Human and Mouse IGL Genes and Alleles
FIGURE 4.1 The CLASSIFICATION concept in IMGT-ONTOLOGY.
• Locus—A locus is a group of immunoglobulin genes that are ordered and localized in the same chromosomal location in a given species. The human and mouse genomes include one main IGL locus on chromosome 22 at 22q11.2 and on chromosome 16 at 13 cM, respectively. Immunoglobulin lambda genes have also been identified in the human genome in other chromosomal locations outside the main locus, thus representing new instances of the locus concept. However, the genes they contain, designated as orphons, are not functional. • Group—A group is a set of genes that share the same gene type (V, J, or C) and participate potentially in the synthesis of a polypeptide of the same chain type. By extension, a group includes the related pseudogenes and orphons. There are three groups—IGLV, IGLJ, and IGLC—for the immunoglobulin lambda genes. • Subgroup—A subgroup is a set of genes that belong to the same group, in a given species, and that share at least 75% identity at the nucleotide level (in the germline configuration for V and J genes). • Gene—A gene is defined as a DNA sequence that can be potentially transcribed and/or translated (this definition includes the regulatory elements in 5¢ and 3¢, and the introns, if present). Instances of the gene concept are gene names. By extension, orphons and pseudogenes are also instances of the gene concept. For each gene, IMGT has defined a reference sequence (Lefranc et al., 1999a). For the V and J genes, the reference sequence corresponds to a germline entity. The rules for the choice of the reference sequences are described at http://imgt.cines.fr, in the IMGT Scientific chart. • Allele—An allele is a polymorphic variant of a gene. Alleles are described, exhaustively and in a standardized way, for the “core” coding regions; that is, the germline V-REGIONs and J-REGIONs, and the C-REGIONs, from immunoglobulin lambda genes. These alleles refer to sequence polymorphisms, with mutations described at
The CLASSIFICATION concept (Figure 4.1) has been used to set up a unique nomenclature of the immunoglobulin genes (Barbié and Lefranc, 1998; Pallarès et al., 1998, 1999; Ruiz et al., 1999; Scaviner et al., 1999; Lefranc, 2000a,b, 2001b; Lefranc and Lefranc, 2001a). A four-letter root designates the group: IGLV, IGLJ, and IGLC for the immunoglobulin lambda genes. Gene names are derived from the four-letter root by adding, if necessary, number(s) and/or letter(s) to allow unambiguous identification of the gene; a single number or letter is used whenever possible. IMGT nomenclature was approved by the HUGO (HUman Genome Organization) Nomenclature Committee, HGNC (http://www.gene.ucl.ac.uk/nomenclature) in 1999 (Wain et al., 2002). All IMGT human immunoglobulin genes have been entered into GDB, Genome Database, Toronto, Canada (http://www.gdb.org); into LocusLink at NCBI (National Center for Biotechnology Information), Bethesda, USA (http://www.ncbi.nlm.nih.gov/LocusLink); and into GeneCards, Weizmann Institute, Israel (http://bioinformatics. weizmann.ac.il/cards/). Links to the IMGT gene cards are provided from GDB, LocusLink, and GeneCards. Links to the accession numbers of the IMGT reference sequences are provided from GDB (Seq@IMGT) and LinkOut at NCBI (http://www.ncbi.nlm.nih.gov/entrez/linkout/). Allele names of the IGL V-REGIONs, J-REGIONs, and C-REGIONs comprise the IMGT gene name followed by an asterisk and a two-figure number. The V-REGIONs, JREGIONs, and C-REGIONs selected as references for the allele polymorphism description have the number *01; other alleles are designated by increasing numbers (*02, *03, . . .) based, if possible, on the chronological order of their publication or confirmation of data by different authors. Note that the number *01 does not mean necessarily that other alleles are already known, but it signifies that any new polymorphic sequence will be described by comparison to that allele *01. IMGT accession numbers are assigned to each allele. Although the IMGT accession numbers are the same as those from the EMBL/GenBank/DDBJ generalist databases, the content of the IMGT/LIGM-DB flat files differs in the expert annotations added by IMGT. IMGT data are available from IMGT/LIGM-DB, IMGT Repertoire, and from Sequence Retrieval System (SRS) sites (available from the IMGT Home page, http://imgt.cines.fr).
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
IGL Genes and the DESCRIPTION Concept The DESCRIPTION concept provides a standardized description of the organization and components of the immunoglobulin sequences, and a characterization of their specific and conserved motifs. Prototypes have been set up to graphically represent the description and configuration of an immunoglobulin gene (Giudicelli et al., 1997). These prototypes give information on the order and expected length (in number of nucleotides) of the labels (Giudicelli et al., 1997; Lefranc et al., 1999a). For example, the prototype V-
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GENE represents a genomic IGLV gene in the germline configuration, whereas V-J-GENE represents genomic IGL V and J genes in the rearranged configuration (Figure 4.2).
The IMGT Unique Numbering A uniform numbering system for the immunoglobulin of all species has been established to facilitate sequence comparison and cross-referencing between experiments from different laboratories, whatever the antigen receptor
FIGURE 4.2 Prototype V-GENE of genomic IGL V gene in the germline configuration and prototype V-J-GENE of genomic V and J genes in the rearranged configuration. Labels (in capital letters) are those used for the sequence description in IMGT (http://imgt.cines.fr). One hundred seventy-seven labels are necessary to describe all structural and functional subregions that compose immunoglobulin sequences (Giudicelli et al., 1997), whereas only seven are available in EMBL, GenBank, or DDBJ.
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(immunoglobulin or T cell receptor), the chain type, or the species. The IMGT unique numbering (Lefranc, 1997, 1999; Lefranc et al., 2003) relies on the high conservation of the structure of the variable regions (V-REGIONs) and domains (V-DOMAINs). This numbering results from the analysis of more than 5,000 sequences, from fish to human. It takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR) (Kabat et al., 1987, 1991), structural data from X-ray diffraction studies (Satow et al., 1986), and the characterization of the hypervariable loops (Chothia and Lesk, 1987). The delimitations of the FR-IMGT and CDR-IMGT regions have been defined, and correspondence between the IMGT numbering and other numberings has been established (Lefranc, 1999; Lefranc and Lefranc, 2001a,b). The IMGT unique numbering has many advantages: • It allows an easy comparison between sequences coding the variable regions, whatever the antigen receptor (immunoglobulins or T cell receptors), the chain type (heavy or light chains for immunoglobulins), or the species. • In the IMGT unique numbering, the conserved amino acids always have the same position: for example, Cystein 23, Tryptophane 41, Leucine 89, Cystein 104. The hydrophobic amino acids of the framework regions are also found in conserved positions. • This unique numbering has allowed the redefinition of the limits of the FR and CDR. The FR-IMGT and CDRIMGT lengths themselves become crucial information characterizing the variable regions belonging to a group, subgroup, or gene. • Framework amino acids (and codons) located at the same position in different sequences can be compared without requiring sequence alignments. This also holds for amino acids belonging to CDR-IMGT of the same length. • The IMGT unique numbering has allowed a standardized IMGT description of mutations for the IMGT description of allele polymorphisms and somatic hypermutations of the variable regions (Lefranc et al., 1998; Lefranc, 1998). • The unique numbering is used as the output of the IMGT/V-QUEST alignment tool (Lefranc, 2003b,c), which analyses variable (germline or rearranged) sequences according to IMGT criteria (Lefranc et al., 1999a). In IMGT/V-QUEST, for example, a variable rearranged lambda sequence is compared to the appropriate sets of V-REGION and J-REGION alleles from the IMGT reference directory. The results show, aligned with the input sequence, the sequences of the most homologous IGL V-REGION and J-REGION alleles. The aligned V-REGION sequences are displayed according to the IMGT unique numbering and with the FR-IMGT and CDR-IMGT delimitations.
The IMGT/JunctionAnalysis tool displays results from position 104 (2nd-CYS) to position 118 (J-PHE of the IGL J-REGION) (Lefranc, 2003b,c). The IMGT unique numbering has been extended to the C-DOMAINs of the immunoglobulins and T cell receptors, and to all domains belonging to the V-set and C-set of the immunoglobulin superfamily (Lefranc et al., 2003). IMGT Collier de Perles The IMGT Colliers de Perles (Lefranc, 1999; Lefranc et al., 1998, 1999a; Ruiz et al., 2000) are two-dimensional graphical representations of the V-REGIONs and VDOMAINs (Figure 4.3) and C-DOMAINs (Figure 4.4), with strands and loops delimitations, according to the IMGT unique numbering. Colliers de Perles 2D representations provide information on the amino acid positions in the betastrands and loops of the lambda variable and constant domains, and in the FR-IMGT and CDR-IMGT of the VREGIONs and V-DOMAINs. They allow a quick visualization of those amino acids important for the structural configuration of the V-REGION, V-DOMAIN and CDOMAIN. For a given V-REGION or V-DOMAIN, the lengths of the three CDR-IMGT are shown in brackets and separated by dots. For example, [8.3.9] means that in the germline IGLV1–36 gene (Figure 4.3), the CDR1-IMGT, CDR2-IMGT, and CDR3-IMGT are 8, 3, and 9 amino acids long, respectively (Figure 4.3); [9.3.10] means that in the rearranged Mcg IGLV2–8*01-IGLJ1*01 gene (Figure 4.4), the CDR1-IMGT, CDR2-IMGT, and CDR3IMGT are 9, 3, and 10 amino acids long, respectively (Figure 4.4).
THE HUMAN IGL GENES Chromosomal Localization of the Human IGL Locus The human IGL locus is located on chromosome 22 (Erikson et al., 1981), on the long arm, at band 22q11.2 (Emanuel et al., 1985) (Figure 4.5). The orientation of the locus has been determined by the analysis of translocations, involving the IGL locus, in leukemia and lymphoma. Sequencing of the long arm of chromosome 22 showed that it encompasses about 35 megabases of DNA and that the IGL locus is localized at six megabases from the centromere (Dunham et al., 1999). Although the correlation between DNA sequences and chromosomal bands has not yet been made, the localization of the IGL locus can be refined to 22q11.2.
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
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FIGURE 4.3 IMGT Collier de Perles of the V-REGION and V-DOMAIN. (a) IMGT Collier de Perles for the human germline IGLV1–36 (Z73653) according to the IMGT unique numbering for V-REGION. The CDR-IMGT of the germline IGLV1–36 have a length of 8, 3, and 9 amino acids, respectively [8.3.9]. Amino acids are shown in the oneletter abbreviation. Hydrophobic amino acids (hydropathy index with positive value) and tryptophan (W) are found at a given position in more than 50% of analyzed immunoglobulin, and T cell receptor sequences are shown in gray. The CDR-IMGT are limited by amino acids shown in squares, which belong to the neighboring FR-IMGT. Hatched circles correspond to missing positions according to the IMGT unique numbering. Arrows indicate the direction of the beta sheets and their different designations in 3D structure. (b) IMGT Collier de Perles of the human Mcg IGLV2–8*01IGLJ1*01 domain (la8j) (from IMGT/3Dstructure-DB) according to the IMGT unique numbering for V-DOMAIN. The IMGT Collier de Perles is displayed on two sheets, with the hydrogen bonds indicated between amino acids of the inner sheet (amino acids in grayed circles with broad contours).
Organization of the Human IGL Locus The human IGL locus at 22q11.2 spans 1,050 kb (Figure 4.6). The human IGL locus consists of 73 to 74 IGLV genes (Frippiat et al., 1995; Kawasaki et al., 1995, 1997; Williams et al., 1996; for review Pallarès et al., 1998; Scaviner et al., 1999; Lefranc and Lefranc, 2001a), localized on 900 kb, and 7 to 11 IGLJ and 7 to 11 IGLC genes depending on the haplotypes, each IGLC gene being preceded by one IGLJ gene (Hieter et al., 1981; Taub et al., 1983; Dariavach et al., 1987; Vasicek and Leder, 1990; Bauer and Blomberg, 1991) (Table 4.1). One enhancer has been identified 8 kb downstream of the IGLC7 gene (Blomberg et al., 1991; Asenbauer et al., 1999; Combriato and Klobeck, 2002).
The Human IGLV Genes Fifty-six to 57 IGLV genes belong to 11 subgroups, whereas 17 pseudogenes that are too divergent to be assigned to subgroups have been assigned to three clans (Table 4.1) (Frippiat et al., 1995; Williams et al., 1996; Kawasaki et al., 1997). (See Pallarès et al., 1998, for an exhaustive list of the human germline IGLV genes with accession numbers, reference sequences, and sequences from the literature; Lefranc and Lefranc, 2001a, for alignments of alleles of the functional and ORF genes.) The most 5¢ IGLV genes occupy the more centromeric position, whereas the IGLC genes, in 3¢ of the locus, are the most telomeric genes in the IGL locus. All human IGLV genes have the same transcriptional orientation and rearrange by a
FIGURE 4.4 IMGT Collier de Perles of the C-DOMAIN. (a) IMGT Collier de Perles of the human IGLC1 domain (X51755) according to the IMGT unique numbering for C-DOMAIN. Amino acids are shown in the one-letter abbreviation. Hydrophobic amino acids (hydropathy index with positive value) and tryptophan (W) are found at a given position in more than 50% of analyzed immunoglobulin, and T cell receptor sequences are shown in gray. The positions 26, 39, and 104 are shown in squares, by homology with the corresponding positions in the V-REGIONs. Positions 45 and 77, which delimit the characteristic CD strand of the C-DOMAINs, and position 118, which corresponds structurally to J-PHE or J-TRP of the J-REGION, are also shown in squares. Hatched circles or squares correspond to missing positions according to the IMGT unique numbering for C-DOMAINs. (b) IMGT Collier de Perles of the human IGLC1 domain according to the IMGT unique numbering for C-DOMAIN. The IMGT Collier de Perles is displayed on two sheets. Amino acids at positions 1 and 3, 45, and 100 are involved in the Mcg, Ke, and Oz serological markers, respectively (see text and Table 4.2).
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FIGURE 4.4 (Continued)
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TABLE 4.1
Complete list of the human IGL genes on chromosome 22 at 22q11.2 Other nomenclatures
IMGT groups IGLC
IGLJ
IGLV
IMGT gene names (Pallarès et al., 1998; Lefranc, 2001)
IMGT functionality
IMGT reference sequence accession numbers
IMGT number of alleles
J00252 J00253
2 4 1 4 2 2 5 2
IGLC1 IGLC2 (*)IGLC2D1 IGLC3 IGLC4 IGLC5 IGLC6 IGLC7
F F F F P P F, P F
IGLJ1 IGLJ2 (*)IGLJ2D1 IGLJ3 IGLJ4 IGLJ5 IGLJ6 IGLJ7
F F F F ORF ORF F F
M15642 X51755 X51755 M18338 X51755
1 1 1 2 1 2 1 2
IGLV1-36 IGLV1-40 IGLV1-41 IGLV1-44 IGLV1-47 IGLV1-50 IGLV1-51 IGLV1-62 IGLV2-5 IGLV2-8 IGLV2-11 IGLV2-14 IGLV2-18 IGLV2-23 IGLV2-28 IGLV2-33 IGLV2-34 IGLV3-1 IGLV3-2 IGLV3-4 IGLV3-6 IGLV3-7 IGLV3-9 IGLV3-10 IGLV3-12 IGLV3-13 IGLV3-15 IGLV3-16 IGLV3-17 IGLV3-19 IGLV3-21 IGLV3-22 IGLV3-24 IGLV3-25 IGLV3-26 IGLV3-27 IGLV3-29 IGLV3-30 IGLV3-31 IGLV3-32 IGLV4-3 IGLV4-60
F F ORF, P F F ORF F P P F F F F F P ORF P F P P P P F, P F F P P F P F F F, P P F P F P P P ORF F F
Z73653 M94116 M94118 Z73654 Z73663 M94112 Z73661 D87022 Z73641 X97462 Z73657 Z73664 Z73642 X14616 X97466 Z73643 D87013 X57826 X97468 D87024 X97465 X97470 X97473 X97464 Z73658 X97463 D87015 X97471 X97472 X56178 X71966 Z73666 X71968 X97474 X97467 D86994 Z73644 Z73646 X97469 Z73645 X57828 Z73667
1 3 2 1 2 1 2 1 2 3 3 4 4 3 1 3 1 1 2 1 2 1 3 2 2 1 1 1 1 1 3 2 2 3 1 1 1 2 2 1 1 3
J00254 J03009 J03010 J03011 X51755 X04457 M15641
Frippiat et al., 1995; Williams et al., 1996
1a 1e 1d 1c 1g 1f 1b 2a1 2c 2e 2a2 2d 2b2 2b1 2f 3r 3q 3a2 3n 3j 3p 3i 3f 3a 3g 3l 3h 3e 3d 3m 3b 3c 3o 3k 3i1 4c 4a
Kawasaki et al., 1997
1-11 1-13 1-14P 1-16 1-17 1-18 1-19 1-23P 1-1P 1-2 1-3 1-4 1-5 1-7 1-8P 1-9 1-10P 2-1 2-2P 2-3P 2-4P 2-5P 2-6 2-7 2-8 2-9P 2-10P 2-11 2-12P 2-13 2-14 2-15 2-16P 2-17 2-18P 2-19 2-20P 2-21P 2-22P 2-23P 5-1 5-4 (continues)
44
TABLE 4.1 (continued) Other nomenclatures
IMGT groups
IMGT gene names (Pallarès et al., 1998; Lefranc, 2001) IGLV4-69 IGLV5-37 (**)IGLV5-39 IGLV5-45 IGLV5-48 IGLV5-52 IGLV6-57 IGLV7-35 IGLV7-43 IGLV7-46 IGLV8-61 IGLV9-49 IGLV10-54 IGLV10-67 IGLV11-55 IGLV(I)-20 IGLV(I)-38 IGLV(I)-42 IGLV(I)-56 IGLV(I)-63 IGLV(I)-68 IGLV(I)-70 IGLV(IV)-53 IGLV(IV)-59 IGLV(IV)-64 IGLV(IV)-65 IGLV(IV)-66-1 IGLV(V)-58 IGLV(V)-66 IGLV(VI)-22-1 IGLV(V)-25-1 IGLV(VII)-41-1
IMGT functionality F F F F ORF F F P F F, P F F F P ORF P P P P P P P P P P P P P P P P P
IMGT reference sequence accession numbers
IMGT number of alleles
Z73648 Z73672 Z73668 Z73670 Z73649 Z73669 Z73673 Z73660 X14614 Z73674 Z73650 Z73675 Z73676 Z73651 D86996 D87007 D87009 X14613 D86996 D87022 D86993 D86993 D86996 D87000 D87022 D87022 D87004 D87000 D87004 X71351 D86994 X99568
2 1 2 3 1 1 1 1 1 3 3 3 3 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Frippiat et al., 1995; Williams et al., 1996 4b 5e 5a 5c 5d 5b 6a 7c 7a 7b 8a 9a 10a 10b
V lambda A
Kawasaki et al., 1997 5-6 4-1 4-2 4-3 4-4 1-22 3-1P 3-2 3-3 3-4 5-2 1-20 1-25P 4-6 1-6P 1-12P 1-15P 1-21P 1-24P 1-26P 1-27P 4-5P 4-7P 4-8P 4-9P 5-3P 5-5P
lambdavg2 lambdavg3 lambdavg1
(*) Allelic polymorphism by gene amplification that concerns the IGLJ and IGLC genes: The additional IGLJ2D1 and IGLC2D1 genes belong to the 8-IGLC gene haplotype (see text). (**) Allelic polymorphism by insertion/deletion that concerns the IGLV5-39 gene. The IGLV genes are designated by a number for the subgroup (Chuchana et al., 1990; Frippiat et al., 1995; Williams et al., 1996) followed by a hyphen and a number for the localization from 3¢ to 5¢ in the locus (for review, see Pallarès et al., 1998; Lefranc, 2001; Lefranc and Lefranc, 2001a). In the IGLV gene name column, the IGLV genes are listed, for each subgroup, according to their position from 3¢ to 5¢ in the locus. (I), (IV), (V) (VI), (VII) refer to the clans for those pseudogenes that could not be assigned to subgroups with functional genes. Clans comprise, respectively: — clan I: IGLV1, IGLV2, IGLV6, and IGLV10 subgroup genes and pseudogenes IGLV(I)-20, -38, -42, -56, -63, -68, and -70; — clan II: IGLV3 subgroup genes; — clan III: IGLV7 and IGLV8 subgroup genes; — clan IV: IGLV5 and IGLV11 subgroup genes and pseudogenes IGLV(IV)-53, -59, -64, -65, and -66-1; — clan V: IGLV4 and IGLV9 subgroup genes and pseudogenes IGLV(V)-58 and -66; — clan VI: pseudogenes IGLV(VI)-22-1 and -25-1; — clan VII: pseudogene IGLV(VII)-41-1. A pseudogene belonging to the IGLV2 subgroup has not been locatized (IGLV2-NL1, Z22209) and is not shown in the table. For IMGT list with links to GDB and LocusLink: http://imgt.cines.fr/cgi-bin/IMGTlect.jv?query=203. Information on individual IGL genes is provided in IMGT/GENE-DB: http://imgt.cines.fr
45
46
Lefranc and Lefranc
Protein display of the allele *01 of each functional and ORF IGL J-REGION of the 7-IGLC gene haplotype is shown in Figure 4.7b. In the 8-IGLC gene haplotype, the additional IGLJ2D1 gene is functional (van der Burg et al., 2002). The potential additional IGLJ genes from the 9- to 11-IGLC gene haplotypes have not yet been characterized and sequenced. The Human IGLC Genes
FIGURE 4.5 Chromosomal localization of the human IGL locus at 22q11.2. A vertical line indicates the localization of the IGL locus at 22q11.2. The arrow indicates the orientation 5¢ Æ 3¢ of the locus and the gene group order in the locus. The arrow is proportional to the size of the locus, indicated in kilobases (kb). The total number of genes in the locus in shown between parentheses. Depending on the haplotype, there are 7 to 11 IGLC genes. In the 7- and 8-IGLC gene haplotype, each IGLC gene is preceded, in 5¢, by one IGLJ gene. Although the additional IGLC genes, in the 9-, 10-, and 11-IGLC gene haplotypes have not yet been sequenced, they are also probably preceded by one IGLJ gene. The number of functional genes define the potential IGL repertoire that comprises, in the 7IGLC gene haplotype, 37 to 43 genes (29–33 IGLV, 4–5 IGLJ, and 4–5 IGLC) per haploid genome.
deletion mechanism. IGLV allelic polymorphisms and RFLP were reported, some in association with diseases (for review, Lefranc et al., 1999b). The IMGT Protein display of the functional and ORF IGLV genes is shown in Figure 4.7a. Lengths of the IGLV CDR-IMGT are as follows: CDR1IMGT: 6 to 9; CDR2-IMGT: 3, 7, 8; and germline CDR3IMGT: 7 to 9, 11 (Scaviner et al., 1999). The expressed IGLV repertoire is mainly due to five IGLV genes: IGLV2–14, IGLV1–40, IGLV2–8, IGLV1–44, and IGLV3–21, which encode 60% of the lambda repertoire (Ignatovich et al., 1997, 1999). The Human IGLJ Genes The human IGLJ group comprises seven mapped genes, in the 7-IGLC gene haplotype (Chang et al., 1986; Dariavach et al., 1987; Udey and Blomberg, 1987, 1988; Vasicek and Leder, 1990; Bauer and Blomberg, 1991; Poul et al., 1991). Five IGLJ genes are functional (IGLJ1, IGLJ2, IGLJ3, IGLJ6, and IGLJ7) and two are ORF (IGLJ4 and IGLJ5) (Table 4.1). The IGLJ4 and IGLJ5 have a noncanonical DONOR-SPLICE and a nonconserved J-HEPTAMER. Moreover, they precede the IGLC4 and IGLC5 pseudogenes (Dariavach et al., 1987; Vasicek and Leder, 1990). The IGLJ6 is functional and can be used in a productive lambda chain when it precedes the rare IGLC6 functional allele (Dariavach et al., 1987; Poul et al., 1991), or in a truncated lambda chain when it precedes the IGLC6 pseudogene alleles (Stiernholm et al., 1995). The IMGT
In the human IGL locus, the IGLC group comprises 7 to 11 genes, depending on the haplotypes (Taub et al., 1983; Ghanem et al., 1988; Kay et al., 1992; Lefranc et al., 1999b). In the 7-IGLC gene haplotype, four to five IGLC genes are functional (Table 4.1). The additional IGLC2D1 gene of the 8-gene haplotype is functional (van der Burg et al., 2002). The additional IGLC genes in the 9-, 10-, and 11-gene haplotypes have not yet been sequenced. The IGL C-REGIONs belong to four isotypic forms that differ in limited amino acid substitutions to produce the serological markers Kern (Ke) (Ponstingl et al., 1968; Hess et al., 1971), Oz (Appella and Ein, 1967; Ein and Fahey, 1967; Ein, 1968), and Mcg (Fett and Deutsch, 1974; Fett and Deutsch, 1975). The isotypes expressed by the different human IGLC genes and the amino acid differences between the four isotypes (Dariavach et al., 1987) are shown in Table 4.2. Mcg+ proteins have Asparagine (Asn) 1 and Threonine (Thr) 3 (according to the IMGT unique numbering for C-DOMAIN) (Figure 4.4), whereas Mcg- proteins have Alanine (Ala) 1 and Serine (Ser) 3. Position 82, initially described as characteristic of the Mcg marker, is not involved. Indeed, the protein MOR is different from the other Mcg- proteins by having Ala 82 (Frangione et al., 1985), and the protein MCP (Mcg-) encoded by the IGLC7 gene (Niewold et al., 1996) has Lys 82. Ke+ proteins have Glycine (Gly) 45, whereas Ke- proteins have Ser 45. Oz+ proteins have Lys 100, whereas Oz- proteins have Arginine (Arg) 100. The IGLC1 gene encodes Mcg+ Ke+ Oz- lambda chains (Table 4.2). The IGLC2 gene encodes Mcg- Ke- Oz- lambda chains. The IGLC3 gene encodes Mcg- Ke- Oz+ (alleles IGLC3*01, *02 and *03) or Mcg- Ke- Oz- (allele IGLC3*04) lambda chains. The IGLC3 Ke- Oz- isotype has not been assigned serologically, but instead on the presence of the characteristic amino acids (Kawasaki et al., 1997). The IGLC6 gene is either functional (allele *01) in a rare haplotype and potentially encodes Mcg- Ke+ Oz- lambda chains (Dariavach et al., 1987) or a pseudogene (alleles *02 to *05) in more frequent haplotypes (Vasicek and Leder, 1990). The pseudogene alleles result from a recent insertion (duplication) of four nucleotides (agct), which leads to a frameshift and premature stop codon and, if expressed, to truncated lambda chains (Stiernholm et al., 1995). The IGLC7 gene encodes the four characteristic amino acids of the Mcg- Ke+ Oz- isotype (Ala 1, Ser 3, Gly 45, and Arg 100). The IMGT Protein display of the allele*01 of each functional and ORF IGL C-
FIGURE 4.6 Representation of the human IGL locus at 22q11.2. The boxes representing the genes are not to scale. Exons are not shown. A, B, and C refer to three distinct V-CLUSTERs based on the IGLV gene subgroup content (Williams et al., 1996). Pseudogenes that could not be assigned to subgroups with functional genes are designated by a roman number between parentheses, corresponding to the clans, followed by a hyphen and a number for the localization from 3¢ to 5¢ in the locus. IGLV(IV)-66-1 has been identified in D87004 by IMGT curators (G. Folch, V. Giudicelli, M.-P. Lefranc
[email protected]). The vestigial sequences have been attributed to the clans VI and VII as IGLV(VI)-22-1 (lvg2) and IGLV(VII)-41-1 (lvg1). IGLV(VI)-25-1 (lvg3) has been identified in D86994 by IMGT curators (N. Bosc, M.-P. Lefranc) between IGLV3–25 and IGLV3–26 (Lefranc and Lefranc, 2001a).
47
A. IGLV gene
FR1-IMGT (1-26) 1 10 20 .........|.........|...... Z73653,IGLV1-36 QSVLTQPPS.VSEAPRQRVTISCSGS M94116,IGLV1-40 QSVLTQPPS.VSGAPGQRVTISCTGS M94118,IGLV1-41 QSVLTQPPS.VSAAPGQKVTISCSGS Z73654,IGLV1-44 QSVLTQPPS.ASGTPGQRVTISCSGS Z73663,IGLV1-47 QSVLTQPPS.ASGTPGQRVTISCSGS M94112,IGLV1-50 QSVLTQPPS.VSGAPGQRVTISCTGS Z73661,IGLV1-51 QSVLTQPPS.VSAAPGQKVTISCSGS X97462,IGLV2-8 QSALTQPPS.ASGSPGQSVTISCTGT Z73657,IGLV2-11 QSALTQPRS.VSGSPGQSVTISCTGT Z73664,IGLV2-14 QSALTQPAS.VSGSPGQSITISCTGT Z73642,IGLV2-18 QSALTQPPS.VSGSPGQSVTISCTGT X14616,IGLV2-23 QSALTQPAS.VSGSPGQSITISCTGT Z73643,IGLV2-33 QSALTQPPF.VSGAPGQSVTISCTGT X57826,IGLV3-1 SYELTQPPS.VSVSPGQTASITCSGD SYELTQPLS.VSVALGQTARITCGGN X97473,IGLV3-9 X97464,IGLV3-10 SYELTQPPS.VSVSPGQTARITCSGD Z73658,IGLV3-12 SYELTQPHS.VSVATAQMARITCGGN X97471,IGLV3-16 SYELTQPPS.VSVSLGQMARITCSGE X56178,IGLV3-19 SSELTQDPA.VSVALGQTVRITCQGD X71966,IGLV3-21 SYVLTQPPS.VSVAPGKTARITCGGN Z73666,IGLV3-22 SYELTQLPS.VSVSPGQTARITCSGD X97474,IGLV3-25 SYELMQPPS.VSVSPGQTARITCSGD D86994,IGLV3-27 SYELTQPSS.VSVSPGQTARITCSGD Z73645,IGLV3-32 SSGPTQVPA.VSVALGQMARITCQGD LPVLTQPPS.ASALLGASIKLTCTLS X57828,IGLV4-3 Z73667,IGLV4-60 QPVLTQSSS.ASASLGSSVKLTCTLS Z73648,IGLV4-69 QLVLTQSPS.ASASLGASVKLTCTLS Z73672,IGLV5-37 QPVLTQPPS.SSASPGESARLTCTLP Z73668,IGLV5-39 QPVLTQPTS.LSASPGASARFTCTLR Z73670,IGLV5-45 QAVLTQPAS.LSASPGASASLTCTLR Z73649,IGLV5-48 QPVLTQPTS.LSASPGASARLTCTLR Z73669,IGLV5-52 QPVLTQPSS.HSASSGASVRLTCMLS Z73673,IGLV6-57 NFMLTQPHS.VSESPGKTVTISCTRS X14614,IGLV7-43 QTVVTQEPS.LTVSPGGTVTLTCASS Z73674,IGLV7-46 QAVVTQEPS.LTVSPGGTVTLTCGSS Z73650,IGLV8-61 QTVVTQEPS.FSVSPGGTVTLTCGLS Z73675,IGLV9-49 QPVLTQPPS.ASASLGASVTLTCTLS Z73676,IGLV10-54 QAGLTQPPS.VSKGLRQTATLTCTGN D86996,IGLV11-55 RPVLTQPPS LSASPGATARLPCTLS M34927,V-PREB
CDR1-IMGT (27-38) 30 ...|........ SSNIGNNA.... SSNIGAGYD... SSDMGNYA.... SSNIGSNT.... SSNIGSNY.... SSNIGAGYV... SSNIGNNY.... SSDVGGYNY... SSDVGGYNY... SSDVGGYNY... SSDVGSYNR... SSDVGSYNL... SSDVGDYDH... KLGDKY...... NIGSKN...... ALPKKY...... NIGSKA...... ALPKKY...... SLRSYY...... NIGSKS...... VLGENY...... ALPKQY...... VLAKKY...... SMEGSY...... SEHSTYT..... SGHSSYI..... SGHSSYA..... SDINVGSYN... SGINVGTYR... SGINVGTYR... SGINLGSYR... SGFSVGDFW... SGSIASNY.... TGAVTSGYY... TGAVTSGHY... SGSVSTSYY... SGYSNYK..... SNNVGNQG.... SDLSVGGKN...
FR2-IMGT (39-55) 40 50 .|.........|..... VNWYQQLPGKAPKLLIY VHWYQQLPGTAPKLLIY VSWYQQLPGTAPKLLIY VNWYQQLPGTAPKLLIY VYWYQQLPGTAPKLLIY VHWYQQLPGTAPKLLIY VSWYQQLPGTAPKLLIY VSWYQQHPGKAPKLMIY VSWYQQHPGKAPKLMIY VSWYQQHPGKAPKLMIY VSWYQQPPGTAPKLMIY VSWYQQHPGKAPKLMIY VFWYQKRLSTTSRLLIY ACWYQQKPGQSPVLVIY VHWYQQKPGQAPVLVIY AYWYQQKSGQAPVLVIY VHWYQQKPGQDPVLVIY AYWYQQKPGQFPVLVIY ASWYQQKPGQAPVLVIY VHWYQQKPGQAPVLVIY ADWYQQKPGQAPELVIY AYWYQQKPGQAPVLVIY ARWFQQKPGQAPVLVIY EHWYQQKPGQAPVLVIY IEWYQQRPGRSPQYIMK IAWHQQQPGKAPRYLMK IAWHQQQPEKGPRYLMK IYWYQQKPGSPPRYLLY IYWYQQKPGSLPRYLLR IYWYQQKPGSPPQYLLR IFWYQQKPESPPRYLLS IRWYQQKPGNPPRYLLY VQWYQQRPGSSPTTVIY PNWFQQKPGQAPRALIY PYWFQQKPGQAPRTLIY PSWYQQTPGQAPRTLIY VDWYQQRPGKGPRFVMR AAWLQQHQGHPPKLLSY MFWYQQKPGSSPRLFLY
CDR2-IMGT (56-65) 60 ....|..... YDD....... GNS....... ENN....... SNN....... RNN....... GNS....... DNN....... EVS....... DVS....... EVS....... EVS....... EGS....... NVN....... QDS....... RDS....... EDS....... SDS....... KDS....... GKN....... YDS....... EDS....... KDS....... KDS....... DSS....... VKSDGSH... LEGSGSY... LNSDGSH... YYSDSDK... YKSDSDK... YKSDSDK... YYSDSSK... YHSDSNK... EDN....... STS....... DTS....... STN....... VGTGGIVG.. RNN....... HYSDSDK...
FR3-IMGT (66-104) 70 80 90 100 ....|.........|.........|.........|.... LLPSGVS.DRFSGSK..SGTSASLAISGLQSEDEADYYC NRPSGVP.DRFSGSK..SGTSASLAITGLQAEDEADYYC KRPSGIP.DRFSGSK..SGTSATLGITGLWPEDEADYYC QRPSGVP.DRFSGSK..SGTSASLAISGLQSEDEADYYC QRPSGVP.DRFSGSK..SGTSASLAISGLRSEDEADYYC NRPSGVP.DQFSGSK..SGTSASLAITGLQSEDEADYYC KRPSGIP.DRFSGSK..SGTSATLGITGLQTGDEADYYC KRPSGVP.DRFSGSK..SGNTASLTVSGLQAEDEADYYC KRPSGVP.DRFSGSK..SGNTASLTISGLQAEDEADYYC NRPSGVS.NRFSGSK..SGNTASLTISGLQAEDEADYYC NRPSGVP.DRFSGSK..SGNTASLTISGLQAEDEADYYC KRPSGVS.NRFSGSK..SGNTASLTISGLQAEDEADYYC TRPSGIS.DLFSGSK..SGNMASLTISGLKSEVEANYHC KRPSGIP.ERFSGSN..SGNTATLTISGTQAMDEADYYC NRPSGIP.ERFSGSN..SGNTATLTISRAQAGDEADYYC KRPSGIP.ERFSGSS..SGTMATLTISGAQVEDEADYYC NRPSGIP.ERFSGSN..PGNTTTLTISRIEAGDEADYYC ERPSGIP.ERFSGSS..SGTIVTLTISGVQAEDEADYYC NRPSGIP.DRFSGSS..SGNTASLTITGAQAEDEADYYC DRPSGIP.ERFSGSN..SGNTATLTISRVEAGDEADYYC ERYPGIP.ERFSGST..SGNTTTLTISRVLTEDEADYYC ERPSGIP.ERFSGSS..SGTTVTLTISGVQAEDEADYYC ERPSGIP.ERFSGSS..SGTTVTLTISGAQVEDEADYYC DRPSRIP.ERFSGSK..SGNTTTLTITGAQAEDEADYYY SKGDGIP.DRFMGSS..SGADRYLTFSNLQSDDEAEYHC NKGSGVP.DRFSGSS..SGADRYLTISNLQLEDEADYYC SKGDGIP.DRFSGSS..SGAERYLTISSLQSEDEADYYC GQGSGVP.SRFSGSKDASANTGILLISGLQSEDEADYYC QQGSGVP.SRFSGSKDASTNAGLLLISGLQSEDEADYYC QQGSGVP.SRFSGSKDASANAGILLISGLQSEDEADYYC HQGSGVP.SRFSGSKDASSNAGILVISGLQSEDEADYYC GQGSGVP.SRFSGSNDASANAGILRISGLQPEDEADYYC QRPSGVP.DRFSGSIDSSSNSASLTISGLKTEDEADYYC NKHSWTP.ARFSGSL..LGGKAALTLSGVQPEDEAEYYC NKHSWTP.ARFSGSL..LGGKAALTLSGAQPEDEAEYYC TRSSGVP.DRFSGSI..LGNKAALTITGAQADDESDYYC SKGDGIP.DRFSVLG..SGLNRYLTIKNIQEEDESDYHC NRPSGIS.ERLSASR..SGNTASLTITGLQPEDEADYYC QLGPGVP.SRVSGSKETSSNTAFLLISGLQPEDEADYYC
CDR3-IMGT (105-115) 110 .....|...... AAWDDSLNG... QSYDSSLSG... LAWDTSPRA... AAWDDSLNG... AAWDDSLSG... KAWDNSLNA... GTWDSSLSA... SSYAGSNNF... CSYAGSYTF... SSYTSSSTL... SLYTSSSTF... CSYAGSSTL... SLYSSSYTF... QAWDSSTA.... QVWDSSTA.... YSTDSSGNH... QVWDSSSDH... LSADSSGTY... NSRDSSGNH... QVWDSSSDH... LSGDEDN..... QSADSSGTY... YSAADNN..... QLIDNHA..... GESHTIDGQVG* ETWDSNT..... QTWGTGI..... MIWPSNAS.... AIWYSSTS.... MIWHSSAS.... MIWHSSAS.... GTWHSNSKT... QSYDSSN..... LLYYGGAQ.... LLSYSGAR.... VLYMGSGI.... GADHGSGSNFV* SAWDSSLSA... QVYESSAN....
QPVLHQPPA.MSSALGTTIRLTCTLR NDHDIGVYS... VYWYQQRPGHPPRFLLR YFSQSDQ... SQGPQVP.PRFSGSQDVARNRGYLSISELQPEDEAMYYC AMGARSSEKEER
B. IGLJ genes 1
...... X04457 M15641 M15642 X51755 X51755 M18338 X51755
,IGLJ1 ,IGLJ2 ,IGLJ3 ,IGLJ4 ,IGLJ5 ,IGLJ6 ,IGLJ7
10 .. .|. YVFGTGTKVTVL VVFGGGTKLTVL VVFGGGTKLTVL FVFGGGTQLIIL WVFGEGTELTVL NVFGSGTKVTVL AVFGGGTQLTVL
C. IGLC genes A AB B BC C CD D DE E EF F FG G --------------> ----------> ------> -------> -----------> -------> -----------> 104 1 10 15 16 20 23 30 36 39 41 45 77 84 85 89 96 97 110 118 121 130 7654321|........|....|123|...|...... ...|.....| |.....|1234567|......|12345677654321|..........|12|....... .....|....... ..|.........| X51755 J00253 K01326 J03011 X51755
,IGLC1 ,IGLC2 ,IGLC3 ,IGLC6 ,IGLC7
(G)QPKANPTVTLFPPSSEELQ...ANKATLVCLIS (G)QPKAAPSVTLFPPSSEELQ...ANKATLVCLIS (G)QPKAAPSVTLFPPSSEELQ...ANKATLVCLIS (G)QPKAAPSVTLFPPSSEELQ...ANKATLVCLIS (G)QPKAAPSVTLFPPSSEELQ...ANKATLVCLVS
DFYP..GAVT DFYP..GAVT DFYP..GPVT DFYP..GAVK DFYP..GAVT
VAWKADGSPVKA..GVETTKPSKQSN......NKYAASSYLSLTPEQW..KSHRSYSC VAWKADSSPVKA..GVETTTPSKQSN......NKYAASSYLSLTPEQW..KSHRSYSC VAWKADSSPVKA..GVETTTPSKQSN......NKYAASSYLSLTPEQW..KSHKSYSC VAWKADGSPVNT..GVETTTPSKQSN......NKYAASSYLSLTPEQW..KSHRSYSC VAWKADGSPVKV..GVETTKPSKQSN......NKYAASSYLSLTPEQW..KSHRSYSC
QVTHE....GSTV QVTHE....GSTV QVTHE....GSTV QVTHE....GSTV RVTHE....GSTV
FIGURE 4.7 IMGT Protein displays of the human IGL genes. (a) Human IGL V-REGIONs. Only the allele *01 of each functional or ORF V-REGION is shown. The FR-IMGT and CDR-IMGT are according to the IMGT unique numbering for V-REGION. Human IGLV genes are listed, for each subgroup, according to their position from 3¢ to 5¢ in the locus. For comparison, the human V-PREB region is displayed at the bottom of the figure (only a part of the nonIg-like segment is shown) (Scaviner et al., 1999). (b) Human IGL J-REGIONs. Only the allele *01 of each functional or ORF J-REGION is shown. (c) Human IGL C-REGIONs. The IGL C-REGIONs correspond to a single C-DOMAIN. The strands and loops are according to the IMGT unique numbering for C-DOMAIN. Amino acids at positions 1 and 3, 45, and 100 are involved in the Mcg, Ke, and Oz serological markers, respectively (see text and Table 4.2).
48
EKTVAPTECS EKTVAPTECS EKTVAPTECS EKTVAPAECS EKTVAPAECS
49
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
REGION of the 7-IGLC gene haplotype is shown in Figure 4.7c. Polymorphisms by duplication of the IGLC2 and/or IGLC3 genes have been described in different populations (Taub et al., 1983; Ghanem et al., 1988) with a total number of 7 to 11 IGLC genes. The restriction fragment length polymorphism (RFLP) alleles correspond to polymorphic 8-, 13-, 18-, 23-, 28-kb EcoRI fragments (Taub et al., 1983; Dariavach et al., 1987; Ghanem et al., 1988; Kay et al., 1992; Lefranc et al., 1999b), which contain two, three, four, five, and six IGLC genes, respectively. So far, only the IGLC2 and IGLC3 genes of the 8-kb EcoRI fragment (7IGLC gene haplotype) (Hieter et al., 1981) and the addi-
TABLE 4.2
tional IGLC2D1 gene of the 13-kb EcoRI fragment (8-IGLC gene haplotype) (van der Burg et al., 2002) have been sequenced.
Human IGL Orphons Six IGL orphons have been identified (Table 4.3) (Frippiat et al., 1997; Lefranc, 2001b). Two IGLV orphons are on chromosome 8 at 8q11.2 and one (belonging to subgroup 8) has been sequenced. Two IGLC orphons and two IGLV orphons have also been characterized on 22q, outside the major IGL locus (Dunham et al., 1999; Lefranc, 2001b) (see also IMGT Repertoire, http://imgt.cines.fr).
Correspondence between serological lambda isotypes and IGLC gene and allele names Amino acid positions (1)
Serological isotype
1 (6) 112
IGLC gene and allele name
3 (8) 114
45 (46) 152
82 (57) 163
100 (83) 190
Mcg+ Ke+ Oz-
IGLC1*01, *02
Asn
Thr
Gly
Lys
Arg
Mcg- Ke- Oz-
IGLC2*01, *02, *03 IGLC3*04 IGLC2D1*01
Ala
Ser
Ser
Thr
Arg
Mcg- Ke- Oz+
IGLC3*01, *02, *03
Ala
Ser
Ser
Thr
Lys
IGLC2*04 IGLC6*01 IGLC7*01, *02
Ala
Ser
Gly
Thr
Arg
Ala
Ser
Gly
Lys
Arg
-
+
-
Mcg Ke Oz
(1) Amino acid positions according to the IMGT unique numbering for C-DOMAIN (in bold) (Figure 4.4), to the IMGT exon numbering (between parentheses) and to the Kabat numbering (in italics). Whereas the serological markers Mcg, Ke, and Oz were assigned to Bence-Jones and myeloma proteins using specific antibodies (Walker et al., 1988), their assignment to the IGLC gene and allele names is based on the presence or absence of characteristic amino acids (Dariavach et al., 1987).
TABLE 4.3 IMGT gene groups
IMGT gene names
IMGT functionality
List of the human IGL orphons
IMGT reference sequences
Accession numbers
IMGT number of alleles
Chromosomal localization
IGLC
IGLC/OR22-1 IGLC/OR22-2
P P
dJ90G24.3 dJ149A16.1
AL008723 AL021937
1 1
22 (16.1 Mb from the centromere) 22 (16.26 Mb from the centromere)
IGLV
IGLV8/OR8-1 IGLV8/OR8-2 IGLV(IV)/OR22-1 IGLV(IV)/OR22-2
P, ORF (1) P P
Orphée1, TL6 Orphée2 bK390C10.1 DJ149A16.4
Y08831, U03636
2 1 1 1
8q11.2 8q11.2 22 (9.4 Mb from the centromere) 22 (16.28 Mb from the centromere)
AL008721 AL021937
Reference sequences in bold have been mapped: “mapped” refers to sequences that have been obtained from clones (phages, cosmids, YACs) either by subcloning or PCR, and does not apply to sequences obtained directly by PCR from genomic DNA. Orphon genes are designated by a subgroup number (if known) followed by a slash, OR (for Orphon), the chromosome number, a dash, and a specific gene number. References and detailed information on the orphons are available in the IMGT Repertoire, http://imgt.cines.fr. (1) Not sequenced.
50
Lefranc and Lefranc
Total Number of Human IGL Genes and Potential Genomic Repertoire The total number of human IGL genes per haploid genome is 87 to 96 (93 to 102 genes, if the orphons are included) (Table 4.4). The potential genomic human IGL repertoire comprises 37 to 43 functional genes: 29 to 33 functional IGLV genes belonging to 10 subgroups, four to five IGLJ, and four to five IGLC functional genes in the 7-IGLC gene haplotype (Table 4.5).
THE MOUSE IGL GENES Chromosomal Localization and Organization of the Mouse IGL Locus The mouse IGL locus is located on chromosome 16 at 13 cM. A complete map of the IGL locus from Mus musculus domesticus and derived laboratory mice was constructed from clone analysis and pulse field gel electrophoresis (PFGE) of large DNA fragments (Figure 4.8) (Storb et al., 1989). The mouse IGL locus spans 240 kb and consists of
TABLE 4.4 Repertoire of the human germline IGLV genes at 22q11.2 Seventy-three–74 IGLV genes on 900 kilobases: 56 to 57 genes belonging to 11 subgroups and 17 pseudogenes assigned to the clans. Twenty-nine to 30 FUNCTIONAL Five ORF Thirty-five PSEUDOGENE Three FUNCTIONAL or PSEUDOGENE One ORF or PSEUDOGENE Potential repertoire: 29 to 33 FUNCTIONAL IGLV genes belonging to 10 subgroups. Subgroup
Functional
ORF
Pseudogene
IGLV1 (B) (C) IGLV2 (A) IGLV3 (A) IGLV4 (A) (C) IGLV5 (B) IGLV6 (C) IGLV7 (B) IGLV8 (C) IGLV9 (B) IGLV10 (C) IGLV11 (C) IGLV(I) (A) (B) (C) IGLV(IV) (C) IGLV(V) (C) IGLV(VI) (A) IGLV(VII) (B) Total
Total
5 — 5 8(+2)* 1 2 3–4** 1 1(+1)* 1 1 1 — — — — — — — —
1(+1)* — 1 1 — — 1 — — — — — 1 — — — — — — —
(+1)* 1 3 12(+2)* — — — — 1(+1)* — — 1 — 1 2 4 5 2 2 1
7 1 9 23 1 2 4–5** 1 3 1 1 2 1 1 2 4 5 2 2 1
29–30(+3)*
5(+1)*
35(+4)*
73–74**
* The following genes have alleles with different functionality: ORF or PSEUDOGENE (IGLV1-41), FUNCTIONAL or PSEUDOGENE (IGLV3-9, IGLV3-22, IGLV7-46). ** An allelic polymorphism by insertion/deletion, which concerns IGLV5-39 (Frippiat et al., 1995). (A), (B), (C) refer to three distinct V-CLUSTERs based on the IGLV gene subgroup content (Williams et al., 1996). (I), (IV), (V), (VI), (VII) refer to the clans for those pseudogenes that cannot be assigned to subgroups with functional genes.
51
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
FIGURE 4.8 Representation of the mouse (Mus musculus) IGL locus on chromosome 16 at 13 cM. The boxes representing the genes are not to scale. Exons are not shown.
TABLE 4.5
Total number of human immunoglobulin lambda (IGL) genes per haploid genome, compared to the total number of kappa (IGK) and heavy (IGH) genes Major loci
Locus IGL IGK IGH
Chromosomal localization
V
D
J
22q11.2 2p11.2 14q32.33
73–74 (40a or) 76 123–129
0 0 27
7–11 5 9
C
Total number of genes in the major locus
Number of orphons
Total number of genes (including orphons)
7–11 1 11b
87–96 (46a or) 82 170–176c
6 25 36d
93–102 (71a or)–107 206–212c,d
a
Number of genes in the rare IGKV haplotype without the distal V-CLUSTER. Allelic IGHC multigene deletions, duplications, and triplications have been described in healthy individuals. The number of IGHC genes may vary from five (deletion I) to probably nineteen (triplication III) per haploid genome (Lefranc and Lefranc, 2001a). c Not included, the seven nonmapped IGHV genes. d Included, the IGHC processed gene, IGHEP2, localized on chromosome 9 (9p24.2–p24.1). b
three IGLV genes, five IGLJ genes, and four IGLC genes organized in two V-J-C-J-C clusters (Figure 4.8). The 5¢ and 3¢ clusters contain two and one IGLV gene(s), respectively. Each IGLC is preceded by one (or two) IGLJ gene(s). Enhancers have been characterized downstream of each cluster (Hagman et al., 1990; Eccles et al., 1990) (Figure 4.8). A search of the Celera database confirmed the order and transcriptional orientation of the IGL genes (IGLV2, the most 5¢ IGLV gene in the mouse locus, is at 15.6 Mb in the Celera contig, whereas IGLC1, the most 3¢ gene in the locus, is at 15.4 Mb) (Gerdes and Wabl, 2002). The Celera mouse
chromosome 16 (Mmu16) draft sequence (Mural et al., 2002) is derived from four mouse strains (A/J, DBA/2J, 129X1/SvJ, 129S1/SvImJ), whereas the public Mouse Genome Sequencing Consortium (MGSC) draft is generated from the C57BL/6J strain (http://mouse.ensembl.org).
The Mouse IGLV Genes In Mus musculus domesticus and derived laboratory mice (BALB/c), the IGL locus only comprises three IGLV genes
52
Lefranc and Lefranc
belonging to two subgroups (Bernard et al., 1978; Tonegawa et al., 1978a,b; Arp et al., 1982; Weiss and Wu, 1987; Sanchez et al., 1990) (Table 4.7). The IGLV1 and IGLV2 genes that belong to subgroup 1 are localized in the 3¢ and 5¢ cluster, respectively. The IGLV3 gene, which is the unique representative of subgroup 2, is localized downstream of IGLV2 in the 5¢ cluster. It shows a stop codon at its end, which considerably reduces the possibility of a productive rearrangement. In derived strains from wild Mus musculus musculus (MBK, PWK, MAI) and Mus spretus (SMZ, STF) mice, IGLV genes that belong to a third IGLV subgroup have been characterized (Table 4.7). This subgroup is absent in Mus musculus domesticus and in other laboratory mice (BALB/c). The first cDNAs from the IGLV3 subgroup were sequenced from two Mus musculus musculus mice from Skive (Denmark) (clone SD26) and Sladeckovce (Czech Republic) (clone CZ81) (Reidl et al., 1992) (Table 4.7). IGLV3 subgroup genes were then identified in some strains from Mus musculus musculus and North African Mus spretus species (Amrani et al., 2002). The number of IGLV3 subgroup genes varies from zero to at least five in Mus musculus musculus strains, and from zero to three in Mus spretus strains, as deduced from RFLP analysis (Table 4.8). The lost Mus spretus SPE strain probably does not have, as the B6.lambdaSEG strain, any gene belonging to the IGLV3 subgroup. Moreover, these strains lack the IGLV1 gene and only have the two IGLV genes (IGLV2 and IGLV3) of the 5¢ cluster (Amrani et al., 2002) (Table 4.7). The IMGT Protein display of the functional and ORF IGLV genes is shown in Figure 4.9A. Lengths of the IGLV CDR-IMGT are as follows: CDR1-IMGT: 7 to 9; CDR2-IMGT: 3, 7; and
germline CDR3-IMGT: http://imgt.cines.fr).
8,
11
(IMGT
Repertoire,
The Mouse IGLJ and IGLC Genes The IGL locus from Mus musculus domesticus and derived laboratory mice contains four IGLC genes belonging to two subgroups: the IGLC1 and IGLC4 genes belong to subgroup 1, whereas the IGLC2 and IGLC3 genes belong to subgroup 2. Each of these genes is preceded by one (or two) IGLJ gene(s). The IGLJ genes (Table 4.9) and IGLC genes (Table 4.10) are arranged in two clusters: J2-C2-J4C4 and J3-J3P-C3-J1-C1 (Bernard et al., 1978; Blomberg et al., 1981; Blomberg and Tonegawa, 1982; Miller et al., 1981, 1982; Selsing et al., 1982; Weiss and Wu, 1987). Probes specific for the IGLC subgroups allowed researchers to estimate the number of IGLC genes per subgroup in different wild Mus musculus musculus and Mus spretus mice and derived strains (Table 4.9). In the B6.lambda SEG strain, the 3¢ cluster V1-J3-J3P-C3-J1-C1 is deleted and the remaining single cluster displays an additional J–C duplication: V2-V3-J2-C2-J4-C4-J5-C5. However, only lambda2 chains can be expressed (with either a rearranged IGLV2-J2 or IGLV3-J2 gene) since IGLC4 is a pseudogene (Table 4.10) and IGLJ5 is an ORF (Table 4.9). This organization is also probably that of the lost strain SPE (Mami and Kindt, 1987) and of the other Mus spretus strains that do not have lambda1 and lambda3 chains (Amrani et al., 2002). The IMGT Protein displays of the allele *01 of each functional and ORF J-REGION and C-REGION are shown in Figure 4.9B and Figure 4.9C, respectively.
TABLE 4.6 Number of functional human immunoglobulin lambda (IGL) genes per haploid genome compared to the number of functional kappa (IGK) and heavy (IGH) genes Chromosomal localization
Locus size in kb (kilobases)
V
D
J
C
Number of functional genes
IGL
22q11.2
1050
29–33
0
4–5
4–5
37–43
29 ¥ 4 = 116 (m) 33 ¥ 5 = 165 (M)
IGK
2p11.2
1820
30–35
0
5
1
36–41
500a
17–19a
0
5
1
23–25a
30 ¥ 5 = 150 (m) 35 ¥ 5 = 175 (M) 17 ¥ 5 = 85 (m)a 19 ¥ 5 = 95 (M)a
1250
38–46
23
6
9b
76–84
Locus
IGH
a
14q32.33
Combinatorial diversity (range per locus)
38 ¥ 23 ¥ 6 = 5244 (m) 46 ¥ 23 ¥ 6 = 6348 (M)
In the rare IGKV haplotype without the distal V-CLUSTER. In haplotypes with multigene deletion, the number of functional IGHC genes is five (deletions I, III, and V), six (deletions IV and VI), or eight (deletion II) per haploid genome (Lefranc and Lefranc, 2001a). In haplotypes with multigene duplication or triplication, the exact number of functional IGHC genes per haploid genome is not known. The range of the theoretical combinatorial diversity indicated takes into account the minimum (m) and the maximum (M) number of functional V, D, and J genes in each of the major IGL, IGK, and IGH loci. b
Mouse (Mus musculus, Mus spretus) IGLV germline genes
TABLE 4.7 Mouse (Mus musculus) IGLV IGLV subgroup
IGLV gene name
IGLV allele name
Fct
IGLV1
IGLV1
IGLV1*01
F
BALB/c
V1
J00590
IGLV1*02 IGLV2*01 IGLV2*02
F F F
BALB/c
IGLV2
BALB/c
M315/eVl1 V2 J558/eVl2
X58417 J00599 X58412
Strain
Reference sequences
Accession numbers
Sequences from the literature BALB/c, A1-13/eVl1[X58409], BALB/c [V00811] [V00815] BALB/c [X58418], BALB/c, M315e/Vl2[X58423], BALB/c [X58424]
IGLV2
IGLV3
IGLV3*01
F
BALB/c
Lg1
M34597
BALB/c, VLx (Vlambdax)[D38129]
IGLV3
IGLV4 IGLV5 IGLV6
IGLV4*01 IGLV5*01 IGLV6*01 IGLV6*02 IGLV6*03 IGLV7*01 IGLV7*02 IGLV8*01 IGLV8*02
[F] [F] [F] [F] [F] [F] [F] [F] [F]
MBK PWK PWK MAI MBK PWK MBK MAI MBK
MBK2 PWK1 PWK3 MAI2 MBK4 PWK2 MBK1 MAI1 MBK3
AF357985° AF357981° AF357979° AF357983° AF357987° AF357980° AF357984° AF357982° AF357986°
SD26[M94349]#c CZ81[M94351]#c
IGLV7 IGLV8
#c: rearranged cDNA. °: genomic DNA, but not known as being germline or rearranged.
Mouse (Mus spretus) IGLV IGLV gene name
IGLV allele name
Fct
IGLV1 IGLV2
(1) IGLV2*01
F
SPE
IGLV2SPE (Vlambda2SPE)
M17529
IGLV2
IGLV3
IGLV3*01
F
B6.lambdaSEG
VlambdaxSEG
AF357988
IGLV3
IGLV4
IGLV4*01 IGLV4*02 IGLV8*01
[F] [F] [F]
SMZ STF SMZ
SMZ1 STF2 SMZ2
AF357978° AF357975° AF357977°
IGLV subgroup IGLV1
IGLV8
Strain
Reference sequences
Accession numbers
Sequences from the literature
B6.lambdaSEG, Vlambda2SEG [AF357989]
STF, STF1[AF357976]°
Functionality (Fct) is shown between brackets when the accession number refers to genomic DNA, but not known as being germline or rearranged. Reference sequences in bold have been mapped: “mapped” refers to sequences that have been obtained from clones (phages, cosmids, YACs) either by subcloning or PCR, and does not apply to sequences obtained directly from genomic DNA. For a given gene name, each horizontal line corresponds to a different allele. (1) The cluster IGLV1-IGLJ3_IGLC3_IGLJ1_IGLC1 is deleted in B6. lambdaSEG and probably also in the lost strain SPE (Amrani et al., 2002). See Tables 4.9 and 4.10 for IGLJ and IGLC genes, respectively.
53
A. Mouse (Mus musculus) IGLV IGLV gene
° ° ° ° °
FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT (1-26) (27-38) (39-55) (56-65) (66-104) (105-115) 1 10 20 30 40 50 60 70 80 90 100 110 .........|.........|...... ...|........ .|.........|..... ....|..... ....|.........|.........|.........|.... .....|......
J00590 ,IGLV1 J00599 ,IGLV2 M34597 ,IGLV3(1) AF357985, IGLV4 AF357981, IGLV5 AF357979, IGLV6 AF357980, IGLV7 AF357982, IGLV8
QAVVTQESA.LTTSPGETVTLTCRSS QAVVTQESA.LTTSPGGTVILTCRSS QLVLTQSSS.ASFSLGASAKLTCTLS TQPSS.VSTSLGSTVKLSCKRS TQPSS.VSTSLGSTVKLSCKRS TQPSS.VSTSLGSTVKLPCKCS TQPSS.VSTSLGSTVKLSCKPS TQPSS.VSTSLGSTVKLPCKRS
TGAVTTSNY... TGAVTTSNY... SQHSTYT..... TGNIGNNY.... TGNIGNNY.... TGNIGSYY.... TGKIGNYF.... TGNIGNDY....
ANWVQEKPDHLFTGLIG ANWVQEKPDHLFTGLIG IEWYQQQPLKPPKYVME VHWYQQYMGRSPTNMIY VNWYQQYMGRSPTNMIY VHWYQQHMGRSPTNMIH MSWYQQHMGRSPTNMIY VHWYQQHMGRSPTNMIY
GTN....... GTS....... LKKDGSH... DDN....... GDD....... SDD....... RDD....... RDD.......
NRAPGVP.ARFSGSL..IGDKAALTITGAQTEDEAIYFC ALWYSNHF.... NRAPGVP.VRFSGSL..IGDKAALTITGAQTEDDAMYFC ALWYSTHF.... STGDGIP.DRFSGSS..SGADRYLSISNIQPEDEAIYIC GVDTIKEQFV*. KRPSGVS.DRFSGSIDSSSNSAFLTINNVQAED QRPTGVS.DRFSGSIDSSSNSAFLTINNVQAED QRPSGVS.DRFSGSIDSSSNSAFLTINNVQAED LRPSGVS.DRFSGSIDSSSNSAFLTINNVQAED QRPSGVS.DRFSGSIDSSSNSAFLTINNVQAED
Mouse (Mus spretus) IGLV IGLV gene
FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT (1-26) (27-38) (39-55) (56-65) (66-104) (105-115) 1 10 20 30 40 50 60 70 80 90 100 110 .........|.........|...... ...|........ .|.........|..... ....|..... ....|.........|.........|.........|.... .....|......
M17529 ,IGLV2 AF357988,IGLV3 ° AF357978, IGLV4 ° AF357977, IGLV8
QAVVTQESA.LTTSPGGTVILTCRSS QPVLTQSSS.ASFSLGASAKLTCTLS TQPSS.VSTSLGSTVKLSCKRS TQPSS.VSTSLGSTVKLPCKRS
TGAVTTSNY... SEHSTYI..... TGNIGNN..... TGNIGNN.....
AIWVQEKTDHLFAGVIG IEWYQQQPLKPPKYVMQ YVHWYQQYMGRSPTNMI YVHWYQQHMGRPPTNMI
DTS....... LKKDGSH... YDD....... YRD.......
NRAPGVP.ARFSGSL..IGDKAALTITGAQTEDDAMYFC ALWYSNHF.... SKGDGIP.DRFSGSS..SGADRYLSISNIQPEDEAIYIC GVDDNIRGQFV. NKRPSGVSDRFSGSIDSSSNSAFLTINNVQAED DQRPSGVSDRFSGSIDSSSNSAFLTINNVQAED
B. Mouse (Mus musculus) IGLJ __________________________________________ IGLJ segments __________________________________________ 1 10 .........|... V00813 J00593 J00583 J00584 J00596
,IGLJ1 ,IGLJ2 ,IGLJ3 ,IGLJ3P ,IGLJ4(1)
WVFGGGTKLTVL. YVFGGGTKVTVL. FIFGSGTKVTVL. GSFSSNGLLYAG. WVFGGGTRLTVL.
Mouse (Mus spretus) IGLJ __________________________________________ IGLJ segments __________________________________________ 1 10 .........|... M16555 ,IGLJ4(1) AF357974,IGLJ5
WVFGGGTRLTVL. WVFGGGTRLTVL.
FIGURE 4.9 IMGT Protein displays of the mouse (Mus musculus, Mus spretus) IGL genes. (a) Mouse IGL VREGIONs. Only the allele *01 of each functional or ORF V-REGION is shown. (1) The last codon of the CDR3-IMGT of the Mus musculus IGLV3 gene is a STOP-CODON, which can disappear during rearrangements. °: genomic DNA, but not known as being germline or rearranged. Partial sequences at both ends. (b) Mouse IGL J-REGIONs. Only the allele *01 of each functional or ORF J-REGION is shown. The sequences of Mus musculus and Mus spretus IGLJ4*01 are identical. (c) Mouse IGL C-REGIONs. The IGLC Protein display is according to the IMGT unique numbering for C-DOMAIN. Only the allele *01 of each functional or ORF J-REGION is shown. N-glycosylation sites (NXS/T, where X is different from P) are underlined. The IGL C-REGIONs correspond to a single C-DOMAIN. The strands and loops are according to the IMGT unique numbering for C-DOMAIN.
54
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
55
C. Mouse (Mus musculus) IGLC ____________________________________________________________________________________________________________________________________________________________ IGLC genes ____________________________________________________________________________________________________________________________________________________________ A AB B BC C CD D DE E EF F FG G --------------> ----------> ------> -------> -----------> -------> ----------> 1 10 15 16 20 23 30 36 39 41 45 77 84 85 89 96 97 104 110 118 121 130 7654321|........|....|123|...|...... ...|.....| |.....|1234567|......|12345677654321|..........|12|....... .....|....... ..|.........| J00587 J00595 J00585
,IGLC1 ,IGLC2 ,IGLC3
(G)QPKSSPSVTLFPPSSEELE...TNKATLVCTIT DFYP..GVVT (G)QPKSTPTLTVFPPSSEELK...ENKATLVCLIS NFSP..SGVT (G)QPKSTPTLTMFPPSPEELQ...ENKATLVCLIS NFSP..SGVT
VDWKVDGTPVTQ..GMETTQPSKQSN......NKYMASSYLTLTARAW..ERHSSYSC QVTHE....GHTV EKSLSRADCS VAWKANGTPITQ..GVDTSNPTKEGN.......KFMASSFLHLTSDQW..RSHNSFTC QVTHE....GDTV EKSLSPAECL VAWKANGTPITQ..GVDTSNPTKEDN.......KYMASSFLHLTSDQW..RSHNSFTC QVTHE....GDTV EKSLSPAECL
Mouse (Mus spretus) IGLC ____________________________________________________________________________________________________________________________________________________________ IGLC genes ____________________________________________________________________________________________________________________________________________________________ A AB B BC C CD D DE E EF F FG G --------------> ----------> ------> -------> -----------> -------> ----------> 104 1 10 15 16 20 23 30 36 39 41 45 77 84 85 89 96 97 110 118 121 130 7654321|........|....|123|...|...... ...|.....| |.....|1234567|......|12345677654321|..........|12|....... .....|....... ..|.........| M16554 ,IGLC2 AF357974,IGLC5
(G)QPKSTPTLTVFPPSSEELK...ENKATLVCLIS NFSP..SGVT VAWKANGTPITQ..GVDTSNPTKEGN.......KFMASSFLHLTSDQW..RSHNSFTC QVTHE....GDTV EKSLSPAECL (G)QPKATPSVNLFPPSSEELK...TKKATLVCMIT EFYA..TAVR MAWKADGTPITQ..DVETTQPPKQS........DNMASSYLLFTAEAW..ESHSSYSC HVTHE....GNTV EKNLSRAECS
FIGURE 4.9 (Continued) TABLE 4.8 Mouse (Mus musculus, Mus spretus) IGLV RFLP Mouse (Mus musculus) Number of IGLV genes IGLV subgroup IGLV1
IGLV gene namea
BALB/c
IGLV1 IGLV2
1 1
MAI
MBK
PWK
2
3
3
2
IGLV2
IGLV3
1
2
2
2
IGLV3
IGLV4–
0
5
5
5
IGLV8
Mouse (Mus spretus) IGLV subgroup IGLV1
IGLV gene namea IGLV1 IGLV2
Number of IGLV genes B6.lambdaSEG 0 1
STF
SMZ
1
3
2
IGLV2
IGLV3
1
1
1
IGLV3
IGLV4,
0
2
3
IGLV8M a
For sequenced genes (see Table 4.7).
Total Number of Mouse IGL Genes and Potential Genomic Repertoire In Mus musculus domesticus and laboratory mice, a total of 12 IGL genes exist per haploid genome. The potential genomic repertoire comprises nine functional genes: three functional IGLV genes that belong to two subgroups, three
IGLJ, and three IGLC functional genes (Tables 4.7, 4.9, and 4.10). In contrast, wild Mus musculus musculus and Mus spretus mice have a more diverse and polymorphic repertoire. Although the organization of the IGL locus in these mice is not known, preliminary data suggest that the total number of IGLV genes may vary from two to at least ten (Table 4.8), and the total number of IGLC may vary from three to at least 10, depending on the strains (Table 4.11). Sequencing will be required to evaluate the functionality of these genes. The phylogenetic tree obtained with IMGT/PhyloGene (Figure 4.10) shows that the mouse IGLV1 subgroup (IGLV1 and IGLV2 genes) is related to the human IGLV7 subgroup, the mouse IGLV2 subgroup (IGLV3 gene) to the human IGLV4 subgroup, and the mouse IGLV3 subgroup (IGLV4 to IGLV8 genes found in wild mice) to the human single IGLV6 subgroup.
CONCLUSION The IMGT classification and description of the human and mouse IGL genes and alleles allow, for the first time, an easy and standardized comparison of the genome and genetics data between species. IMGT/V-QUEST and IMGT/JunctionAnalysis online tools, based on IMGT reference sequence data sets, facilitate the analysis of the human and mouse lambda repertoire in normal and pathological situations at the allele level. Moreover, the IMGT unique numbering for V-DOMAIN and C-DOMAIN provides interesting insights into the 3D structure and function of the immunoglobulin lambda chains between mouse and human. In mouse, it was demonstrated that a selective 50-fold
TABLE 4.9
Mouse (Mus musculus, Mus spretus) IGLJ germline genes
Mouse (Mus musculus) IGLJ IGLJ gene name
IGLJ allele name
IGLJ1
IGLJ1*01
F
BALB/c
J1
V00813
BALB/c, M315J1 [X58419], J1 [Js00586], BALB/c [X58411]
IGLJ2
IGLJ2*01
F
BALB/c
J2
J00593
BALB/c, M315J2 [X58420], BALB/c [X58414]
IGLJ3
IGLJ3*01
F
BALB/c
J3
J00583
BALB/c M315J3 [X58421], BALB/c [X58411]
IGLJ3P
IGLJ3P*01
ORF
BALB/c
Pseudo J3
J00584
IGLJ4
IGLJ4*01
ORF
BALB/c
PseudoJL4
J00596
BALB/c, M315J4 [X58422], BALB/c [X58414]
IGLJ allele name
Strain
Reference sequences
Accession numbers
Sequences from the literature
Fct
IGLJ4
IGLJ4*01
ORF
SPE
J4SPE
M16555
IGLJ5
IGLJ5*01
ORF
B6.lambda SEG
J4SEG2
AF357974
Fct
Reference sequences
Strain
Accession numbers
Sequences from the literature
Mouse (Mus spretus) IGLJ IGLJ gene name IGLJ2
Fct: IMGT functionality. Reference sequences in bold have been mapped: “mapped” refers to sequences that have been obtained from clones (phages, cosmids, YACs) either by subcloning or PCR, and does not apply to sequences obtained directly by PCR from genomic DNA.
TABLE 4.10 Mouse (Mus musculus) IGLC
Mouse (Mus musculus, Mus spretus) IGLC genes and alleles
IGLC subgroup
IGLC gene name
IGLC allele name
Fct
Strain
IGLC1
IGLC1 IGLC4
IGLC1*01 IGLC1*02 IGLC4*01
F P P
BALB/c BALB/c BALB/c
IGLC5
—
IGLC2 IGLC3
IGLC2*01 IGLC3*01
F F
IGLC gene name
IGLC allele name
Fct
Strain
IGLC1 IGLC4
— IGLC4*01
P
B6.lambdaSEG SPE
IGLC5
IGLC4*02 IGLC5*01
P F
B6.lambdaSEG B6.lambdaSEG
IGLC2*01 IGLC2*02 —
F F
SPE B6.lambdaSEG B6.lambdaSEG
IGLC2
Reference sequences
Accession numbers
Clambda1
BALB/c [X58411]
J558/aCl4
J00587 V00814 X58416
Clambda2 Clambda3
J00595 J00585
[J00592], BALB/c [X58414] BALB/c [X58415], BALB/c [X58411]
Sequences from the literature
BALB/c, M315/eCL4 [X58410], BALB/c, Clambda4 [J00598], BALB/c [X58414]
BALB/c BALB/c BALB/c
Mouse (Mus spretus) IGLC IGLC subgroup IGLC1
IGLC2
IGLC2 IGLC3
Reference sequences
Accession numbers
Clambda4S (Clambda4SPE) Clambda4SEG1 Clambda4SEG2
M16628
Clambda2SPE Clambda2SEG
M16554 AF357973
Sequences from the literature
AF357972 AF357974
Fct: IMGT functionality. Reference sequences in bold have been mapped: “mapped” refers to sequences that have been obtained from clones (phages, cosmids, YACs) either by subcloning or PCR, and does not apply to sequences obtained directly by PCR from genomic DNA. A dash indicates the absence of a gene.
56
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse
57
Mouse (Mus musculus, Mus spretus) IGLC RFLP Mouse (Mus musculus) TABLE 4.11
IGLC subgroup
IGLC gene namea
Number of IGLC genes BALB/c
MAI
MBK
PWK
IGLC1
IGLC1 IGLC4 IGLC5
1 1 0
2
3
3
2
IGLC2
IGLC2 IGLC3
1 1
2
7
6
6
Mouse (Mus spretus) IGLC subgroup
IGLC gene namea
Number of IGLC genes B6.lambdaSEG
STF
SMZ
IGLC1
IGLC1 IGLC4 IGLC5
0 1 1
2
2
3
IGLC2
IGLC2 IGLC3
1 0
1
4
5
a
For sequenced genes (see Table 4.10).
decrease in lambda1, observed in the SJL and related BSVS and FVB strains, is due to a single point mutation that changes a glycine to a valine at position 45 in the IGLC1 gene (Sun et al., 2002). Interestingly, that position is at the beginning of the CD transversal strand of the C-DOMAIN and corresponds to the position that, in the human IGLC genes, is involved in the Kern serological marker. This correlation is of particular interest since the mutation in the mouse lambda chain leads to a defect in B cell receptor signaling.
Acknowledgments We are grateful to the IMGT team members for their helpful contribution. We thank Valérie Thouvenin-Contet for her assistance in the preparation of the manuscript. IMGT is funded by the European Union’s 5th PCRDT (QLG2-2000-01287) program, the Centre National de la Recherche Scientifique (CNRS), and the Ministère de la Recherche et de l’Education Nationale.
References Amrani, Y. M., Voegtlé, D., Montagutelli, X., Cazenave, P. A., and Six, A. (2002). The Ig light chain restricted B6.k-lSEG mouse strain suggests that the IGL locus genomic organization is subject to constant evolution. Immunogenetics 54, 106–119. Appella, E., and Ein, D. (1967). Two types of lambda polypeptide chains in human immunoglobulins based on an amino acid substitution at position 190. Proc Natl Acad Sci U S A 57, 1449–1454.
FIGURE 4.10 Phylogenetic tree of human and mouse IGLV genes, using IMGT/PhyloGene (Elemento and Lefranc, 2003). The tree, constructed using a distance matrix and the neighbor-joining algorithm, is displayed with branch lengths and rooted using the midpoint procedure.
Arp, B., McMullen, M. D., and Storb, U. (1982). Sequences of immunoglobulin lambda 1 genes in a lambda 1 defective mouse strain. Nature 298, 184–187. Asenbauer, H., Combriato, G., and Klobeck, H. G. (1999). The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription. Eur J Immunol 29, 713–724. Barbié, V., and Lefranc, M.-P. (1998). The human immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp Clin Immunogenet 15, 171–183. Bauer, T. R. Jr., and Blomberg, B. (1991). The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7. J Immunol 146, 2813–2820. Bernard, O., Hozumi, N., and Tonegawa, S. (1978). Sequences of mouse immunoglobulin light chain genes before and after somatic changes. Cell 15, 1133–1144. Blomberg, B., and Tonegawa, S. (1982). DNA sequences of the joining regions of mouse lambda light chain immunoglobulin genes. Proc Natl Acad Sci U S A 79, 530–533. Blomberg, B., Rudin, C. M., and Storb, U. (1991). Identification and localization of an enhancer for the human lambda L chain Ig gene complex. J Immunol 147, 2354–2358. Blomberg, B., Traunecker, A., Eisen, H., and Tonegawa, S. (1981). Organisation of four mouse l light chain immunoglobulin genes. Proc Natl Acad Sci U S A 78, 3765–3769.
58
Lefranc and Lefranc
Chang, H., Dmitrovsky, E., Hieter, P. A., Mitchell, K., Leder, P., Turoczi, L., Kirsch, I. R., and Hollis, G. F. (1986). Identification of three new Ig lambda-like genes in man. J Exp Med 163, 425–435. Chothia, C., and Lesk, A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196, 901–917. Chuchana, P., Blancher, A., Brockly, F., Alexandre, D., Lefranc, G., and Lefranc, M.-P. (1990). Definition of the human immunoglobulin variable lambda (IGLV) gene subgroups. Eur J Immunol 20, 1317–1325. Combriato, G., and Klobeck, H. G. (2002). Regulation of human Ig lambda light chain gene expression by NF-kappa B. J Immunol 168, 1259–1266 Dariavach, P., Lefranc, G., and Lefranc, M.-P. (1987). Human immunoglobulin C lambda 6 gene encodes the Kern+Oz-lambda chain and C lambda 4 and C lambda 5 are pseudogenes. Proc Natl Acad Sci U S A 84, 9074–9078. Dunham, I., Shimizu, N., Roe, B. A., Chissoe, S., Hunt, A. R., Collins, J. E., Bruskiewich, R., Beare, D. M., Clamp, M., Smink, L. J., Ainscough, R., Almeida, J. P., Babbage, A., Bagguley, C., Bailey, J., Barlow, K., Bates, K. N., Beasley, O., Bird, C. P., Blakey, S., Bridgeman, A. M., Buck, D., Burgess, J., Burrill, W. D., O’Brien, K. P., et al. (1999). The DNA sequence of human chromosome 22. Nature 402, 489–495. Eccles, S., Sarner, N., Vidal, M., Cox, A., and Grosveld, F. (1990). Enhancer sequences located 3’ of the mouse immunoglobulin lambda locus specify high-level expression of an immunoglobulin lambda gene in B cells of transgenic mice. New Biol 2, 801–811. Ein, D. (1968). Nonallelic behavior of the Oz groups in human lambda immunoglobulin chains. Proc Natl Acad Sci U S A 60, 982–985. Ein, D., and Fahey, J. L. (1967). 2 types of lambda polypeptide chains in human immunoglobulins. Science 156, 947–948. Elemento, O., and Lefranc, M.-P. (2003). IMGT/PhyloGene: An online software package for phylogenetic analysis of immunoglobulin and T cell receptor genes. Dev Comp Immunol 27, 763–779. Emanuel, B. S., Cannizzaro, L. A., Magrath, I., Tsujimoto, Y., Nowell, P. C., and Croce, C. M. (1985). Chromosomal orientation of the lambda light chain locus: V lambda proximal to C lambda in 22q11. Nucleic Acids Res 13, 381–387. Erikson, J., Martinis, J., and Croce, C. M. (1981). Assignment of the genes for human lambda immunoglobulin chains to chromosome 22. Nature 294, 173–175. Fett, J. W., and Deutsch, H. F. (1974). Primary structure of the Mcg lambda chain. Biochemistry 13, 4102–4114. Fett, J. W., and Deutsch, H. F. (1975). A new lambda-chain gene. Immunochemistry 12, 643–652. Frangione, B., Moloshok, T., Prelli, F., and Solomon, A. (1985). Human lambda light-chain constant region gene CMor lambda: The primary structure of lambda VI Bence Jones protein Mor. Proc Natl Acad Sci U S A. 82, 3415–3419. Frippiat, J.-P., Dard, P., Marsh, S., Winter, G., and Lefranc, M.-P. (1997). Immunoglobulin lambda light chain orphons on human chromosome 8q11.2. Eur J Immunol 27, 1260–1265. Frippiat, J.-P., Williams, S. C., Tomlinson, I. M., Cook, G. P., Cherif, D., Le Paslier, D., Collins, J. E., Dunham, I., Winter, G., and Lefranc, M.-P. (1995). Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum Mol Genet 4, 983–991. Gerdes, T., and Wabl, M. (2002). Physical map of the mouse l light chain and related loci. Immunogenetics 54, 62–65. Ghanem, N., Dariavach, P., Bensmana, M., Chibani, J., Lefranc, G., and Lefranc, M.-P. (1988). Polymorphism of immunoglobulin lambda constant region genes in populations from France, Lebanon and Tunisia. Exp Clin Immunogenet 5, 186–195. Giudicelli, V., and Lefranc, M.-P. (1999). Ontology for immunogenetics: IMGT-ONTOLOGY. Bioinformatics 12, 1047–1054. Giudicelli, V., Chaume, D., Bodmer, J., Muller, W., Busin, C., Marsh, S., Bontrop, R., Lemaitre, M., Malik, A., and Lefranc, M.-P. (1997). IMGT,
the international ImMunoGeneTics database. Nucleic Acids Res 25, 206–211. Hagman, J., Rudin, C. M., Haasch, D., Chaplin, D., and Storb, U. (1990). A novel enhancer in the immunoglobulin lambda locus is duplicated and functionally independent of NF kappa B. Genes Dev 4, 978–992. Hess, M., Hilschmann, N., Rivat, L., Rivat, C., and Ropartz, C. (1971). Isotypes in human immunoglobulin lambda-chains. Nat New Biol 234, 58–61. Hieter, P. A., Hollis, G. F., Korsmeyer, S. J., Waldmann, T. A., and Leder, P. (1981). Clustered arrangement of immunoglobulin lambda constant region genes in man. Nature 294, 536–540. Ignatovich, O., Tomlinson, I. M., Jones, P. T., and Winter, G. (1997). The creation of diversity in the human immunoglobulin V(lambda) repertoire. J Mol Biol 268, 69–77. Ignatovich, O., Tomlinson, I. M., Popov, A. V., Bruggemann, M., and Winter, G. (1999). Dominance of intrinsic genetic factors in shaping the human immunoglobulin Vlambda repertoire. J Mol Biol 294, 457–465. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., and Gottesman, K. S. (1987). Sequences of proteins of immunological interest, 4th ed. Washington, D.C.: Public Health Service. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991). Sequences of proteins of immunological interest. Washington, D.C.: Public Health Service. NIH Publication 91–3242. Kawasaki, K., Minoshima, S., Schooler, K., Kudoh, J., Asakaw, S., de Jong, P. J., and Shimizu, N. (1995). The organization of the human immunoglobulin lambda gene locus. Genome Res 5, 125–135. Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Schmeits, J. L., Wang, J., and Shimizu, N. (1997) One-megabase sequence analysis of the human immunoglobulin lambda gene locus. Genome Res 7, 250–261. Kay, P. H., Moriuchi, J., Ma, P. J., and Saueracher, E. (1992). An unusual allelic form of the immunoglobulin lambda constant region genes in the Japanese. Immunogenetics 35, 341–343. Lefranc, M.-P. (1997). Unique database numbering system for immunogenetic analysis. Immunol Today 8, 509. Lefranc, M.-P. (1998). IMGT (ImMunoGeneTics) locus on focus. A new section of Experimental and Clinical Immunogenetics. Exp Clin Immunogenet 15, 1–7. Lefranc, M.-P. (1999). The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domains. Immunologist 7, 132–136. Lefranc, M.-P. (2000a). Locus maps and genomic repertoire of the human immunoglobulin genes. Immunologist 8/3, 80–88. Lefranc, M.-P. (2000b). Nomenclature of the human immunoglobulin genes. Curr Protocols Immunol A.1P.1–A.1P.37. Lefranc, M.-P. (2000c). IMGT ImMunoGeneTics database. Int BIOforum 4, 98–100. Lefranc, M.-P. (2001a). IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 29, 207–209. Lefranc, M.-P. (2001b). Nomenclature of the human immunoglobulin lambda (IGL) genes. Exp Clin Immunogenet 18, 242–254. Lefranc, M.-P. (2003a). IMGT, the international ImMunoGeneTics database®. Nucleic Acids Res 31, 307–310. Lefranc, M.-P. (2003b). IMGT, the international ImMunoGeneTics information system®. In Methods in molecular biology. Antibody engineering: Methods and protocols, Benny K. C. Lo, ed. Humana Press, Totowa, N.J., USA. Lefranc, M.-P. (2003c). IMGT® databases, web resources and tools for immunoglobulin and T cell receptor sequence analysis, http://imgt.cines.fr. Leukemia. 17, 260–266. Lefranc, M.-P., and Lefranc, G. (2001a). The immunoglobulin FactsBook (London: Academic Press), pp. 1–458. ISBN: 012441351X. Lefranc, M.-P., and Lefranc, G. (2001b). The T cell receptor FactsBook. London: Academic Press), pp. 1–398. ISBN: 012441351X.
4. Immunoglobulin Lambda (IGL) Genes of Human and Mouse Lefranc, M.-P., Pallarès, N., and Frippiat, J.-P. (1999b). Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus. Hum Genet 104, 361–369. Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Contet, V., and Lefranc, G. (2003). IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27, 55–77. Lefranc, M.-P., Giudicelli, V., Busin, C., Bodmer, J., Muller, W., Bontrop, R., Lemaitre, M., Malik, A., and Chaume, D. (1998). IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 26, 297–303. Lefranc, M.-P., Giudicelli, V., Ginestoux, C., Bodmer, J., Müller, W., Bontrop, R., Lemaitre, M., Malik, A., Barbié, V., and Chaume, D. (1999a). IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27, 209–212. Mami, F., and Kindt, T. J. (1987). C lambda 2 and C lambda 4 immunoglobulin light chain genes in a wild-derived inbred mouse strain. J Immunol 138, 3980–3985. Miller, J., Bothwell, A., and Storb, U. (1981). Physical linkage of the constant region genes for immunoglobulin light chain lI and lII. Proc Natl Acad Sci U S A. 78, 3829–3833. Miller, J., Selsing, E., and Storb, U. (1982). Structural alterations in J regions of mouse immunoglobulin lambda genes are associated with differential gene expression. Nature 295, 428–430. Mural, R. J., Adams, M. D., Myers, E. W., Smith, H. O., Gabor Miklos, G. L., Wides, R., et al. (2002). A comparison of whole-genome shotgun derived mouse chromosome 16 and the human genome. Science 296, 1661–1671. Niewold, T. A., Murphy, C. L., Weiss, D. T. and Solomon, A. (1996). Characterization of a light chain product of the human JC lambda 7 gene complex. J Immunol 157, 4474–4477. Pallarès, N., Frippiat, J.-P., Giudicelli, V., and Lefranc, M.-P. (1998). The human immunoglobulin lambda variable (IGLV) genes and joining (IGLJ) segments. Exp Clin Immunogenet 15, 8–18. Pallarès, N., Lefebvre, S., Contet, V., Matsuda, F., and Lefranc, M.-P. (1999). The human immunoglobulin heavy variable (IGHV) genes. Exp Clin Immunogenet 16, 36–60. Ponstingl, H., Hess, M., and Hilschmann, N. (1968). Complete aminco acid sequence of Bence Jones protein Kern. A new subgroup of the immunoglobulin L-chains of lambda-type. Hoppe Seylers Z Physiol Chem 349, 867–871. Poul, M.-A., Zhang, X. M., Ducret, F., and Lefranc, M.-P. (1991). The IGLJ6 joining segment as a STS in the human immunoglobulin lambda light chain constant region gene locus (located at 22q11). Nucleic Acids Res 19, 4785. Reidl, L. S., Kinoshita, C. M., and Steiner, L. A. (1992). Wild mice express an Ig V lambda gene that differs from any V lambda in BALB/c but resembles a human V lambda subgroup. J Immunol 149, 471–480. Ruiz, M., Pallarès, N., Contet, V., Barbié, V., and Lefranc, M.-P. (1999). The human immunoglobulin heavy diversity (IGHD) and joining (IGHJ) segments. Exp Clin Immunogenet 16, 173–184. Ruiz, M., Giudicelli, V., Ginestoux, C., Stoehr, P., Robinson, J., Bödmer, J., Marsh, S., Bontrop, R., Lemaître, M., Lefranc, G., Chaume, D., and Lefranc, M.-P. (2000). IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 28, 219–221. Sanchez, P., Marche, P. N., Rueff-Juy, D., and Cazenave, P. A. (1990). Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species. J Immunol 144, 2816–2820.
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Satow, Y., Cohen, G. H., Padlan, E. A. and Davies, D. R. (1986). Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. J Mol Biol 190, 593–604. Scaviner, D., Barbié, V., Ruiz, M., and Lefranc, M.-P. (1999). Protein displays of the human immunoglobulin heavy, kappa and lambda variable and joining regions. Exp Clin Immunogenet 16, 234–240. Selsing, E., Miller, J., Wilson, R., and Storb, U. (1982). Evolution of mouse immunoglobulin lambda genes. Proc Natl Acad Sci U S A 79, 4681–4685. Stiernholm, N. B., Verkoczy, L. K., and Berinstein, N. L. (1995). Rearrangement and expression of the human psi C lambda 6 gene segment results in a surface Ig receptor with a truncated light chain constant region. J Immunol 154, 4583–4591. Storb, U., Haasch, D., Arp, B., Sanchez, P., Cazenave, P. A., and Miller, J. (1989). Physical linkage of the mouse l genes by pulse field gel electrophoresis suggests that the rearrangement process favours proximate target sequences. Mol Cell Biol 9, 711–718. Sun, T., Clark, M. R., and Storb, U. (2002). A point mutation in the constant region of Ig lambda 1 prevents normal B Cell development due to defective BCR signalling. Immunity 16, 245–255. Taub, R. A., Hollis, G. F., Hieter, P. A., Korsmeyer, S. J., Waldmann, T. A., and Leder, P. (1983). Variable amplification of immunoglobulin lambda light-chain genes in human populations. Nature 304, 172– 174. Tonegawa, S., Brack, C., Hozumi, N., and Pirrotta, V. (1978a). Organization of immunoglobulin genes. Cold Spring Harb Symp Quant Biol 42, 921–931. Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, O., and Gilbert, W. (1978b). Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain. Proc Natl Acad Sci U S A 75, 1485–1489. Udey, J. A., and Blomberg, B. B. (1987). Human lambda light chain locus: organization and DNA sequences of three genomic J regions. Immunogenetics 25, 63–70. Udey, J. A., and Blomberg, B. B. (1988). Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences. Nucleic Acids Res 16, 2959–2969. van der Burg, M., Barendregt, B. H., van Gastel-Mol, E. J., Tumkaya, T., Langerak, A. W., and van Dongen, J. J. (2002). Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Ozpolymorphism in the human Ig lambda locus. J Immunol 169, 271– 276. Vasicek, T. J., and Leder, P. (1990). Structure and expression of the human immunoglobulin lambda genes. J Exp Med 172, 609–620. Wain, H. M., Bruford, E. A., Lovering, R. C., Lush, M. J., Wright, M. W., and Povey, S. (2002). Guidelines for human gene momenclature. Genomics 79, 464–470. Walker, M. R., Solomon, A., Weiss, D. T., Deutsch, H. F., and Jefferis, R. (1988). Immunogenic and antigenic epitopes of Ig. XXV. Monoclonal antibodies that differentiate the Mcg+/Mcg- and Oz+/Oz-C region isotypes of human lambda L chains. J Immunol 140, 1600– 1604. Weiss, S., and Wu, G. E. (1987). Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains. EMBO J 6, 927–932. Williams, S. C., Frippiat, J.-P., Tomlinson, I. M., Ignatovich, O., Lefranc, M.-P., and Winter, G. (1996). Sequence and evolution of the human germline V lambda repertoire. J Mol Biol 264, 220–232.
C
H
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5 The Mechanism of V(D)J Recombination JOANN SEKIGUCHI
FREDERICK W. ALT
MARJORIE OETTINGER
Department of Internal Medicine, Divison of Molecular Medicine and Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA
Howard Hughes Medical Institute, The Children’s Hospital, The Center for Blood Research, Boston, Massachusetts, USA
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
most if not all of the regulation of the recombination reaction is imposed. The broken DNA generated by RAG cleavage can be resolved through several different pathways (Figure 5.1). The first is standard V(D)J joining, in which the hairpins are opened and joined imprecisely to each other to form a coding joint (CJ) and the two signal ends ligated heptamer to heptamer, to generate a signal joint (SJ). Joining is mediated by components of the nonhomologous end joining (NHEJ) DNA repair pathway (reviewed by Bassing et al., 2002). Alternatively, two nonstandard products of V(D)J recombination can be generated by the joining of signal ends to coding ends (Lewis et al., 1988). Rejoining of a signal to its original coding flank yields an open and shut joint, whereas joining of a signal to its reaction partner’s coding flank generates a hybrid joint (HJ). Finally, RAG1/2 bound to the signal ends can catalyze the transpositional attack of the signal ends on unrelated target DNA (Agrawal et al., 1998; Hiom et al., 1998). The mechanisms and factors involved in each of these reactions are considered separately later in this chapter. The past several years have seen an explosion in the understanding of the cleavage mechanism and the functional properties of the RAG proteins, in large part due to the ability to carry out V(D)J cleavage with purified proteins. Great strides in understanding the repair stage of the reaction have also been made. Six key factors required for repairing the broken molecules to generate signal and coding joints have been identified and some of their biochemical properties defined. This chapter considers in detail what is known about the V(D)J recombination reaction, including the biochemistry of the cleavage reactions, the activities of the RAG proteins, and the components of the NHEJ DNA repair pathway.
A quarter-century ago, the revolutionary discovery was made that DNA in lymphoid cells encoding the antigen receptors is altered from that of other somatic tissues and germline cells (Hozumi and Tonegawa, 1976). This DNA rearrangement is at the heart of the ability of B and T cells to generate a highly diverse array of antigen receptor molecules, thus allowing a virtually unlimited set of antigen molecules to be recognized with a high degree of specificity. A series of site-specific recombination events, collectively termed V(D)J recombination, serves to assemble antigen receptor genes from arrays of gene segments (for additional reviews on this topic, see Bassing et al., 2002; Fugmann et al., 2000; Gellert, 2002; Lewis, 1994). In addition to the combinatorial diversity that results from this assembly process, the V(D)J reaction itself contributes to the diversity by joining the gene segments imprecisely. This junctional diversity is achieved precisely at the complementaritydetermining region 3 (CDR3) of the antigen receptor, a major determinant of the specific interaction between antigen receptor and antigen. In broad terms, rearrangement is initiated by the lymphoid-specific V(D)J recombinase composed of the recombination activating gene 1 and 2 (RAG1 and RAG2) proteins (Oettinger et al., 1990; Schatz et al., 1989). Together, RAG1 and RAG2 bind to the recombination signal sequences (RSS) that flank each gene segment and introduce a double-strand break (DSB) between the RSS and the flanking coding DNA (Figure 5.1) (reviewed by Gellert, 2002). The DNA ends generated by cleavage are asymmetrical, with the coding end covalently sealed into a hairpin and the signal end present as a 5¢ phosphorylated blunt DNA end (Roth et al., 1992; Roth et al., 1993; Schlissel et al., 1993). This first stage of V(D)J rearrangement is the point at which
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FIGURE 5.1 RAG-mediated DNA rearrangements. (A) RAG1/2 initiate V(D)J recombination by nicking the RSS sequences adjacent to the coding segments, leaving a 3¢-OH on the coding flanks. RAG1/2 activate the hydroxyl group to attack the opposite DNA strand to form a hairpin coding end and blunt, 5¢ phosphorylated signal end. (B) Standard inversional V(D)J recombination is catalyzed by the NHEJ proteins to form modified coding joints (CJ) and precise signal joints (SJ). (C) Nonstandard V(D)J recombination is catalyzed by the NHEJ proteins to form open-and-shut and hybrid joints (HJ). Nucleotide loss or addition can be observed within the junctions. (D) RAG-mediated transeseterification reactions occur in the absence of the NHEJ proteins. The reactions catalyzed by the RAGs include transposition and formation of incomplete open-and-shut joints and HJs. RSSs, triangles; coding segments, rectangles.
ANTIGEN RECEPTOR GENE ASSEMBLY Immunoglobulin (Ig) genes and T cell receptor (TCR) genes exist in the germline as linear arrays of clustered gene segments. Seven antigen receptor loci exist: TCR a, b, g, and d, and IgH, k, and l. All loci contain V (variable) and J (joining) segments, and three (TCR b and d and IgH) also contain D (diversity) segments between the V and J clusters. The heterodimeric immune receptors are always composed of one polypeptide derived from a locus containing V, D, and J elements and one from a locus with just V and J elements. At each locus, the variable region exon consisting of VJ or VDJ elements is then fused to a C (constant) region through RNA splicing (Figure 5.2). Each recombinationally active gene segment is flanked by an RSS that consists of a dyad symmetric heptamer, an A/T rich nonamer, and a spacer region of conserved length (12 or 23 bp +/-1) but generally nonconserved sequence
(Figure 5.3). The consensus sequences for the heptamer (CACAGTG) and nonamer (ACAAAAACC) are also optimal for rearrangement, but considerable deviation from the consensus is tolerated, with few segments flanked by an RSS that fits the consensus sequence precisely (Lewis, 1994). The length of the spacer plays a crucial role in the reaction: efficient V(D)J recombination requires a pair of signals, one with a 12- and the other with a 23-bp spacer (Tonegawa, 1983). This relatively simple restriction, the 12/23 rule, has important biological outcomes. First, because all segments of a particular type (e.g., Vk segments) are flanked by one type of signal, and all the segments to which they could be joined (Jk) are flanked by the opposite type, this arrangement ensures that joining is restricted to events that could be biologically productive. Second, because a signal pair is required to induce the catalytic activity of the RAG proteins, the chance of introducing a double-strand break in the absence of a partner DNA to join to is greatly reduced. This restriction
5. The Mechanism of V(D)J Recombination
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FIGURE 5.3 The 12/23 rule. Two V region coding segments (Vk and Jk) are depicted as rectangles and are flanked by a 12-bp and a 23-bp spacer RSS. The consensus sequences of the conserved heptamer and nonamer are shown. Coupled cleavage of the 12/23 RSSs by RAG1/RAG2 occurs at the 5¢ end of the RSS heptamers (arrows). The coding ends are joined after further modification, and the heptamers at signal ends are joined precisely.
FIGURE 5.2 Immunoglobulin heavy (IgH) chain gene assembly and expression. The events involved in the assembly of the IgH chain into a complete immunoglobulin molecule are depicted. H chain gene assembly begins with the rearrangement of a DH segment to a JH segment, followed by rearrangement of a VH segment to the pre-assembled D-JH segment. Transcripts originating from the VH promoter that encode the V-D-JH and Cm gene segments are differentially spliced and give rise to both the membrane and secreted forms of IgM. An IgH chain protein combines with an IgL chain protein to form a typical monomeric subunit of an Ig molecule.
therefore decreases the potential for RAG-induced genomic instability. The RSS sequences are all that is required to render a piece of DNA a substrate for V(D)J recombination. As shown in Figure 5.4, the orientation of the signal sequences with respect to each other determines the outcome of the reaction. Rearrangement can result in retention of the coding joint in the chromosome and deletion of a circular molecule containing the signal joint. Alternatively, recombination can lead to inversion of the DNA between the RSSs with retention of both the SJ and CJ in the chromosome. Both of these arrangements are found in vivo (Fujimoto and Yamagishi, 1987; Okazaki et al., 1987; Zachau, 1993). As an experi-
FIGURE 5.4 Deletional and inversional V(D)J recombination. (A) The intervening sequences between the recombining coding segments can be deleted when the 12/23 RSSs are oriented, as depicted, to form a CJ on the chromosome and a SJ on an extrachromosomal circle. (B) RSSs oriented in the same direction along the chromosome, as depicted, lead to inversional V(D)J recombination in which both CJ and SJ remain on the chromosome.
mental convenience, plasmid-based synthetic recombination substrates can be generated that retain either the signal or the coding joint on the plasmid, allowing for recovery of the joined molecule and a detailed analysis of the junction (Hesse et al., 1987; Lewis et al., 1985). Such substrates also permit a detailed analysis of the sequence requirements for a functional RSS and flanking coding DNA. An outline of a standard assay for V(D)J recombination in tissue culture cells is shown in Figure 5.5.
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FIGURE 5.5 In vitro V(D)J recombination assay. Extrachromosomal plasmid recombination substrates contain two RSSs (triangles) that can undergo site-specific recombination (Hesse et al., 1987). The substrates harbor a prokaryotic promoter and a drug resistance or color marker gene (i.e., chloramphenicol resistance [CAT] or LacZ genes). These elements are separated by a prokaryotic transcription terminator flanked by the RSS sequences. The plasmid recombination substrates are introduced into cells along with constructs expressing RAG1 and RAG2. Recombination between the RSSs deletes the transcription terminator sequence, thus allowing transcription through the selectable marker genes. The efficiency of recombination can be measured via transformation into bacteria and selection on media containing the appropriate antibiotics. Coding and RSS joining can be measured based on the orientation of the RSSs within the substrate.
MUTATIONAL ANALYSIS OF RECOMBINATION SIGNAL SEQUENCES The RSSs are remarkably well conserved among vertebrates, with the same motifs found from sharks to humans. Using the assay outlined in Figure 5.5, the RSS requirements for V(D)J recombination in vitro have been extensively explored (Akamatsu et al., 1994; Akira et al., 1987; Connor et al., 1995; Hesse et al., 1987; Hesse et al., 1989; Nadel et al., 1998; Ramsden and Wu, 1991). The first three
nucleotides of the heptamer sequence are the most crucial, with considerable variation tolerated at the remaining four positions. The CAC sequence also shows the greatest degree of conservation among naturally occurring RSSs. Within the nonamer, alterations in positions 5, 6, and 7 cause the greatest decrease in recombination, but generally the sequence of the nonamer is less critical than the heptamer. In fact, recombination between a signal pair in which one signal contains only a heptamer, while the other has the consensus sequence, is still observable (down 20- to 50-fold) (Hesse et al., 1989). Nucleotide substitutions have similar effects when incorporated into a 12-RSS or a 23-RSS, suggesting that the recognition of these two signals by the recombinase is similar. Early thoughts that the dyad symmetry of the RSS would allow for pairing between signals as part of the joining process have not proved to be correct, as such homology can be disrupted without affecting recombination of a signal pair (Hesse et al., 1989). Although the spacer sequence is not well-conserved, there is mounting evidence that its sequence can considerably influence RSS usage (for example, see Jung et al., 2003; Nadel et al., 1998). Although the initial description of the sequence requirements for an RSS was determined in cell culture experiments, these same requirements are seen in vitro. The actual RSS sequence used at an antigen receptor locus is rarely the consensus sequence. This variation may influence segment usage, with segments flanked by RSSs closer to the consensus favored over others. For example, the RSSs at Vk are generally closer to consensus than those at Vl, perhaps providing one level of explanation for the favored usage of Vk segments. This preference (up to 100-fold) can be reproduced with kappa and lambda RSSs in synthetic substrates (Feeney et al., 2000; Ramsden and Wu, 1991). However, as discussed later, restrictions on RSS usage go beyond this 12/23 regulation.
“BEYOND 12/23” RESTRICTION OF V(D)J REARRANGEMENTS As indicated above, the organization of 12- and 23-RSSs, which flank V, D, and J segments within the Ig and TCR loci, facilitates proper rearrangement. However, additional restrictions on RSS usage must exist. For example, the simple 12/23 rule cannot fully account for the rearrangement patterns observed at the TCRb locus. At this locus, the Vs are flanked with 23-RSSs and Js with 12-RSSs, whereas the D has a 5¢ 12-RSS and a 3¢ 23-RSS. Direct Vb to Jb joining is rarely observed in vivo, although it would be in accordance with the 12/23 rule. The vast majority of TCRb variable region genes are generated via D to Jb and then Vb to DJb rearrangements (Born et al., 1985; Ferrier et al., 1990; Sleckman et al., 2000).
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These observations have been explained by further studies showing that some pairs of functional RSSs are restricted from recombining with each other. This restriction “beyond the 12/23 rule” (B12/23) was first suggested by placing transgenic recombination substrates in the TCRb locus, and more definitively shown by gene-targeted mutation (Bassing et al., 2000; Sleckman et al., 2000; Wu et al., 2003). Targeted replacement of the 5¢ Db1 12-RSS with the Jb 12-RSS prevented Vb rearrangement. However, replacement of the Jb 12-RSS with the 5¢ Db1 12-RSS in a simplified locus (lacking the Db2Jb2 cluster) in which Db1 had been deleted allowed direct Vb–Jb joining but only to the Jb that contained the replaced RSS (Bassing et al., 2000). Thus, the 5¢ Db1 12-RSS, but not the Jb 12-RSS, is an acceptable target for the rearrangement of the Vb segments. The notion that particular RSSs restrict specific rearrangements in a B12/23 manner was further illustrated by studies in which the Vb14 23-RSS was replaced with the 3¢ Db1 23-RSS (Wu et al., 2003). The Vb14/3¢DbRSS replacement dramatically increased the usage of Vb14, predominantly due to an increase in the relative level of primary Vb14 to DJb rearrangement. However, the 3¢Db1 23-RSS replacement also broke the TCRb locus B12/23 restriction and allowed direct Vb14 to Jb1 rearrangement. Thus, the Vb14 23-RSSs contributes to B12/23 restriction by preventing Vb14 to Jb1 rearrangement; furthermore, the high recombination potential of the 3¢ Db1 23-RSS may have evolved to ensure that complete Db to Jb rearrangements occur prior to Vb rearrangement. The B12/23 restrictions observed in vivo at the TCRb locus were recapitulated in nonlymphoid cells using extrachromosomal V(D)J recombination substrates containing various combinations of Vb 23-RSSs, 5¢ Db 12-RSSs, 3¢ Db 23-RSSs, and Jb 12-RSSs, as well as with in vitro cleavage assays using purified RAG proteins (Jung et al., 2003; Tillman et al., 2003). All Vb 23-RSSs analyzed in these studies preferred the 5¢ Db1 12-RSS over the Jb1 12 RSSs. Thus, consistent utilization of the Db gene segment is largely ensured by the constraints imposed on the formation of functional cleavage complexes (discussed later) containing an RSS pair, the RAG proteins, and HMG1.
INFLUENCE OF CODING FLANKS In addition to the RSS, the first two or three nucleotides of the coding flank immediately abutting the heptamer of the RSS can have considerable effects on the efficiency of the recombination reaction. Although most sequences are essentially neutral, certain nucleotide combinations can be favorable or unfavorable to the reaction. A run of T’s (5¢ to 3¢ toward the heptamer) substantially reduces recombination (Boubnov et al., 1995; Ezekiel et al., 1995; Ramsden and Wu, 1991). Some dinucleotides such as 5¢ TG 3¢ favor
recombination and are termed “good flanks”), while others such as 5¢ AC 3¢ and 5¢ GG 3¢ are unfavorable (“bad flanks”) (Sadofsky et al., 1995). The effect of the coding flank sequence appears to be primarily at the cleavage phase of the reaction (Cuomo et al., 1996; Ramsden et al., 1996), although there may be some effect on end-processing and the later joining stage of the reaction.
THE BIOCHEMISTRY OF V(D)J CLEAVAGE V(D)J cleavage requires only RAG1 and RAG2, a divalent metal ion (Mn2+ or Mg2+), and a DNA substrate containing the RSS (McBlane et al., 1995). In addition, the nonspecific DNA bending protein HMG1 (or HMG2) can serve to augment the reaction, as discussed here. No external source of energy is needed (McBlane et al., 1995; van Gent et al., 1995). With these simple components, the cleavage reaction can be reproduced, including the RSS selectivity (Cuomo et al., 1996; Ramsden et al., 1996) and the requirement for a 12/23 signal pair (Eastman et al., 1996; Kim and Oettinger, 1998; van Gent et al., 1997; van Gent et al., 1996). In general, the reactions have been studied using truncated “core” portions of the RAG proteins (mouse RAG1 amino acids 384 to 1,008 of 1,040 and RAG2 amino acids 1 to 382 of 527). These endonucleolytically active core portions have been more readily purifiable than their fulllength counterparts (McBlane et al., 1995; Sawchuk et al., 1997). When expressed in tissue culture cells, the core proteins permit V(D)J recombination to occur (Cuomo and Oettinger, 1994; Kirch et al., 1996; Sadofsky et al., 1994; Sadofsky et al., 1993; Silver et al., 1993) though some differences from the full-length proteins have been seen. All enzymatic activities of the RAG proteins require the cooperation of RAG1 and RAG2; individual activities of either protein have not been described. Cleavage itself occurs in two separable steps (McBlane et al., 1995). In the first step, a nick is introduced on the top strand adjacent to the recombination signal, leaving a 3¢ hydroxyl on the coding side and a 5¢ phosphoryl group on the signal end (see Figure 5.1). In the second step, the 3¢ hydroxyl from the top strand attacks the phosphodiester bond at the same position on the opposing strand, resulting in the formation of the covalently sealed hairpinned coding end, and the blunt signal end. The energy required for the formation of the new bond is derived from the breakage of the old one. Stereochemical studies have shown that this conservative reaction occurs with the inversion of chirality, indicating that a covalent bond between the RAG proteins and DNA is not formed (van Gent et al., 1996). This distinguishes the RAG cleavage reaction from that of a number of other site-specific recombinases, such as Cre, Flp, and Lambda-Int, which rely on a covalent intermediate. Instead, the direct transesterifi-
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cation mechanism used by RAG1/2 is similar to that of bacterial transposases and HIV integrase (Engelman et al., 1991; Mizuuchi and Adzuma, 1991; van Gent et al., 1996). The in vivo recombination reaction is largely a coupled process. That is, two signal sequences are required not just for a complete recombination event, but also for the initiating cleavage events. In addition, a 12/23 signal pair is preferred by approximately 50-fold over a 12–12 pair (Steen et al., 1996). The in vitro reaction can reproduce both this requirement for a signal pair and approximately the same extent of 12/23 preference as observed in vivo. Both these properties are intrinsic features of the RAG proteins, and the full extent of 12/23 preference can be observed upon the addition of HMG1 and nonspecific competitor DNA (Eastman et al., 1996; Kim and Oettinger, 1998; van Gent et al., 1997; van Gent et al., 1996). However, by altering the reaction conditions (substituting Mn2+ for Mg2+) cleavage can be uncoupled, thus allowing a break at a single RSS to be made. Studying the reaction under these conditions allows an examination of requirements for DNA recognition and cleavage as distinct from the requirements for synaptic complex assembly.
RSS Requirements for Cleavage A simple oligonucleotide containing an RSS can serve as a substrate for V(D)J cleavage (McBlane et al., 1995). By modifying this substrate, the precise DNA sequence requirements for binding and cleavage have been determined and compared with the rules for V(D)J recombination derived from in vivo experiments. Both a 12- and a 23-RSS can be recognized and individually cleaved by the RAG proteins. However, a 12-RSS is cleaved more efficiently than a 23-RSS. As with V(D)J recombination in vivo, a consensus RSS serves at the optimal for cleavage (Cuomo et al., 1996; Ramsden et al., 1996). The first three nucleotides of the heptamer are again the most sensitive to mutation. However, substitutions at these nucleotides primarily affect hairpin formation, influencing the initial nicking step to a much lesser extent. In the complete absence of a heptamer, the nonamer alone can still direct some nicking (but no hairpinning). This somewhat imprecise and very inefficient nicking occurs where the boundary of the heptamer would have been in a 12- or 23-RSS (that is, 19 or 30 nucleotides from the end of the nonamer), as if the proteins reach out a defined distance from the bound nonamer. Substrates containing a heptamer alone also work, with cleavage reduced by only a few-fold. The length of the spacer sequence is also important (Cuomo et al., 1996; Ramsden et al., 1996). The length difference between the 12- and 23-RSS is almost precisely one helical turn. This disposition suggests that having the two recognition elements, the heptamer and nonamer, in the same rotational phase is important for binding and cleavage.
Alteration of the length of the spacer supports this view. Adding an extra one-half helical turn (18- or 29-bp) substantially inhibits cleavage, below that seen with an isolated heptamer, whereas adding one full turn (33- or 34-bp) permits a substantial level of cleavage. Taken together these results suggest that each element can function on its own and act together synergistically with the proper spacing, but they conflict when the spacing is wrong. Alteration of the structure of the substrate DNA suggests that DNA unpairing and structural distortion might play a role in V(D)J cleavage (Cuomo et al., 1996; Ramsden et al., 1996). Unpairing of the first few nucleotides of coding sequence, when those bases are unfavorable for cleavage, can significantly enhance hairpin formation, suggesting that unpairing of the DNA sequence may be an important part of the cleavage reaction. More dramatically, with the coding flank remaining as duplex DNA, the RSS of a nucleotide substrate can be made single-stranded and still serve to direct site-specific binding and hairpin formation. In this reaction, only the heptamer appears important. This reaction is very efficient, again suggesting that DNA unwinding, perhaps due to RAG binding, may play an important role in the cleavage reaction. The specific sequence of the heptamer may have evolved not only to serve as a specific binding site for the RAG proteins, but also to be readily unpaired. The CACA/GTGT sequence of the heptamer is considerably distorted both in free solution and in crystals (Cheung et al., 1984; Patel et al., 1987; Timsit et al., 1991). Good flanks extend this unusual structure (purine/pyrimidine alternation) (Sadofsky et al., 1995), perhaps explaining their effect.
RAG1/2-RSS BINDING RAG1 and RAG2 together are required for highly specific binding to DNA; RAG1/2 prefers an RSS sequence over nonspecific DNA by ~50-fold in competition experiments (Hiom and Gellert, 1997). Two distinct species of RAG1/2 bound to a single RSS can be resolved by gel retardation experiments (Mundy et al., 2002; Swanson, 2002). These two complexes differ in protein content, but not in other properties. The RAG1/2-DNA complex, once formed, is highly stable. It remains bound (and active for cleavage) up to 8 hours after assembly, and resists very high levels of competitor DNA (Akamatsu and Oettinger, 1998; Hiom and Gellert, 1997; Li et al., 1997; Mundy et al., 2002). Both the heptamer and nonamer (with proper spacing) are required for maximal binding (Akamatsu and Oettinger, 1998; Hiom and Gellert, 1997; Nagawa et al., 1998; Swanson and Desiderio, 1998). Footprinting of the RAG1/2 complex on a single RSS reveals contacts in both the heptamer and nonamer (Akamatsu and Oettinger, 1998; Nagawa et al., 1998; Swanson and Desiderio, 1998), and photocrosslinking experiments indicate that the heptamer is
5. The Mechanism of V(D)J Recombination
touched by both RAG proteins (Eastman et al., 1999; Mo et al., 1999; Swanson and Desiderio, 1998). RAG1 binds to DNA on its own, but with considerably lower affinity and specificity, and appears only to contact the nonamer (Akamatsu and Oettinger, 1998; Mo et al., 1999; Swanson and Desiderio, 1998). Thus, in the presence of RAG2, the DNA contacts of RAG1 appear to change. Moreover, the enhancements of chemical cleavage observed with dimethyl sulfate (DMS) protection and phenanthroline-copper (OP-Cu) DNA footprinting support the idea that some DNA unwinding occurs near the heptamer–coding DNA border and that this is a result of the binding of RAG1 together with RAG2 (Akamatsu and Oettinger, 1998; Mo et al., 1999; Swanson and Desiderio, 1998). Although RAG1/2 together can bind to a single RSS, it is the synaptic “paired complex” (PC) containing a 12/23 signal pair that is competent to generate double-strand breaks under restrictive coupled-cleavage (Mg2+) conditions (Hiom and Gellert, 1998). The synaptic PC is a very stable species, resistant to high levels of nonspecific competitor DNA (Hiom and Gellert, 1998). Footprint analysis of this complex shows greatly enhanced protection of the heptamer sequence over that seen in a single-site complex (Nagawa et al., 2002). PC contains a dimer of RAG2 and either a dimer or tetramer of RAG1 (Landree et al., 2001; Mundy et al., 2002; Swanson, 2002); the ambiguity in RAG1 content arises from similar experiments that yield differing results (Mundy et al., 2002; Swanson, 2002), though additional experiments support the conclusion that RAG1 binds as a tetramer (Godderz et al., 2003). At this step of synaptic complex assembly the 12/23 rule is at least largely enforced with a 12/23 pair greatly preferred over a 12/12 or 23/23 pair (Hiom and Gellert, 1998; Mundy et al., 2002). It has also been suggested that the cleavage step itself may also contribute to 12/23 restriction (West and Lieber, 1998; Yu and Lieber, 2000). Although the 12/23 rule is enforced at the binding step, it is only hairpin formation that is subject to this control, because nicking can occur without synapsis. Although it was generally expected that each RSS would serve as a half-site, binding half the content of RAG1/2 that would later be present in the PC, this turns out not to be the case (Jones and Gellert, 2002). The RAG protein content of the slower mobility SC2 complex does not differ from PC (Mundy et al., 2002). Instead, SC2 and PC differ only by the addition of the second RSS containing DNA (Mundy et al., 2002). In other words, the RAG proteins bind to one signal first (with a strong preference for the 12RSS, as shown in competition experiments), then recognize and bind the second signal (Jones and Gellert, 2002). Because SC2 is not competent to form hairpins under restrictive Mg2+ conditions, even though all the necessary RAG1/2 proteins are present (Mundy et al., 2002), it is highly likely that binding to the second signal induces some conformational change in
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the RAG1/2 complex to render it catalytically active. Such a process would help to regulate cleavage in vivo, thus reducing the chance of the inappropriate introduction of double-strand breaks into the genome.
RAG1/2 POST-CLEAVAGE COMPLEX After cleavage, the RAG1/2 complex remains bound to the DNA. Stable post-cleavage complexes of RAG1/2 bound to a pair of signal ends (Agrawal and Schatz, 1997; Jones and Gellert, 2001) or to all four DNA ends (two hairpin and two coding ends) (Hiom and Gellert, 1998) have been observed in vitro. The finding that cleaved products can be resolved to form SJ, CJ, and hybrid or open and shut joints (a joining of coding end to signal end) suggests that all four ends are held together in a complex in vivo as well (Lewis et al., 1988). Several lines of evidence suggest that the bound RAG proteins participate in the later resolution stages of the reaction. First, deproteinization of the signal ends is required prior to joining by NHEJ factors in vitro (Leu et al., 1997; Ramsden et al., 1997). Second, there are mutants of both RAG1 and RAG2 that cleave the RSS but fail to support complete V(D)J recombination, thus suggesting the RAGs are involved in joining (Huye et al., 2002; Qiu et al., 2001; Tsai et al., 2002; Yarnell Schultz et al., 2001). Third, bluntend joining in yeast is normally imprecise, but following V(D)J cleavage, blunt signals are rejoined precisely, suggesting that the RAGs play a role in this process (Clatworthy et al., 2003). Fourth, the nonstandard resolution products of RAG cleavage observed in vitro indicate a role for the RAG proteins post-cleavage. RAG1/2 complexed with cleaved signal ends can bind to unrelated target DNA in the target capture step of transposition (discussed later), and the formation of a hybrid joint (in vitro but not necessarily in vivo) can be formed by a RAG-mediated attack of a signal end on a hairpin coding end (discussed later). It has been suggested that the RAG proteins bound to the cleaved ends may serve as a scaffold and may recruit the NHEJ factors to facilitate end-processing and joining (Huye et al., 2002; Tsai et al., 2002). In this regard, mutations that affect the ability of the RAGs to maintain the broken ends in stable postcleavage complexes may lead to misrepair of the DSBs, and thereby may have the potential to cause oncogenic chromosomal aberrations (Huye et al., 2002; Tsai et al., 2002).
A ROLE FOR HMG1 (OR HMG2) IN V(D)J RECOMBINATION Although RAG1 and RAG2 are the only lymphoidspecific proteins required for cleavage, the high mobility group protein 1 (HMG1) or HMG2 may be a generally
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important co-factor for RAG-mediated activities. HMG1 and -2 are ubiquitously expressed, abundant nuclear proteins that bind to DNA without sequence specificity and can bend linear DNA. HMG1 and -2 are also associated with chromatin, and appear to play important roles in the assembly of nucleoprotein complexes involved in DNA repair and transcription (reviewed by Thomas and Travers, 2001). The addition of HMG1 to the V(D)J cleavage reaction generally has little effect on the efficiency of cleavage of a 12RSS but substantially increases cleavage at a 23RSS (Sawchuk et al., 1997; van Gent et al., 1997). The nonspecific binding or bending activity of HMG1 suggests its role may be to facilitate the distortion of the DNA to allow for the same components of an active RAG complex to bind the two different signal sequences. Formation of the synaptic complex is greatly enhanced by the presence of HMG1 (or HMG2) (Hiom and Gellert, 1998; West and Lieber, 1998; Swanson, 2002), and the addition of HMG1 increases the preference for a 12/23 pair over a 12/12 pair (van Gent et al., 1997; Hiom and Gellert, 1998; Kim and Oettinger, 1998). HMG1 (or -2) is also required for RAG-mediated transposition, both for paired complex formation and for capturing target DNA (Agrawal et al., 1998; Hiom et al., 1998; Swanson, 2002) (discussed later). Finally, the addition of HMG1 augments V(D)J cleavage of RSS sequences assembled into nucleosomes, suggesting it may play an important role in facilitating RAG binding at endogenous loci (Kwon et al., 1998). However, an in vivo role for HMG1 or -2 during V(D)J recombination has not yet been established.
A CLOSER LOOK AT RAG1 AND RAG2 The RAG genes were originally identified based on their ability, when expressed in fibroblasts, to induce the V(D)J recombination of an artificial recombination substrate (Oettinger et al., 1990; Schatz et al., 1989). As such they are the only lymphoid-specific factors required to induce V(D)J recombination even in a nonlymphoid cell where this reaction would not normally occur, because the other required factors are generally expressed and can be recruited to complete the joining reaction. In the absence of RAG1 or RAG2, no V(D)J recombination can occur and thus mice with targeted disruptions of either gene lack mature B and T cells (Mombaerts et al., 1992; Shinkai et al., 1992). However, such mice do not exhibit any defects outside of the immune system, indicating that RAG function is limited to the lymphoid lineage. RAG1 and RAG2 share no sequence similarity. However, the genomic structure of the RAG genes is highly unusual. In all species examined, the two genes are adjacent and convergently transcribed. Although adjacent, their lack of
sequence similarity indicates that they did not arise via gene duplication. In most species (human, mouse, chicken, Xenopus) they also share the unusual feature of encoding the entire structural gene within a single exon. Two exceptions have been found: the zebra fish and trout RAG1 coding sequences contain introns (Hansen and Kaattari, 1995; Willett et al., 1997). This unusually compact structure of the RAG genomic locus led to the suggestion that the RAG genes might have evolved from (been co-opted from) a primordial transposon (Oettinger et al., 1990; Thompson, 1995), a presumption strengthened by biochemical demonstrations of RAG transposase activity (discussed later) (Agrawal et al., 1998; Hiom et al., 1998). Although the RAG genes do not show sequence similarity to each other, the RAG genes are highly conserved between species. Between pufferfish and human, there is 66% and 61% amino acid similarity for the RAG1 and RAG2 proteins respectively (Peixoto et al., 2000). Interestingly, whereas RAG1 is highly conserved across the entire structural gene, that conservation is biphasic, with the region between 411 and 1,036 even more highly conserved (75% amino acid identity) (Peixoto et al., 2000). This region roughly corresponds to the “core portion” of RAG1, the minimal region required for catalysis. A comparison of the two RAG proteins with known structures and structural motifs has led to the proposal that RAG2 has two distinct domains separated by a “hinge” region (Aravind and Koonin, 1999; Callebaut and Mornon, 1998), and studies with limited proteolysis confirm that two protease resistant domains exist (Arbuckle et al., 2001; Kim et al., 2003). The core portion of RAG2 (the domain required for catalysis) is proposed to fold into a structure resembling a six-bladed propeller where each blade contains a kelch motif (Aravind and Koonin, 1999; Callebaut and Mornon, 1998). Each kelch motif, originally identified in a Drosophila regulatory protein, would contain a four-stranded twisted antiparallel beta sheet. In other cases, such structures are involved in protein–protein interactions, suggesting that this domain may not only bind DNA but allow for interaction with RAG1 or an additional RAG2, a proposal consistent with studies of RAG1/RAG2 protein–protein interaction (Corneo et al., 2000; Gomez et al., 2000; Landree et al., 1999). The C-terminal region of RAG2 contains an acidic portion (amino acids 352 to 410, 42% acidic), a Cys-His rich PHD motif (aa 420 to 480), and a binding site for CDK2 (thr490) (Lee and Desiderio, 1999). Amino acids from 383 to the end of the protein (aa 527) are absent in the recombinationally active RAG2 core protein so that the study of the functions of these regions has been limited. Recent success in purifying the full-length protein has led to the observation that the presence of the C-terminal domain of RAG2 diminishes RAG1/RAG2 mediated transposition following V(D)J cleavage (Elkin et al., 2003; Tsai and Schatz, 2003). Thr490
5. The Mechanism of V(D)J Recombination
is involved in regulation and is required for the proper cellcycle control of RAG2 protein levels (Lee and Desiderio, 1999). Thr490 phosphorylation leads to translocation of RAG2 from the nucleus to the cytoplasm, where it is degraded by the ubiquitin–proteosome system during S phase (Mizuta et al., 2002). PHD motifs are found in a number of regulatory proteins, many of them thought to affect chromatin structure or bind to chromatin components. Such a role is of interest given the indications that the absence of the C-terminal domain of RAG2 leads to a decrease in assembly of particular antigen receptor loci (Akamatsu et al., 2003; Kirch et al., 1998; Liang et al., 2002). Identifiable sequence motifs within RAG1 are minimal, though it is notable for containing several putative zinc fingers. Limited proteolysis has defined three distinct domains, the N-terminal (and dispensable) domain and two in the active core (Arbuckle et al., 2001). The most Cterminal domain displays DNA binding activity on its own (Arbuckle et al., 2001), although additional sites within the core are required for formation of a functional RAG1/RAG2 DNA complex. The structure of the N-terminal part of RAG1 has been solved and contains one zinc finger of the RING family and two additional zinc finger motifs (Bellon et al., 1997; Freemont et al., 1991; Rodgers et al., 1996). Such RING motifs are often found in proteins that serve as ubiquitin ligases, so-called E3 proteins. Recently it has been shown that this domain of RAG1 does indeed have E3 ubiquitin ligase activity (Yurchenko et al., 2003). Although this domain is not required for RAG1 enzymatic activity, its deletion is associated with some alterations in V(D)J recombination, and the E3 activity implies a regulatory role in V(D)J recombination for this domain. A simple search for homologies or motifs that might identify the active site within RAG1 or RAG2 did not yield obvious candidates. However, the knowledge that the RAG proteins cleave DNA using the same chemistry as transposases suggested that the RAG active site might share similarities with these enzymes. Many transposases use a triad of Asp and Glu residues, often termed a DDE motif, to bind a divalent metal at the active site (Rice et al., 1996). Mutational analysis of acidic residues in RAG1 and RAG2 led to the identification of three residues in RAG1—D600, D708, and E962—that are absolutely required for V(D)J recombination in vivo and V(D)J cleavage in vitro, but not for DNA binding (Fugmann et al., 2000; Kim et al., 1999; Landree et al., 1999). A role in metal binding has been established for D600 and D708 (Kim et al., 1999; Landree et al., 1999). Mutations in either of these residues eliminates hydroxyl radical cleavage activity of RAG1 (Kim et al., 1999). In addition, as has been seen for other transposases (Sarnovsky et al., 1996), the substitution of Asp with Cys restores some cleavage in Mn++ but not Mg++ (Kim et al., 1999; Landree et al., 1999). These same experiments failed to show that E962 was directly involved in metal binding, and its func-
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tion remains unclear. RAG1 may not actually contain a classical DDE motif, as this third amino acid is at a greater distance from the first two than is typically seen and appears to be located in a separate domain of RAG1 (Arbuckle et al., 2001), whereas all three residues of the known DDE motifs are contained in a single domain. Despite exhaustive mutagenesis of RAG2, no acidic residues were seen to be required (Landree et al., 1999). Other RAG2 point mutations do disrupt catalysis (Qiu et al., 2001), suggesting that RAG2 helps to establish the full active site. A crystal structure would be most useful in understanding how the active site is formed. Naturally occurring mutations in the RAG proteins are responsible for some forms of human severe combined immunodeficiency (SCID) (Schwarz et al., 1996; Villa et al., 1998; Villa et al., 2001). Complete immunodeficiencies, where the patients lack both B and T cells arise when RAG activity is absent. Partial loss-of-function mutations can give rise to a partial SCID phenotype or the related immunodeficiency, Omenn syndrome, in which T cells are more severely affected than B cells (Villa et al., 1998; Villa et al., 2001). Several of the RAG2 SCID mutations cluster along one surface of the predicted kelch propeller, leading to the suggestion that this region is involved in interactions with RAG1 (Corneo et al., 2000). Additional mutations have helped to further define the regions in which these two proteins interact with each other and with DNA (Fugmann and Schatz, 2001; Gomez et al., 2000; Landree et al., 1999). As indicated here, the core domains of RAG1 and RAG2 are sufficient to mediate V(D)J recombination in vivo. However, some notable differences occur between the behavior of the full-length proteins and the core versions. First, the frequency of V(D)J recombination on exogenous or integrated substrates in fibroblast cells is lower with the core than full-length proteins (Cuomo and Oettinger, 1994; Kirch et al., 1996; Sadofsky et al., 1995; Sadofsky et al., 1994; Silver et al., 1993). Second, recombination by the core RAG proteins in fibroblast cells leads to a greater accumulation of signal ends than is observed with full-length proteins (Steen et al., 1999). Third, the absence of the amino terminus of RAG1 results in reduced D to J rearrangement, with differential effects observed on the assembly of endogenous T cell receptor and immunoglobulin genes (Noordzij et al., 2000; Roman et al., 1997; Santagata et al., 2000). Fourth, mice that express core RAG1 in the absence of wild type RAG1 exhibit reduced frequency of both D-toJH and VH-to-DJH chromosomal rearrangements in RAG1c/c mice, which most likely reflects a decrease in overall V(D)J recombination efficiency (Dudley et al., 2003). Fifth, in both pro-B cell lines and mice that express core RAG2, Dh-to-Jh joining is mildly lowered, whereas Vh-to-DJh joining is severely reduced (Akamatsu et al., 2003; Kirch et al., 1998; Liang et al., 2002). Thus, the noncore regions of
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RAG1 and RAG2 clearly play important roles in ensuring efficient V(D)J recombination in vivo.
Hybrid Joining Although normal V(D)J recombination results in the formation of signal and coding joints, nonstandard joining products, hybrid joints (HJ), and open-and-shut joints are also observed in vivo (Lewis, 1994; Lewis et al., 1988). Such products arise when a signal end is rejoined to a coding end; rejoining to the original coding flank yields an open-and-shut joint (detectable if base loss or addition occurs), whereas rejoining to the partner flank yields a hybrid joint. These products are detectable both in artificial recombination substrates, where they can account for up to 10% of the recombination products, and at lower frequency in the antigen receptor loci themselves (Lewis et al., 1988). A reaction that would lead to the generation of hybrid or open-and-shut joints can be carried out with purified proteins in vitro (Melek et al., 1998). In this reaction, the RAG proteins initially perform a standard coupled cleavage reaction, giving rise to hairpin coding ends and blunt signal ends. Following this cleavage, the RAG proteins then catalyze the attack of the free hydroxyl of the RSS end onto the coding hairpin at or near the tip, thereby joining the signal end to coding DNA on one strand (see Figure 5.1). Attack on the original coding DNA would yield an open and-shut joint, while an attack on the partner coding end would result in HJ formation. Such a reaction would give rise to a perfect or near-perfect HJ, with no further nucleotide loss or addition. These in vitro products are joined only on the strand where the transesterification occurred (Melek et al., 1998), and can be generated by the core and, to a lesser extent, the fulllength versions of RAG1 and RAG2 (Elkin et al., 2003; Tsai and Schatz, 2003). The core RAG proteins can mediate formation of this same type of precise but incomplete HJ in NHEJ-deficient cells (Bogue et al., 1997; Han et al., 1999; Sekiguchi et al., 2001). In contrast, the full-length RAGs form very few HJs in NHEJ-deficient cells and the majority of the joints contain large deletions and appear to be repaired by an alternative DNA repair pathway; thus, the full-length RAGs do not efficiently facilitate HJ formation in a cellular context (Sekiguchi et al., 2001). These results led to the proposal that the noncore regions suppress the ability of the RAGs to catalyze HJ in vivo (Sekiguchi et al., 2001). Consistent with this notion, in vitro experiments subsequently demonstrated that the full-length RAGs exhibit decreased HJ activity in comparison to core RAGs (Elkin et al., 2003; Tsai and Schatz, 2003). However, because the full-length RAGs can form detectable levels of precise HJs in vitro, additional cellular factors may be responsible for further influencing the pathways of HJ formation in vivo.
Transposition Mediated by RAG1/2 Whereas RAG1/2 serves as an endonuclease in V(D)J cleavage, RAG1/2 can also perform transposition, inserting the cleaved RSSs into unrelated DNA (reviewed by Fugmann, 2001). Several observations led to the experiments that demonstrated RAG1/2 transposase activity. First were the stereochemical studies of hairpin formation which, as discussed earlier, demonstrated that these were formed by direct transesterification rather than by a reaction requiring a covalent intermediate (van Gent et al., 1996). This type of conservative DNA strand transfer (the generation of the hairpin bond requires the breakage of the opposing DNA strand) is typical of transposases (Engelman et al., 1991; Mizuuchi and Adzuma, 1991). Second was the demonstration that purified core RAG1/2 could form hybrid joints in vitro (Melek et al., 1998). This type of hybrid joint formation uses the same chemistry as transposition. Efficient transposition can be achieved with the purified core RAG proteins (Agrawal et al., 1998; Hiom et al., 1998). Transposition requires a 12/23 RSS pair, presumably to activate the RAG proteins, and relies on the same active site as is used for RSS cleavage. Transposition need not be coupled to RSS cleavage, as precut RSS ends can also be used. Although an RSS pair is required for transposition, both double-ended and single-ended insertions can be observed (Figure 5.6). As with other transposases that attack the two DNA strands at staggered positions, the DNA insertion sites for the two RSS ends in a coupled attack on the opposite strands of DNA are offset, in this case by 3 to 5 bp. The target DNA sites are generally GC-rich and a preference for insertion into DNA that can form a hairpin loop has been reported. Both intra- and intermolecular transposition can occur. Despite the efficiency of transposition in vitro, attempts to detect RAG-mediated transposition by expressing RAG proteins in cultured mammalian cells have been unsuccessful. However, two examples of RAG-mediated transposition of TCR a signal ends into the HPRT gene in a T cell isolate from a normal individual have recently been described, indicating that transposition in mammalian cells is not totally excluded (Messier et al., 2003). Regulatory mechanisms likely exist in lymphocytes to suppress the propagation of transposable elements, because frequent transposition events involving the rearranging antigen receptor loci would be highly detrimental to the host genome. Indeed, the lack of efficient full-length RAG-mediated HJ formation (which is mechanistically similar to transposition) in NHEJdeficient cells suggests that this RAG activity is downregulated in a cellular context (Sekiguchi et al., 2001). Furthermore, in vitro experiments using full-length RAG1 and RAG2 have clearly demonstrated that the noncore regions significantly inhibit RAG-mediated transposition (Elkin et al., 2003; Tsai and Schatz, 2003). In addi-
5. The Mechanism of V(D)J Recombination
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FIGURE 5.6 RAG-mediated transposition. (A) Two-ended transposition. Upon cleavage of the 12/23 RSSs by RAG1/2, the RSS ends can be used in an attack on another, nonspecific DNA duplex (dashed lines). This coupled attack leads to the integration of the RSS flanked DNA fragment at positions staggered by 3 to 5 bp, resulting in target site duplication at the integration site. (B) One-ended transposition. RAG1/2 can also mediate attack of a single RSS end on a DNA molecule (depicted as a duplex circle).
tion, RAG-mediated induction of transposition has been demonstrated in yeast, indicating that the proteins are fully capable of carrying out transposition in vivo (Clatworthy et al., 2003). Therefore, active mechanisms must be in place within mammalian cells to channel RSS ends toward signal joint formation and to inhibit transposition and, in vivo, the full-length RAGs have evolved regulatory mechanisms to significantly downregulate this activity. It is not surprising that RAG-mediated transposition is prevented in developing lymphoid cells, because active transposition could lead to harmful genomic alterations, such as the generation of potentially oncogenic chromosomal translocations or inactivation of essential or tumor suppressor genes. Although many lymphoid tumors are associated with translocations initiated by V(D)J cleavage, the vast majority of these tumors appear to result from intrachromosomal V(D)J recombination or by misrepair of RAG-generated DSBs at antigen receptor loci. As discussed later, lymphomas resulting from the latter class of translocations can be eliminated by removal of the RAG genes. However, other than the one example mentioned, there is still no evidence for RAG-mediated transposition as a major pathway leading to translocations. However, generation of mice harboring
FIGURE 5.7 The NHEJ pathway joins RAG-liberated coding and signal ends. The Ku heterodimer, XRCC4, and Lig4 are required for both coding and signal joining, whereas DNA-PKcs and Artemis are more important for coding joining. The RAGs also play an important role during the joining phase of V(D)J recombination in the context of post-cleavage synaptic complexes.
appropriate RAG mutations may help to elucidate the potential existence of such a pathway in vivo.
CODING AND SIGNAL JOINT FORMATION REQUIRES THE NHEJ PATHWAY The DNA ends generated by the RAG1/2 endonuclease cleavage reaction are joined by generally expressed cellular DNA repair machinery. The coding and RSS ends produced by RAG cleavage form different substrates for the joining phase of the V(D)J recombination; however, both types of end structures are fused by the ubiquitously expressed nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair (Figure 5.7) (reviewed by Bassing et al., 2002). In this regard, hairpinned coding ends must be opened and further processed before joining, whereas blunt RSS ends can be directly fused. Extensive nucleotide sequence analyses of endogenous joints have shown that hairpin coding ends normally are opened at or near the apex. Cleavage of a hairpin away from the apex leaves an over-
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hanging flap, which if incorporated into the joint results in a P (palindromic) nucleotide addition (Figure 5.8) (Lafaille et al., 1989; McCormack et al., 1989). These additions are one source of “junctional diversity” (reviewed by Lewis, 1994). The opened hairpin ends can be further modified by nuclease action, which can remove a self-complementary overhang or cut further into the original coding sequence. Finally, the lymphoid-specific terminal deoxynucleotidyl transferase (TdT) enzyme can add nontemplated (N) nucleotides to the ends (Alt and Baltimore, 1982; Gilfillan et al., 1993; Komori et al., 1993). N regions are believed to play a major role in the somatic diversification of the repertoire of antigen receptor variable regions (Davis et al., 1997). Finally, additional junctional diversity comes from the nucleolytic activities that remove potential coding end nucleotides. Thus, the joining phase of the V(D)J recombination provides a major source of diverse junctional sequences for V(D)J coding joins (Figure 5.8). In this regard, the region of the Ig sequence encodes CDR3 and also
encodes an analogous region of the TCR chains; thus, such junctional diversification mechanisms provide a major source of antigen receptor diversity (Davis et al., 1997).
Double Strand Break Repair by Nonhomologous DNA End-Joining DNA double strand breaks (DSBs) can be introduced by external agents such as ionizing radiation (IR) or radiomimetic drugs, by normal cellular metabolism, and in the context of specific developmental programs such as V(D)J recombination. DSBs are one of the most dangerous lesions that a cell can suffer, potentially leading to adverse consequences such as cell death or chromosomal translocations that can contribute to cancer. In this context, mammalian cells employ two different pathways to repair DNA double strand breaks (DSBs). Homologous recombination leads to accurate repair of DSBs by copying intact information from a homologous DNA template and is generally
FIGURE 5.8 Processing of coding ends prior to joining. Subsequent to RAG1/2 cleavage and concomitant formation of blunt, 5¢ phosphorylated signal and hairpin coding ends, several different events can modify the coding ends prior to ligation. P elements may be added if the hairpins are opened at sites away from the apex, the TdT enzyme can add nontemplated N nucleotides to the open coding ends, and the coding ends can undergo deletion. The events are depicted here as independent, but can occur concurrently during V(D)J rearrangements.
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thought to be most prominently used in the S and G2 phases of the cell cycle, when such templates are most readily available (reviewed by Thompson and Schild, 2001). On the other hand, NHEJ rejoins broken ends irrespective of sequence, can result in deletions or insertions at the junctions, and appears most prominent in the G1 phase of the cell cycle, the phase during which RAG activity is also predominant (Lin and Desiderio, 1995; Takata et al., 1998; reviewed by Jackson, 2002). The NHEJ pathway is known to involve at least six proteins, including Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, DNA Ligase IV (Lig4), and Artemis. The first five proteins were linked, directly or indirectly, to the NHEJ pathway by studies of mutant cells that were both sensitive to IR and defective in V(D)J recombination (reviewed by Bassing et al., 2002; Taccioli and Alt, 1995). Artemis was identified as the gene mutated in one form of human SCID (Moshous et al., 2001); see below). Notably, after finding their role in NHEJ in mammalian cells, Ku70, Ku80, XRCC4, and Lig4 homologs were found to participate in a NHEJ pathway conserved in yeast (Boulton and Jackson, 1996; Boulton and Jackson, 1996; Feldmann et al., 1996; Herrmann et al., 1998; Mages et al., 1996; Milne et al., 1996; Schar et al., 1997; Siede et al., 1996; Teo and Jackson, 1997; Wilson et al., 1997). However, DNA-PKcs and Artemis appear to have evolved more recently in vertebrates and, as described later, appear to play a more restricted role in the NHEJ reaction (Jeggo and O’Neill, 2002). The recent identification of a human SCID cell line not defective in any of these genes indicates that additional factors may also be required (Dai et al., 2003).
and Carroll, 1991) actually involved a mutation in the carboyl terminus of the large DNA-PKcs gene (Araki et al., 1997; Blunt et al., 1996). The roles for two additional NHEJ proteins, Lig4 and Ku70, in NHEJ and V(D)J joining were implicated based on their interaction with XRCC4 and Ku80, respectively (Critchlow et al., 1997; Grawunder et al., 1997; Mimori et al., 1986). Subsequent gene-targeted mutation studies definitively showed that Lig4 and Ku70 were required both for normal DNA DSB repair and V(D)J recombination (Frank et al., 1998; Grawunder et al., 1998; Gu et al., 1997; Gu et al., 1997; Ouyang et al., 1997). Artemis deficiency in humans leads to radiosensitivity and a V(D)J recombination defect (Moshous et al., 2001) and its similar role in mice was confirmed by gene-targeted mutation analyses (Rooney et al., 2003; Rooney et al., 2002). To date, Artemis is the only NHEJ factor identified that has been implicated in human SCID, possibly because other known NHEJ factors may be more necessary for cellular proliferation and survival in humans than in mice (Li et al., 2002).
Identification of Mammalian NHEJ Proteins
Ku70 and Ku80 form a heterodimer, Ku, which possesses DNA end-binding activity (Mimori and Hardin, 1986). Purified Ku protein was found to promote the association of two DNA molecules in vitro; thus, it was proposed to possess end bridging or alignment activity (Ramsden and Gellert, 1998). The crystal structure of Ku bound to DNA revealed that the Ku heterodimer forms a ring that encircles duplex DNA and positions the DNA helix in a defined path, thus providing structural evidence in support of an end alignment function (Walker et al., 2001). Upon binding to DNA ends, Ku associates with and activates the serine–threonine protein kinase activity intrinsic to DNA-PKcs, thus forming the trimeric DNA-PK holoenzyme (Khanna and Jackson, 2001); one potential role for this function may be inferred from the interaction between DNA-PKcs and Artemis. Studies in yeast have supported the notion that Ku may serve an end-protection function as well (Lee et al., 1998). Additional Ku functions during V(D)J recombination have been suggested based on in vitro studies (reviewed by Featherstone and Jackson, 1999; Tuteja and Tuteja, 2000) and may include end remodeling, or recruitment of factors
The importance of the NHEJ pathway during V(D)J joining was established by discoveries that certain IR sensitive mutant rodent cells also exhibit a severe impairment in ability to join RAG-induced DSBs (Bosma and Carroll, 1991; Taccioli et al., 1993). Through the analysis of different complementation groups of radiosensitive Chinese hamster ovary (CHO) cell lines (Taccioli et al., 1993), two known proteins, Ku80 and DNA-PKcs, were identified as NHEJ factors (Blunt et al., 1995; Kirchgessner et al., 1995; Smider et al., 1994; Taccioli et al., 1994; Taccioli et al., 1994). A complementation cloning approach utilizing an additional IR-sensitive CHO line led to the identification of XRCC4, a previously unknown gene, as another NHEJ factor (Li et al., 1995). Subsequently, the roles for these proteins in V(D)J recombination in vivo were confirmed by gene-targeted mutation studies (Gao et al., 1998; Gao et al., 1998; Kurimasa et al., 1999; Nussenzweig et al., 1996; Taccioli et al., 1998; Zhu et al., 1996), as well as by the fact that the spontaneously arising scid mutation in mice (Bosma
Functions of NHEJ Proteins The functions of the various NHEJ proteins are beginning to emerge, both from biochemical characterization of their activities as well as by analyses of the steps in the V(D)J reaction that are impaired in cells carrying homozygous inactivating mutations of genes encoding individual factors. Ku70 and Ku80
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in addition to DNA-PKcs, including the XRCC4/Lig4 complex (Chen et al., 2000; Nick McElhinny et al., 2000); however, it has not yet been proved that Ku plays such roles in vivo (Doherty and Jackson, 2001). XRCC4 and DNA Ligase IV Although several other DNA ligases are present in mammalian cells, they cannot compensate for a defect in Lig4 activity during V(D)J joining or general DNA DSB repair. Cells deficient for Lig4 (or XRCC4) are severely impaired for both coding and RSS joining and are markedly radiosensitive (Frank et al., 1998; Gao et al., 1998; Grawunder et al., 1998). XRCC4 binds to Lig4, and this interaction stimulates Lig4 in vitro (Critchlow et al., 1997; Grawunder et al., 1997) and stabilizes it in vivo (Bryans et al., 1999). However, it is possible that XRCC4 may have additional functions during NHEJ, because it can bind nonspecifically to DNA in the absence of Lig4 (Modesti et al., 1999). The crystal structure of XRCC4 indicates that it forms a stable dimer that interacts with Lig4 (Junop et al., 2000; Sibanda et al., 2001). DNA-PKcs and Artemis A number of observations indicate that DNA-Pkcs functions primarily in coding joint formation. A role for DNAPKcs in coding versus RSS joining was originally indicated by the finding that cells from SCID mice, later found to be DNA-PKcs–deficient (Blunt et al., 1995; Blunt et al., 1996; Miller et al., 1995), were much more severely impaired for coding versus RSS joining (Blackwell et al., 1989; Lieber, 1998; Malynn et al., 1988). Moreover, hairpin coding ends were found to accumulate in DNA-PKcs–deficient developing lymphocytes (Roth et al., 1992), suggesting a difficulty in processing them. Also, unusually large P nucleotide additions are found in the rare coding joints recovered from DNA-PKcs–deficient cells, suggesting that the hairpins have been improperly opened further from the apex than is normal (Lewis, 1994). More recently, these findings generally have been reproduced in DNA-PKcs–deficient cells and mice generated by gene-targeted mutation and which lack DNA-PKcs protein (Gao et al., 1998; Kurimasa et al., 1999; Taccioli et al., 1998). DNA-PKcs is a serine–threonine protein kinase containing a phosphatidylinositol 3 kinase (PI3K) catalytic domain that is activated upon interaction with Ku bound to DNA ends (Smith and Jackson, 1999). DNA-PK phosphorylates a variety of targets in vitro, including p53, transcription factors, WRN, XRCC4, Ku, and Artemis (Ma et al., 2002; Smith and Jackson, 1999) and is capable of autophosphorylation (Chan et al., 2002; Chan and Lees-Miller, 1996; Merkle et al., 2002); however, in addition to Artemis (see below), the physiological relevance of its in vitro substrates is unclear. In addition to its roles in the context of Ku and
Artemis complexes, DNA-PKcs itself may be capable of synapsing broken DNA ends (DeFazio et al., 2002), and thus may also serve a structural role during end joining. Finally, DNA-PKcs may function outside the NHEJ pathway (Gurley and Kemp, 2001; Sekiguchi et al., 2001), playing roles that may overlap with those of the ataxia telangiectasia mutated (ATM) protein. ATM, like DNA-PKcs, is a serine–threonine protein kinase with a PI3 kinase domain and is involved in controlling cellular responses to DNA DSBs (reviewed by Shiloh, 2001). Thus, the novel NHEJindependent roles for DNA-PKcs may involve damage signaling related to checkpoint control (Jackson, 2002). The discovery and characterization of Artemis provided a major insight into one potential in vivo function of DNAPKcs. Artemis is a 77.6 kDa protein that is a member of the metallo-b-lactamase superfamily (Callebaut et al., 2002; Moshous et al., 2001), of which some members appear to be involved in the repair of interstrand cross-links (ICL) in mice and yeast. The RS-SCID patients having Artemis mutations lack B and T lymphocytes and show increased radiosensitivity of bone marrow cells and skin fibroblasts (Cavazzana-Calvo et al., 1993; Moshous et al., 2001; Nicolas et al., 1996; Nicolas et al., 1998). Moreover, transient V(D)J recombination substrate studies showed that RSSCID fibroblasts are more defective for coding than RSS joins, much like DNA-PKcs deficient cells (Moshous et al., 2001; Moshous et al., 2000; Nicolas et al., 1998). In addition, a large proportion of the rare coding joints recovered from Artemis-deficient ES cells contain longer than average P nucleotide additions, reminiscent of those recovered from DNA-PKcs-deficient cells (Rooney et al., 2003). Thus, it was proposed that Artemis may function to open hairpin DNA coding ends (Moshous et al., 2001). Strong support for this notion came from in vitro studies showing that DNAPKcs forms a complex with and phosphorylates Artemis, leading to the activation of an endonuclease activity that can cleave RAG-generated hairpins (Ma et al., 2002). Thus, these results led to the hypothesis that a DNA-PKcs/Artemis complex, perhaps recruited by Ku, opens coding hairpin ends in vivo (Karanjawala et al., 2002; Ma et al., 2002). Indeed, in support of this notion, hairpin coding ends accumulate in Artemis-deficient thymocytes (Rooney et al., 2002), as they also do in Ku and DNA-PKcs–deficient thymocytes, consistent with a role for the entire Ku/DNA–PKcs/Artemis complex in this reaction (Gao et al., 1998; Roth et al., 1992; Zhu et al., 1996; Zhu and Roth, 1995). The more limited role of the DNA-PKcs/Artemis complex in V(D)J recombination, that of opening of coding end hairpins, as opposed to the four evolutionarily conserved factors that are required for both RSS and coding joins, also may give further insight into the evolution and function of these proteins. Thus, the four conserved factors may be components of a conserved complex that forms a
5. The Mechanism of V(D)J Recombination
basic end-ligation function. DNA-PKcs and Artemis, as also suggested by other lines of evidence (Gao et al., 1998; Rooney et al., 2003, see below), may have evolved more recently to function to process ends that cannot be simply ligated (e.g., blocked ends or hairpins) to a form that can be joined by the basic end-ligation apparatus. Other Activities Several additional activities are predicted to be required during V(D)J joining. One such activity is a DNA polymerase responsible for filling in short gaps at coding junctions that may be generated by end modifications. Eukaryotic DNA polymerases of the pol X family [e.g., Pol4 in S. cerevisiae (Wilson and Lieber, 1999) and Pol m in mammals (Mahajan et al., 2002)] have been implicated as potential candidates for such a V(D)J polymerase. Human pol m, which has homology to TdT, has been found to interact with Ku and requires Ku, XRCC4, and Lig4 for stable DNA binding in vitro (Mahajan et al., 2002). Pol m is upregulated and forms foci upon exposure of cells to IR, suggesting a role in general DNA DSB repair (Mahajan et al., 2002). A subset of mice deficient in pol m exhibit a significant depletion of B cells in peripheral lymphoid organs, thus indicating a function for pol m during B cell development (Bertocci et al., 2002). However, currently no compelling in vivo evidence exists to point to a specific DNA polymerase that functions during V(D)J recombination; thus, the identity of the V(D)J polymerase remains unknown. It is evident that normal V(D)J recombination involves a nucleolytic activity that deletes nucleotides at the coding ends. Several potential candidates include the Mre11/Rad50/Nbs1 (MRN), RAG1/RAG2, and Artemis/ DNA–PKcs complexes, which may fulfill the role of a V(D)J nuclease during processing of open coding hairpin ends. The MRN complex is required for DNA DSB repair in vivo and in vitro and has been demonstrated to possess endo and exonuclease activities (D’Amours and Jackson, 2002). Nbs1 has been found in foci at V(D)J induced breaks (Chen et al., 2000), and mice expressing a hypomorphic allele of Nbs1 exhibit defects in lymphocyte development (Kang et al., 2002). However, mutations in Nbs1 that result in DNA DSB repair defects do not have any obvious effects on coding junction sequences in vitro or in vivo (Harfst et al., 2000; Kang et al., 2002; Yeo et al., 2000). Thus, the Mre11/Rad50/Nbs1 complex may play a more indirect role in V(D)J recombination, such as in DSB detection and/or signaling. In vitro, truncated forms of RAG1/RAG2 have been demonstrated to open hairpin coding ends and cleave 3¢ flap structures (Besmer et al., 1998; Santagata et al., 1999; Shockett and Schatz, 1999). Although it appears that the RAGs do not play a significant role in hairpin end opening in vivo (Zhu and Roth, 1995; Zhu et al., 1996; Rooney et al., 2002), they may play a role
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in further processing the open hairpins. Artemis/DNA–PKcs possesses endonuclease activity on 5¢ and 3¢ single strand overhanging flaps and Artemis, in the absence of DNA-PKcs, has intrinsic 5¢ to 3¢ exonuclease activity on single strand DNA (Ma et al., 2002); thus, it may play roles in coding end processing in addition to nicking hairpins. In support of this notion, rare coding joints recovered from Artemis-deficient ES cells in transient transfection V(D)J recombination assays exhibit significantly less nucleotide loss at the junctions compared to those recovered from wild type ES cells (Rooney et al., 2003). A clearer picture of which of these factors, if any, are involved in coding end processing awaits additional detailed studies. As mentioned previously, a major source of junctional diversity comes from the addition of nongermline encoded nucleotides at V-D, D-J, and some V-J junctions, which are referred to as N regions (Alt and Baltimore, 1982). Although it was proposed early on that N regions were added by TdT, this was proved by gene-targeted mutations studies that clearly demonstrated the absence of N regions in TdTdeficient lymphocytes (Gilfillan et al., 1993; Komori et al., 1993). TdT is not expressed substantially during fetal development and therefore most Ig and TCR junctions formed in the fetal repertoire lack N regions (reviewed by Benedict et al., 2000; Komori et al., 1996). In addition, junctions formed in the absence of N region addition also often used short homologies to form “canonical” junctions that appear very frequently in the absence of TdT [e.g., in fetal repertories; (Benedict et al., 2000; Komori et al., 1996)]. Thus, TdT expression during B and T cell development in the adult diversifies repertoires both by N region addition and by the diminution of canonical junctions, which form much less frequently in the presence of N regions.
Mice Deficient in the NHEJ Factors Mice deficient for all known lymphoid-specific and general V(D)J recombination factors have been generated by gene-targeted mutation (reviewed by Bassing et al., 2002; Rooney et al., 2002). RAG-1 or -2 deficient mice have a severe combined immune deficiency (SCID) due to the inability to initiate V(D)J recombination. Other than a complete block in B and T cell development at the progenitor stage, RAG-deficient mice do not exhibit any other phenotypes. This very specific phenotype is consistent with the notion that RAGs evolved only for their role in effecting antigen receptor variable region gene assembly in developing lymphocytes (Shinkai et al., 1992). Mice deficient for TdT, the only other known lymphocyte-specific V(D)J recombination factor besides RAG1 and 2, exhibit relatively normal V(D)J recombination levels; however, V(D)J coding junctions lack N-region additions (Gilfillan et al., 1993; Komori et al., 1993). This phenotype is consistent with the
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nonessential role for TdT in V(D)J recombination, which involves the diversification of V(D)J junctions via the addition of nontemplated N nucleotides to coding ends. Deficiencies in the NHEJ factors also result in impaired lymphocyte development. However, the NHEJ mutant mice and cells exhibit phenotypes beyond defects in V(D)J recombination owing to the importance of the NHEJ pathway in general DSB repair. Classical SCID mice, which express a nearly full length but catalytically inactive form of DNA-PKcs, and Artemis-deficient mice exhibit a “leaky” SCID phenotype with some T and B cells appearing in older mice (e.g., Bosma et al., 1988; Rooney et al., 2002; Taccioli et al., 1998). This leaky V(D)J joining occurs at very low levels and is catalyzed by the basic NHEJ pathway (or by an alternative repair pathway) following the opening of coding end hairpins by some lower level activity. However, this interpretation is somewhat complicated by the fact that some lines of DNA-PKcs–deficient mice generated by gene-targeted mutation to completely lack DNA-PKcs protein have not been found to be leaky (Gao et al., 1998). Thus, these and certain other minor phenotypic differences between complete DNA-PKcs knock-out mice and SCID mice (e.g. Bosma et al., 2002; Manis et al., 2002) may reflect some residual activity of the latter. DNA-PKcs and Artemis deficiencies result in variable cellular IR sensitivity (Gao et al., 1998; Rooney et al., 2003). Thus, ES cells harboring targeted inactivating mutations in either DNA-PKcs or Artemis are not radiosensitive; but murine embryonic fibroblasts (MEFs) homozygous for the same mutations are significantly more IR sensitive than wildtype MEFs, suggesting potentially redundant factors in ES cells (Gao et al., 1998; Rooney et al., 2003). Also, whereas DNA-PKcs– and Artemis-deficient ES cells do not display substantial IR sensitivity, they do display more significant sensitivity to bleomycin, which is a radiomimetic drug (Rooney et al., 2003). As bleomycin and IR may lead to a different spectrum of broken ends (Povirk, 1996), these findings are consistent with the notion, outlined earlier, that DNA-PKcs and Artemis are employed in NHEJ for repairing a specific subset of DNA damage that requires processing prior to ligation. Other than variable cellular IR sensitivity, DNA-PKcs– and Artemis-deficient mice have no other obvious consistent phenotype. Ku-deficient mice also have a SCID phenotype (Gu et al., 1997; Nussenzweig et al., 1996; Ouyang et al., 1997). In this regard, the SCID phenotype of Ku70-deficient mice is leaky (Gu et al., 1997; Ouyang et al., 1997), likely due to the lowlevel joining of RAG-induced DSBs, similar to that observed in DNA-PKcs– and Artemis-deficient mice. However, unlike DNA-PKcs and Artemis deficiencies, Ku deficiency results in mice that are significantly smaller than littermates (Gu et al., 1997; Nussenzweig et al., 1996; Ouyang et al., 1997) and that show increased apoptosis of
newly generated, post-mitotic neurons in embryos (Gu et al., 2000). In addition, Ku-deficient cells exhibit growth defects, premature senescence, and IR sensitivity (Gu et al., 1997; Nussenzweig et al., 1996; Ouyang et al., 1997). The phenotypic differences between Ku- and DNA-PKcs–deficient mice reinforce the notion that Ku possesses functions separate from any it may effect in the context of the DNA-PK holoenzyme. Finally, Ku70-, but not Ku80-, deficient mice also show an increased incidence of thymic lymphomas (Gu et al., 1997; Li et al., 1998). The reason for this difference is not clear but could relate to relative leakiness of the defects, as leaky SCID mice also are more prone to T cell lymphomas on certain backgrounds (Custer et al., 1985; Jhappan et al., 1997). XRCC4- and Lig4-deficiency leads to late embryonic lethality accompanied by severe neuronal apoptosis throughout the central nervous system (Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998). In addition to cellular defects analogous to those of Ku-deficient mice, XRCC4and Lig4-deficient embryos exhibit a complete block in B and T cell development in fetal lymphoid organs. Notably, the breeding of XRCC4- or Lig4-deficient mice into a p53 deficient background rescues their embryonic lethality and neuronal apopotosis defects, but not their V(D)J recombination or lymphocyte development defects (Frank et al., 2000; Gao et al., 2000). Thus, the embryonic lethality and severe neuronal apoptosis of XRCC4- or Lig4-deficient mice appears to result from a p53-dependent response to unrepaired DSBs and not from the inability to repair the breaks via NHEJ per se (Frank et al., 2000; Gao et al., 2000). Conversely, defective lymphocyte development appears to result primarily from the inability to repair the RAGinitiated DSBs to generate the functional antigen receptor genes necessary to drive further development. However, it is also clear that XRCC4- or Lig4-deficient progenitor lymphocytes pools are severely depleted due to a p53-dependent response to the unrepaired RAG-initiated DSBs (Frank et al., 2000; Gao et al., 2000). XRCC4-, Lig4-, and Ku-deficient mice appear quite similar in a p53-deficient background (Difilippantonio et al., 2002; Difilippantonio et al., 2000; Frank et al., 2000; Gao et al., 2000; Lim et al., 2000; Zhu et al., 2002), suggesting that the major differences in Ku- versus XRCC4- or Lig4deficient phenotypes are likely quantitative, as indicated by the greater “leakiness” in NHEJ and a somewhat lower level of apoptotic cell death in Ku-deficient mice which, for example, allows the generation of a functional nervous system (Gu et al., 2000; Sekiguchi et al., 1999). Thus, deficiencies in the evolutionarily conserved NHEJ factors, Ku, XRCC4, and Lig4, exhibit similar phenotypes, albeit with varying severity, whereas, DNA-PKcs and Artemis have milder phenotypes consistent with their involvement in a more limited set of NHEJ functions.
5. The Mechanism of V(D)J Recombination
Recognition of RAG-Initiated DSBs by the DNA Repair and Cell Cycle Checkpoint Machinery Initiation of V(D)J recombination by RAG1/2 is tightly coupled to the cell cycle, as evidenced by the accumulation of RAG-generated DSBs in G0/G1 cells and the periodic accumulation of the RAG2 protein during G0/G1 and its subsequent degradation at the G1–S transition (Desiderio et al., 1996; Schlissel et al., 1993). This form of regulation would be optimal to ensure joining via NHEJ. Developing lymphocytes containing unrepaired RAG-initiated DSBs normally undergo programmed cell death resulting from induction of the p53-dependent cell cycle checkpoint (reviewed by Lu and Osmond, 2000). Likewise, progenitor populations of developing lymphocytes are dramatically reduced in NHEJ-deficient animals. This decrease appears to be caused by the extensive apoptosis of progenitors harboring unrepaired RAG-induced DSBs, as p53-deficiency leads to the increased survival and proliferation of NHEJdeficient lymphocyte progenitors (Difilippantonio et al., 2000; Frank et al., 2000; Gao et al., 2000; Guidos et al., 1996), which in turn leads to the development of aggressive progenitor-B cell lymphomas. Thus, the efficient recognition of RAG-generated DNA ends by the NHEJ pathway is clearly imperative to avoid DSB detection and induction of p53-dependent apoptosis. Normally, coding ends are joined rapidly (Ramsden and Gellert, 1995; Zhu and Roth, 1995); however, in contrast, RSS ends persist throughout G1 and are joined at the G1/S transition (Ramsden and Gellert, 1995; Roth et al., 1992; Schlissel et al., 1993). However, such persistent expression does not lead to p53 induction in normal progenitor populations (Guidos et al., 1996). Thus, the prolonged presence of RSS ends appears to escape detection by the cell cycle checkpoint machinery, possibly by sequestration in a stable postsynaptic cleavage complex. In addition to p53, other proteins that monitor DNA damage and repair also appear to interplay with the V(D)J recombination reaction. The ATM protein is noteworthy in this regard. Although ATM is not directly involved in V(D)J recombination (Barlow et al., 1996; Elson et al., 1996; Hsieh et al., 1993; Xu et al., 1996), deficiency for this protein leads to lymphoid malignancies (reviewed by Khanna et al., 2001), which, in mice, are predominantly T cell lymphomas that frequently harbor translocations involving their TCRa/d locus (Barlow et al., 1996; Liyanage et al., 2000; Petiniot et al., 2000). Histone H2AX is another class of factor that has been implicated in some aspect of the V(D)J rearrangement process, which may include linkage with DNA repair and/or checkpoint pathways. H2AX is a histone H2A variant that phosphorylates upon DNA damage, such as is induced by IR, and is found in a phosphorylated form within foci of repair factors at DNA DSBs (Rogakou et al., 1998). Targeted muta-
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tional studies have shown that H2AX, which is phosphorylated by ATM and related kinases (Burma et al., 2001; Ward and Chen, 2001), is required for normal DNA DSB repair and for maintenance of genomic stability (Bassing et al., 2002; Celeste et al., 2002). Moreover, phosphorylated H2AX has also been observed to co-localize in foci with Nbs1 at DSBs induced during V(D)J recombination (Chen et al., 2000). Although mice deficient for H2AX do not exhibit severe defects in lymphocyte development (Bassing et al., 2002; Celeste et al., 2002), the localization data suggestes that H2AX may be involved in monitoring V(D)J rearrangements in the context of DNA checkpoints to suppress oncogenic translocations, possibly through modulation of chromatin structure. In this regard, very recent findings have shown that p53-deficient mice that are also deficient or haplo-insufficient for H2AX, are prone to lymphomas, including B lineage lymphomas with translocations that appear to involve RAGgenerated DSBs (Bassing et al., 2003; Celeste et al., 2003).
NHEJ Factors and Suppression of RAGInitiated Translocations In addition to their roles in V(D)J recombination and DNA DSB repair, the NHEJ factors also play important roles in maintaining genomic stability. A number of different types of chromosomal aberrations are observed in cells lacking a functional NHEJ pathway, including chromosome fragments, fusions, and translocations (reviewed by Ferguson and Alt, 2001). The importance of the NHEJ factors as genomic caretakers is highlighted by the fact that NHEJ-deficiencies, including inactivating mutations in Ku, XRCC4, Lig4, Artemis, and the classical SCID mutation in DNA-PKcs, in combination with deficiencies in the p53 cell cycle checkpoint protein in mice, predispose to lymphomagenesis (Difilippantonio et al., 2000; Frank et al., 2000; Gao et al., 2000; Gladdy et al., 2003; Lim et al., 2000; Nacht et al., 1996; Rooney, Sekiguchi, and Alt, in preparation). Equally notable is that fact that in all cases, the predominant tumor is a pro-B cell lymphoma that has translocations and amplifications involving the c-myc and IgH loci (Difilippantonio et al., 2000; Gao et al., 2000). Various lines of evidence have shown that the initiating lesions that cause the oncogenic chromosomal aberrations in these NHEJ/ p53-deficient pro-B lymphomas are RAG-induced DSBs (Difilippantonio et al., 2002; Gladdy et al., 2003; Vanasse et al., 1999; Zhu et al., 2002). Thus, the translocations involve JH region sequences, and the introduction of RAG mutation into these mutant backgrounds eliminates the occurrence of pro-B lymphomas bearing the hallmark chromosomal anomalies (Difilippantonio et al., 2002; Gladdy et al., 2003; Vanasse et al., 1999; Zhu et al., 2002). These findings implicate a pro-B cell lymphomagenesis model in which RAG-initiated DSBs in p53/NHEJ pro-B
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cells are neither repaired nor eliminated via a G1 checkpoint. Thus, these mutant pro-B cells can progress into S phase where the RAG-initiated DSBs at their JH locus are replicated. Subsequently, these replicate to generate dicentric chromosomes which, in the p53-deficient background, can rapidly generate the amplification of genes conferring a selective growth advantage via a breakage bridge fusion mechanism (Difilippantonio et al., 2002; Zhu et al., 2002). Although it is unclear if this mechanism contributes to human B cell lymphomas, it might be involved in advanced human solid tumors and potentially in some B lineage tumors, including advanced stage myelomas (Mills et al., in press). Finally, Ku, DNA-PKcs, and Artemis also may play roles in telomere maintenance, as cells deficient in these factors lead to defects in telomere capping (Bailey et al., 2001; Bailey et al., 1999; Espejel et al., 2002; Goytisolo et al., 2001; Rooney et al., 2003; Samper et al., 2000). In addition, Ku and DNA-PKcs deficiencies may also result in dysregulation of telomere length (d’Adda di Fagagna et al., 2001; de Lange, 2002; Espejel et al., 2002; Hsu et al., 2000; Samper et al., 2000).
Acknowledgments F.W.A. is an Investigator of the Howard Hughes Medical Institute. J.S. is a Special Fellow of the Leukemia and Lymphoma Society. This work was supported by NIH grants AI35714 and NCI CA92625 (FWA) and GM48025 (MAO).
References Agrawal, A., Eastman, Q. M., and Schatz, D. G. (1998). Nature 394, 744–751. Agrawal, A., and Schatz, D. G. (1997). Cell 89, 43–53. Akamatsu, Y., Monroe, R., Dudley, D. D., Elkin, S. K., Gartner, F., Talukder, S. R., Takahama, Y., Alt, F. W., Bassing, C. H., and Oettinger, M. A. (2003). Proc Natl Acad Sci U S A 100, 1209–1214. Akamatsu, Y., and Oettinger, M. A. (1998). Mol Cell Biol 18, 4670–4678. Akamatsu, Y., Tsurushita, N., Nagawa, F., Matsuoka, M., Okazaki, K., Imai, M., and Sakano, H. (1994). J Immunol 153, 4520–4529. Akira, S., Okazaki, K., and Sakano, H. (1987). Science 238, 1134–1138. Alt, F. W., and Baltimore, D. (1982). Proc Natl Acad Sci U S A 79, 4118–4122. Araki, R., Fujimori, A., Hamatani, K., Mita, K., Saito, T., Mori, M., Fukumura, R., Morimyo, M., Muto, M., Itoh, M., Tatsumi, K., and Abe, M. (1997). Proc Natl Acad Sci U S A 94, 2438–2443. Aravind, L., and Koonin, E. V. (1999). J Mol Biol 287, 1023–1040. Arbuckle, J. L., Fauss, L. A., Simpson, R., Ptaszek, L. M., and Rodgers, K. K. (2001). J Biol Chem 276, 37093–37101. Bailey, S. M., Cornforth, M. N., Kurimasa, A., Chen, D. J., and Goodwin, E. H. (2001). Science 293, 2462–2465. Bailey, S. M., Meyne, J., Chen, D. J., Kurimasa, A., Li, G. C., Lehnert, B. E., and Goodwin, E. H. (1999). Proc Natl Acad Sci U S A 96, 14899–14904. Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M., Collins, F., Shiloh, Y., Crawley, J. N., Ried, T., Tagle, D., and Wynshaw-Boris, A. (1996). Cell 86, 159–171. Barnes, D. E., Stamp, G., Rosewell, I., Denzel, A., and Lindahl, T. (1998). Curr Biol 8, 1395–1398.
Bassing, C. H., Alt, F. W., Hughes, M. M., D’Auteuil, M., Wehrly, T. D., Woodman, B. B., Gartner, F., White, J. M., Davidson, L., and Sleckman, B. P. (2000). Nature 405, 583–586. Bassing, C. H., Chua, K. F., Sekiguchi, J., Suh, H., Whitlow, S. R., Fleming, J. C., Monroe, B. C., Ciccone, D. N., Yan, C., Vlasakova, K., Livingston, D. M., Ferguson, D. O., Scully, R., and Alt, F. W. (2002). Proc Natl Acad Sci U S A 28, 28. Bassing, C. H., Swat, W., and Alt, F. W. (2002). Cell 109, S45–S55. Bassing, C. H., Suh, H., Ferguson, D. O., Chua, K. F., Manis, J., Eckersdorff, M., Gleason, M., Bronson, R., Lee, C., and Alt, F. W. (2003). Cell 114, 359–370. Bellon, S. F., Rodgers, K. K., Schatz, D. G., Coleman, J. E., and Steitz, T. A. (1997). Nat Struct Biol 4, 586–591. Benedict, C. L., Gilfillan, S., Thai, T. H., and Kearney, J. F. (2000). Immunol Rev 175, 150–157. Bertocci, B., De Smet, A., Flatter, E., Dahan, A., Bories, J. C., Landreau, C., Weill, J. C., and Reynaud, C. A. (2002). J Immunol 168, 3702–3706. Besmer, E., Mansilla-Soto, J., Cassard, S., Sawchuk, D. J., Brown, G., Sadofsky, M., Lewis, S. M., Nussenzweig, M. C., and Cortes, P. (1998). Mol Cell 2, 817–828. Blackwell, T. K., Ferrier, P., Malynn, B. A., Pollock, R. R., Covey, L. R., Suh, H. Y., Heinke, L. B., Fulop, G. M., Phillips, R. A., Yancopoulos, G. D., et al. (1989). Curr Top Microbiol Immunol 152, 85–94. Blunt, T., Finnie, N. J., Taccioli, G. E., Smith, G. C., Demengeot, J., Gottlieb, T. M., Mizuta, R., Varghese, A. J., Alt, F. W., Jeggo, P. A., et al. (1995). Cell 80, 813–823. Blunt, T., Gell, D., Fox, M., Taccioli, G. E., Lehmann, A. R., Jackson, S. P., and Jeggo, P. A. (1996). Proc Natl Acad Sci U S A 93, 10285–10290. Bogue, M. A., Wang, C., Zhu, C., and Roth, D. B. (1997). Immunity 7, 37–47. Born, W., Yague, J., Palmer, E., Kappler, J., and Marrack, P. (1985). Proc Natl Acad Sci U S A 82, 2925–2929. Bosma, G. C., Fried, M., Custer, R. P., Carroll, A., Gibson, D. M., and Bosma, M. J. (1988). J Exp Med 167, 1016–1033. Bosma, G. C., Kim, J., Urich, T., Fath, D. M., Cotticelli, M. G., Ruetsch, N. R., Radic, M. Z., and Bosma, M. J. (2002). J Exp Med 196, 1483–1495. Bosma, M. J., and Carroll, A. M. (1991). Annu Rev Immunol 9, 323–350. Boubnov, N. V., Wills, Z. P., and Weaver, D. T. (1995). Nucleic Acids Res 23, 1060–1067. Boulton, S. J., and Jackson, S. P. (1996). Nucleic Acids Res 24, 4639–4648. Boulton, S. J., and Jackson, S. P. (1996). EMBO J 15, 5093–5103. Bryans, M., Valenzano, M. C., and Stamato, T. D. (1999). Mutat Res 433, 53–58. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., and Chen, D. J. (2001). J Biol Chem 276, 42462–42467. Callebaut, I., and Mornon, J. P. (1998). Cell Mol Life Sci 54, 880–891. Callebaut, I., Moshous, D., Mornon, J. P., and de Villartay, J. P. (2002). Nucleic Acids Res 30, 3592–3601. Cavazzana-Calvo, M., Le Deist, F., De Saint Basile, G., Papadopoulo, D., De Villartay, J. P., and Fischer, A. (1993). J Clin Invest 91, 1214 –1218. Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M. J., Redon, C., Pilch, D. R., Olaru, A., Eckhaus, M., Camerini-Otero, R. D., Tessarollo, L., Livak, F., Manova, K., Bonner, W. M., Nussenzweig, M. C., and Nussenzweig, A. (2002). Science 296, 922–927. Celeste, A., Difilippantonio, S., Difilippantonio, M. J., Fernandez-Capetillo, O., Pilch, D. R., Sedelnikova, O. A., Eckhaus, M., Reid, T., Bonner, W. M., and Nussenzweig, A. (2003). Cell 114, 371–383. Chan, D. W., Chen, B. P., Prithivirajsingh, S., Kurimasa, A., Story, M. D., Qin, J., and Chen, D. J. (2002). Genes Dev 16, 2333–2338. Chan, D. W., and Lees-Miller, S. P. (1996). J Biol Chem 271, 8936–8941.
5. The Mechanism of V(D)J Recombination Chen, H. T., Bhandoola, A., Difilippantonio, M. J., Zhu, J., Brown, M. J., Tai, X., Rogakou, E. P., Brotz, T. M., Bonner, W. M., Ried, T., and Nussenzweig, A. (2000). Science 290, 1962–1965. Chen, L., Trujillo, K., Sung, P., and Tomkinson, A. E. (2000). J Biol Chem 275, 26196–26205. Cheung, S., Arndt, K., and Lu, P. (1984). Proc Natl Acad Sci U S A 81, 3665–3669. Clatworthy, A., Valencia, M. A., Haber, J. E., and Oettinger, M. A. (2003). Mol Cell 12, 489–499. Connor, A. M., Fanning, L. J., Celler, J. W., Hicks, L. K., Ramsden, D. A., and Wu, G. E. (1995). J Immunol 155, 5268–5272. Corneo, B., Moshous, D., Callebaut, I., de Chasseval, R., Fischer, A., and de Villartay, J. P. (2000). J Biol Chem 275, 12672–12675. Critchlow, S. E., Bowater, R. P., and Jackson, S. P. (1997). Curr Biol 7, 588–598. Cuomo, C. A., Mundy, C. L., and Oettinger, M. A. (1996). Mol Cell Biol 16, 5683–5690. Cuomo, C. A., and Oettinger, M. A. (1994). Nucleic Acids Res 22, 1810–1814. Custer, R. P., Bosma, G. C., and Bosma, M. J. (1985). Am J Pathol 120, 464–477. d’Adda di Fagagna, F., Hande, M. P., Tong, W. M., Roth, D., Lansdorp, P. M., Wang, Z. Q., and Jackson, S. P. (2001). Curr Biol 11, 1192–1196. D’Amours, D., and Jackson, S. P. (2002). Nat Rev Mol Cell Biol 3, 317–327. Dai, Y., Kysela, B., Hanakahi, L. A., Manolis, K., Riballo, E., Stumm, M., Harville, T. O., West, S. C., Oettinger, M. A., and Jeggo, P. A. (2003). Proc Natl Acad Sci U S A 100, 2462–2467. Davis, M. M., Lyons, D. S., Altman, J. D., McHeyzer-Williams, M., Hampl, J., Boniface, J. J., and Chien, Y. (1997). Ciba Found Symp 204, 94–100; discussion 100–104. de Lange, T. (2002). Oncogene 21, 532–540. DeFazio, L. G., Stansel, R. M., Griffith, J. D., and Chu, G. (2002). EMBO J 21, 3192–3200. Desiderio, S., Lin, W. C., and Li, Z. (1996). Curr Top Microbiol Immunol 217, 45–59. Difilippantonio, M. J., Petersen, S., Chen, H. T., Johnson, R., Jasin, M., Kanaar, R., Ried, T., and Nussenzweig, A. (2002). J Exp Med 196, 469–480. Difilippantonio, M. J., Zhu, J., Chen, H. T., Meffre, E., Nussenzweig, M. C., Max, E. E., Ried, T., and Nussenzweig, A. (2000). Nature 404, 510–514. Doherty, A. J., and Jackson, S. P. (2001). Curr Biol 11, R920–R924. Dudley, D., Sekiguchi, J., Zhu, C., Sadofsky, M. J., Whitlow, S., Devido, J., Monroe, R. J., Bassing, C. H., and AH, F. W. (2003). J Exp Med 198, 1439–1450. Eastman, Q. M., Leu, T. M., and Schatz, D. G. (1996). Nature 380, 85–88. Eastman, Q. M., Villey, I. J., and Schatz, D. G. (1999). Mol Cell Biol 19, 3788–3797. Elkin, S. K., Matthews, A. G., and Oettinger, M. A. (2003). EMBO J 22. Elson, A., Wang, Y., Daugherty, C. J., Morton, C. C., Zhou, F., CamposTorres, J., and Leder, P. (1996). Proc Natl Acad Sci U S A 93, 13084–13089. Engelman, A., Mizuuchi, K., and Craigie, R. (1991). Cell 67, 1211–1221. Espejel, S., Franco, S., Rodriguez-Perales, S., Bouffler, S. D., Cigudosa, J. C., and Blasco, M. A. (2002). EMBO J 21, 2207–2219. Espejel, S., Franco, S., Sgura, A., Gae, D., Bailey, S. M., Taccioli, G. E., and Blasco, M. A. (2002). EMBO J 21, 6275–6287. Ezekiel, U. R., Engler, P., Stern, D., and Storb, U. (1995). Immunity 2, 381–389. Featherstone, C., and Jackson, S. P. (1999). Curr Biol 9, R759–R761. Feeney, A. J., Tang, A., and Ogwaro, K. M. (2000). Immunol Rev 175, 59–69. Feldmann, H., Driller, L., Meier, B., Mages, G., Kellermann, J., and Winnacker, E. L. (1996). J Biol Chem 271, 27765–27769.
79
Ferguson, D. O., and Alt, F. W. (2001). Oncogene 20, 5572–5579. Ferrier, P., Krippl, B., Blackwell, T. K., Furley, A. J., Suh, H., Winoto, A., Cook, W. D., Hood, L., Costantini, F., and Alt, F. W. (1990). EMBO J 9, 117–125. Frank, K. M., Sekiguchi, J. M., Seidl, K. J., Swat, W., Rathbun, G. A., Cheng, H. L., Davidson, L., Kangaloo, L., and Alt, F. W. (1998). Nature 396, 173–177. Frank, K. M., Sharpless, N. E., Gao, Y., Sekiguchi, J. M., Ferguson, D. O., Zhu, C., Manis, J. P., Horner, J., DePinho, R. A., and Alt, F. W. (2000). Mol Cell 5, 993–1002. Freemont, P. S., Hanson, I. M., and Trowsdale, J. (1991). Cell 64, 483–484. Fugmann, S. D. (2001). Immunol Res 23, 23–39. Fugmann, S. D., and Schatz, D. G. (2001). Mol Cell 8, 899–910. Fugmann, S. D., Villey, I. J., Ptaszek, L. M., and Schatz, D. G. (2000). Mol Cell 5, 97–107. Fujimoto, S., and Yamagishi, H. (1987). Nature 327, 242–243. Gao, Y., Chaudhuri, J., Zhu, C., Davidson, L., Weaver, D. T., and Alt, F. W. (1998). Immunity 9, 367–376. Gao, Y., Ferguson, D. O., Xie, W., Manis, J., Sekiguchi, J., Frank, K. M., Chaudhuri, J. J. H., DePinho, R. A., and Alt, F. W. (2000). Nature 404, 897–900. Gao, Y., Sun, Y., Frank, K. M., Dikkes, P., Fujiwara, Y., Seidl, K. J., Sekiguchi, J. M., Rathbun, G. A., Swat, W., Wang, J., Bronson, R. T., Malynn, B. A., Bryans, M., Zhu, C., Chaudhuri, J., Davidson, L., Ferrini, R., Stamato, T., Orkin, S. H., Greenberg, M. E., and Alt, F. W. (1998). Cell 95, 891–902. Gellert, M. (2002). Annu Rev Biochem 71, 101–132. Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C., and Mathis, D. (1993). Science 261, 1175–1178. Gladdy, R. A., Taylor, M. D., Williams, C. J., Grandal, I., Karaskova, J., Squire, J. A., Rutka, J. T., Guidos, C. J., and Danska, J. S. (2003). Cancer Cell 3, 37–50. Godderz, L. J., Rahman, N. S., Risinger, G. M., Arbuckle, J. L., and Rodgers, K. K. (2003). Nucleic Acids Res 31, 2014–2023. Gomez, C. A., Ptaszek, L. M., Villa, A., Bozzi, F., Sobacchi, C., Brooks, E. G., Notarangelo, L. D., Spanopoulou, E., Pan, Z. Q., Vezzoni, P., Cortes, P., and Santagata, S. (2000). Mol Cell Biol 20, 5653–5664. Goytisolo, F. A., Samper, E., Edmonson, S., Taccioli, G. E., and Blasco, M. A. (2001). Mol Cell Biol 21, 3642–3651. Grawunder, U., Wilm, M., Wu, X., Kulesza, P., Wilson, T. E., Mann, M., and Lieber, M. R. (1997). Nature 388, 492–495. Grawunder, U., Zimmer, D., Fugmann, S., Schwarz, K., and Lieber, M. R. (1998). Mol Cell 2, 477–484. Gu, Y., Jin, S., Gao, Y., Weaver, D. T., and Alt, F. W. (1997). Proc Natl Acad Sci U S A 94, 8076–8081. Gu, Y., Seidl, K. J., Rathbun, G. A., Zhu, C., Manis, J. P., van der Stoep, N., Davidson, L., Cheng, H. L., Sekiguchi, J. M., Frank, K., StanhopeBaker, P., Schlissel, M. S., Roth, D. B., and Alt, F. W. (1997). Immunity 7, 653–665. Gu, Y., Sekiguchi, J., Gao, Y., Dikkes, P., Frank, K., Ferguson, D., Hasty, P., Chun, J., and Alt, F. W. (2000). Proc Natl Acad Sci U S A 97, 2668–2673. Guidos, C. J., Williams, C. J., Grandal, I., Knowles, G., Huang, M. T., and Danska, J. S. (1996). Genes Dev 10, 2038–2054. Gurley, K. E., and Kemp, C. J. (2001). Curr Biol 11, 191–194. Han, J. O., Steen, S. B., and Roth, D. B. (1999). Mol Cell 3, 331–338. Hansen, J. D., and Kaattari, S. L. (1995). Immunogenetics 42, 188– 195. Harfst, E., Cooper, S., Neubauer, S., Distel, L., and Grawunder, U. (2000). Mol Immunol 37, 915–929. Herrmann, G., Lindahl, T., and Schar, P. (1998). EMBO J 17, 4188–4198. Hesse, J. E., Lieber, M. R., Gellert, M., and Mizuuchi, K. (1987). Cell 49, 775–783. Hesse, J. E., Lieber, M. R., Mizuuchi, K., and Gellert, M. (1989). Genes Dev 3, 1053–1061.
80
Sekiguchi et al.
Hiom, K., and Gellert, M. (1997). Cell 88, 65–72. Hiom, K., and Gellert, M. (1998). Mol Cell 1, 1011–1019. Hiom, K., Melek, M., and Gellert, M. (1998). Cell 94, 463–470. Hozumi, N., and Tonegawa, S. (1976). Proc Natl Acad Sci U S A 73, 3628–3632. Hsieh, C. L., Arlett, C. F., and Lieber, M. R. (1993). J Biol Chem 268, 20105–20109. Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. C., Campisi, J., Kohwi-Shigematsu, T., and Chen, D. J. (2000). Genes Dev 14, 2807–2812. Huye, L. E., Purugganan, M. M., Jiang, M. M., and Roth, D. B. (2002). Mol Cell Biol 22, 3460–3473. Jackson, S. P. (2002). Carcinogenesis 23, 687–696. Jeggo, P., and O’Neill, P. (2002). DNA Repair (Amst) 1, 771–777. Jhappan, C., Morse, H. C. 3rd, Fleischmann, R. D., Gottesman, M. M., and Merlino, G. (1997). Nat Genet 17, 483–486. Jones, J. M., and Gellert, M. (2001). Proc Natl Acad Sci U S A 98, 12926–12931. Jones, J. M., and Gellert, M. (2002). EMBO J 21, 4162–4171. Jung, D., Bassing, C. H., Fugmann, S. D., Cheng, H. L., Schatz, D. G., and Alt, F. W. (2003). Immunity 18, 65–74. Junop, M. S., Modesti, M., Guarne, A., Ghirlando, R., Gellert, M., and Yang, W. (2000). EMBO J 19, 5962–5970. Kang, J., Bronson, R. T., and Xu, Y. (2002). EMBO J 21, 1447–1455. Karanjawala, Z. E., Adachi, N., Irvine, R. A., Oh, E. K., Shibata, D., Schwarz, K., Hsieh, C. L., and Lieber, M. R. (2002). DNA Repair (Amst) 1, 1017–1026. Khanna, K. K., and Jackson, S. P. (2001). Nat Genet 27, 247–254. Khanna, K. K., Lavin, M. F., Jackson, S. P., and Mulhern, T. D. (2001). Cell Death Differ 8, 1052–1065. Kim, D. R., Dai, Y., Mundy, C. L., Yang, W., and Oettinger, M. A. (1999). Genes Dev 13, 3070–3080. Kim, D. R., and Oettinger, M. A. (1998). Mol Cell Biol 18, 4679–4688. Kirch, S. A., Rathbun, G. A., and Oettinger, M. A. (1998). EMBO J 17, 4881–4886. Kirch, S. A., Sudarsanam, P., and Oettinger, M. A. (1996). Eur J Immunol 26, 886–891. Kirchgessner, C. U., Patil, C. K., Evans, J. W., Cuomo, C. A., Fried, L. M., Carter, T., Oettinger, M. A., and Brown, J. M. (1995). Science 267, 1178–1183. Komori, T., Okada, A., Stewart, V., and Alt, F. W. (1993). Science 261, 1171–1175. Komori, T., Pricop, L., Hatakeyama, A., Bona, C. A., and Alt, F. W. (1996). Int Rev Immunol 13, 317–325. Kurimasa, A., Ouyang, H., Dong, L. J., Wang, S., Li, X., Cordon-Cardo, C., Chen, D. J., and Li, G. C. (1999). Proc Natl Acad Sci U S A 96, 1403–1408. Kwon, J., Imbalzano, A. N., Matthews, A., and Oettinger, M. A. (1998). Mol Cell 2, 829–839. Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y., and Tonegawa, S. (1989). Cell 59, 859–870. Landree, M. A., Kale, S. B., and Roth, D. B. (2001). Mol Cell Biol 21, 4256–4264. Landree, M. A., Wibbenmeyer, J. A., and Roth, D. B. (1999). Genes Dev 13, 3059–3069. Lee, J., and Desiderio, S. (1999). Immunity 11, 771–781. Lee, S. E., Moore, J. K., Holmes, A., Umezu, K., Kolodner, R. D., and Haber, J. E. (1998). Cell 94, 399–409. Leu, T. M., Eastman, Q. M., and Schatz, D. G. (1997). Immunity 7, 303–314. Lewis, S., Gifford, A., and Baltimore, D. (1985). Science 228, 677–685. Lewis, S. M. (1994). Adv Immunol 56, 27–150. Lewis, S. M. (1994). Semin Immunol 6, 131–141. Lewis, S. M., Hesse, J. E., Mizuuchi, K., and Gellert, M. (1988). Cell 55, 1099–1107.
Li, G., Nelsen, C., and Hendrickson, E. A. (2002). Proc Natl Acad Sci U S A 99, 832–837. Li, G. C., Ouyang, H., Li, X., Nagasawa, H., Little, J. B., Chen, D. J., Ling, C. C., Fuks, Z., and Cordon-Cardo, C. (1998). Mol Cell 2, 1–8. Li, W., Swanson, P., and Desiderio, S. (1997). Mol Cell Biol 17, 6932–6939. Li, Z., Otevrel, T., Gao, Y., Cheng, H. L., Seed, B., Stamato, T. D., Taccioli, G. E., and Alt, F. W. (1995). Cell 83, 1079–1089. Liang, H. E., Hsu, L. Y., Cado, D., Cowell, L. G., Kelsoe, G., and Schlissel, M. S. (2002). Immunity 17, 639–651. Lieber, M. R. (1998). Am J Pathol 153, 1323–1332. Lim, D. S., Vogel, H., Willerford, D. M., Sands, A. T., Platt, K. A., and Hasty, P. (2000). Mol Cell Biol 20, 3772–3780. Lin, W. C., and Desiderio, S. (1995). Immunol Today 16, 279–289. Liyanage, M., Weaver, Z., Barlow, C., Coleman, A., Pankratz, D. G., Anderson, S., Wynshaw-Boris, A., and Ried, T. (2000). Blood 96, 1940–1946. Lu, L., and Osmond, D. G. (2000). Immunol Rev 175, 158–174. Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002). Cell 108, 781–794. Mages, G. J., Feldmann, H. M., and Winnacker, E. L. (1996). J Biol Chem 271, 7910–7915. Mahajan, K. N., Nick McElhinny, S. A., Mitchell, B. S., and Ramsden, D. A. (2002). Mol Cell Biol 22, 5194–5202. Malynn, B. A., Blackwell, T. K., Fulop, G. M., Rathbun, G. A., Furley, A. J. W., Ferrier, P., Heinke, L. B., Phillips, R. A., Yancopoulos, G. D., and Alt, F. W. (1988). Cell 54, 453–460. Manis, J. P., Dudley, D., Kaylor, L., and Alt, F. W. (2002). Immunity 16, 607–617. McBlane, J. F., van Gent, D. C., Ramsden, D. A., Romeo, C., Cuomo, C. A., Gellert, M., and Oettinger, M. A. (1995). Cell 83, 387–395. McCormack, W. T., Tjoelker, L. W., Carlson, L. M., Petryniak, B., Barth, C. F., Humphries, E. H., and Thompson, C. B. (1989). Cell 56, 785–791. Melek, M., Gellert, M., and van Gent, D. C. (1998). Science 280, 301– 303. Merkle, D., Douglas, P., Moorhead, G. B., Leonenko, Z., Yu, Y., Cramb, D., Bazett-Jones, D. P., and Lees-Miller, S. P. (2002). Biochemistry 41, 12706–12714. Messier, T. L., O’Neill, J. P., Hou, S. M., Nicklas, J. A., and Finette, B. A. (2003). EMBO J 22, 1381–1388. Miller, R. D., Hogg, J., Ozaki, J. H., Gell, D., Jackson, S. P., and Riblet, R. (1995). Proc Natl Acad Sci U S A 92, 10792–10795. Milne, G. T., Jin, S., Shannon, K. B., and Weaver, D. T. (1996). Mol Cell Biol 16, 4189–4198. Mimori, T., and Hardin, J. A. (1986). J Biol Chem 261, 10375–10379. Mimori, T., Hardin, J. A., and Steitz, J. A. (1986). J Biol Chem 261, 2274–2278. Mizuta, R., Mizuta, M., Araki, S., and Kitamura, D. (2002). J Biol Chem 277, 41423–41427. Mizuuchi, K., and Adzuma, K. (1991). Cell 66, 129–140. Mo, X., Bailin, T., and Sadofsky, M. J. (1999). J Biol Chem 274, 7025–7031. Modesti, M., Hesse, J. E., and Gellert, M. (1999). EMBO J 18, 2008–2018. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaioannou, V. E. (1992). Cell 68, 869–877. Moshous, D., Callebaut, I., de Chasseval, R., Corneo, B., Cavazzana-Calvo, M., Le Deist, F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A., and de Villartay, J. P. (2001). Cell 105, 177–186. Moshous, D., Li, L., Chasseval, R., Philippe, N., Jabado, N., Cowan, M. J., Fischer, A., and de Villartay, J. P. (2000). Hum Mol Genet 9, 583–588. Mundy, C. L., Patenge, N., Matthews, A. G., and Oettinger, M. A. (2002). Mol Cell Biol 22, 69–77. Nacht, M., Strasser, A., Chan, Y. R., Harris, A. W., Schlissel, M., Bronson, R. T., and Jacks, T. (1996). Genes Dev 10, 2055–2066.
5. The Mechanism of V(D)J Recombination Nadel, B., Tang, A., Escuro, G., Lugo, G., and Feeney, A. J. (1998). J Exp Med 187, 1495–1503. Nagawa, F., Ishiguro, K., Tsuboi, A., Yoshida, T., Ishikawa, A., Takemori, T., Otsuka, A. J., and Sakano, H. (1998). Mol Cell Biol 18, 655–663. Nagawa, F., Kodama, M., Nishihara, T., Ishiguro, K., and Sakano, H. (2002). Mol Cell Biol 22, 7217–7125. Nick McElhinny, S. A., Snowden, C. M., McCarville, J., and Ramsden, D. A. (2000). Mol Cell Biol 20, 2996–3003. Nicolas, N., Finnie, N. J., Cavazzana-Calvo, M., Papadopoulo, D., Le Deist, F., Fischer, A., Jackson, S. P., and de Villartay, J. P. (1996). Eur J Immunol 26, 1118–1122. Nicolas, N., Moshous, D., Cavazzana-Calvo, M., Papadopoulo, D., de Chasseval, R., Le Deist, F., Fischer, A., and de Villartay, J. P. (1998). J Exp Med 188, 627–634. Noordzij, J. G., Verkaik, N. S., Hartwig, N. G., de Groot, R., van Gent, D. C., and van Dongen, J. J. (2000). Blood 96, 203–209. Nussenzweig, A., Chen, C., da Costa Soares, V., Sanchez, M., Sokol, K., Nussenzweig, M. C., and Li, G. C. (1996). Nature 382, 551–555. Oettinger, M. A., Schatz, D. G., Gorka, C., and Baltimore, D. (1990). Science 248, 1517–1523. Okazaki, K., Davis, D. D., and Sakano, H. (1987). Cell 49, 477–485. Ouyang, H., Nussenzweig, A., Kurimasa, A., Soares, V. C., Li, X., CordonCardo, C., Li, W., Cheong, N., Nussenzweig, M., Iliakis, G., Chen, D. J., and Li, G. C. (1997). J Exp Med 186, 921–929. Patel, D. J., Shapiro, L., and Hare, D. (1987). In Unusual DNA structures, R. D. Wells and S. C. Harvey, eds. (New York: Springer), pp. 115–161. Peixoto, B. R., Mikawa, Y., and Brenner, S. (2000). Gene 246, 275–283. Petiniot, L. K., Weaver, Z., Barlow, C., Shen, R., Eckhaus, M., Steinberg, S. M., Ried, T., Wynshaw-Boris, A., and Hodes, R. J. (2000). Proc Natl Acad Sci U S A 97, 6664–6669. Povirk, L. F. (1996). Mutat Res 355, 71–89. Qiu, J. X., Kale, S. B., Yarnell Schultz, H., and Roth, D. B. (2001). Mol Cell 7, 77–87. Ramsden, D. A., and Gellert, M. (1995). Genes Dev 9, 2409–2420. Ramsden, D. A., and Gellert, M. (1998). EMBO J 17, 609–614. Ramsden, D. A., McBlane, J. F., van Gent, D. C., and Gellert, M. (1996). EMBO J 15, 3197–3206. Ramsden, D. A., Paull, T. T., and Gellert, M. (1997). Nature 388, 488–491. Ramsden, D. A., and Wu, G. E. (1991). Proc Natl Acad Sci U S A 88, 10721–10725. Rice, P., Craigie, R., and Davies, D. R. (1996). Curr Opin Struct Biol 6, 76–83. Rodgers, K. K., Bu, Z., Fleming, K. G., Schatz, D. G., Engelman, D. M., and Coleman, J. E. (1996). J Mol Biol 260, 70–84. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. (1998). J Biol Chem 273, 5858–5868. Roman, C. A., Cherry, S. R., and Baltimore, D. (1997). Immunity 7, 13–24. Rooney, S., Alt, F. W., Lombard, D., Whitlow, S., Eckersdorff, M., Fleming, J., Fugmann, S., Ferguson, D. O., Schatz, D. G., and Sekiguchi, J. (2003). J Exp Med 197, 553–565. Rooney, S., Sekiguchi, J., Zhu, C., Cheng, H. L., Manis, J., Whitlow, S., DeVido, J., Foy, D., Chaudhuri, J., Lombard, D., and Alt, F. W. (2002). Mol Cell 10, 1379–1390. Roth, D. B., Menetski, J. P., Nakajima, P. B., Bosma, M. J., and Gellert, M. (1992). Cell 70, 983–991. Roth, D. B., Zhu, C., and Gellert, M. (1993). Proc Natl Acad Sci U S A 90, 10788–10792. Sadofsky, M., Hesse, J. E., van Gent, D. C., and Gellert, M. (1995). Genes Dev 9, 2193–2199. Sadofsky, M. J., Hesse, J. E., and Gellert, M. (1994). Nucleic Acids Res 22, 1805–1809. Sadofsky, M. J., Hesse, J. E., McBlane, J. F., and Gellert, M. (1993). Nucleic Acids Res 21, 5644–5650. Samper, E., Goytisolo, F. A., Slijepcevic, P., van Buul, P. P., and Blasco, M. A. (2000). EMBO Rep 1, 244–252.
81
Santagata, S., Besmer, E., Villa, A., Bozzi, F., Allingham, J. S., Sobacchi, C., Haniford, D. B., Vezzoni, P., Nussenzweig, M. C., Pan, Z. Q., and Cortes, P. (1999). Mol Cell 4, 935–947. Santagata, S., Gomez, C. A., Sobacchi, C., Bozzi, F., Abinun, M., Pasic, S., Cortes, P., Vezzoni, P., and Villa, A. (2000). Proc Natl Acad Sci U S A 97, 14572–14577. Sarnovsky, R. J., May, E. W., and Craig, N. L. (1996). EMBO J 15, 6348–6361. Sawchuk, D. J., Weis-Garcia, F., Malik, S., Besmer, E., Bustin, M., Nussenzweig, M. C., and Cortes, P. (1997). J Exp Med 185, 2025–2032. Schar, P., Herrmann, G., Daly, G., and Lindahl, T. (1997). Genes Dev 11, 1912–1924. Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989). Cell 59, 1035–1048. Schlissel, M., Constantinescu, A., Morrow, T., Baxter, M., and Peng, A. (1993). Genes Dev 7, 2520–2532. Schwarz, K., Gauss, G. H., Ludwig, L., Pannicke, U., Li, Z., Lindner, D., Friedrich, W., Seger, R. A., Hansen-Hagge, T. E., Desiderio, S., Lieber, M. R., and Bartram, C. R. (1996). Science 274, 97–99. Sekiguchi, J., Ferguson, D. O., Chen, H. T., Yang, E. M., Earle, J., Frank, K., Whitlow, S., Gu, Y., Xu, Y., Nussenzweig, A., and Alt, F. W. (2001). Proc Natl Acad Sci U S A 98, 3243–3248. Sekiguchi, J. A., Whitlow, S., and Alt, F. W. (2001). Mol Cell 8, 1383–1390. Sekiguchi, J. M., Gao, Y., Gu, Y., Frank, K., Sun, Y., Chaudhuri, J., Zhu, C., Cheng, H.-L., Manis, J., Ferguson, D., Davidson, L., and Alt, F. W. (1999). Cold Spring Harb Symp Quant Biol 64, 169–181. Shiloh, Y. (2001). Biochem Soc Trans 29, 661–666. Shinkai, Y., Rathbun, G., Lam, K.-P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M., and Alt, F. W. (1992). Cell 68, 855–867. Shockett, P. E., and Schatz, D. G. (1999). Mol Cell Biol 19, 4159–4166. Sibanda, B. L., Critchlow, S. E., Begun, J., Pei, X. Y., Jackson, S. P., Blundell, T. L., and Pellegrini, L. (2001). Nat Struct Biol 8, 1015–1019. Siede, W., Friedl, A. A., Dianova, I., Eckardt-Schupp, F., and Friedberg, E. C. (1996). Genetics 142, 91–102. Silver, D. P., Spanopoulou, E., Mulligan, R. C., and Baltimore, D. (1993). Proc Natl Acad Sci U S A 90, 6100–6104. Sleckman, B. P., Bassing, C. H., Hughes, M. M., Okada, A., D’Auteuil, M., Wehrly, T. D., Woodman, B. B., Davidson, L., Chen, J., and Alt, F. W. (2000). Proc Natl Acad Sci U S A 97, 7975–7980. Smider, V., Rathmell, W. K., Lieber, M. R., and Chu, G. (1994). Science 266, 288–291. Smith, G. C., and Jackson, S. P. (1999). Genes Dev 13, 916–934. Steen, S. B., Gomelsky, L., and Roth, D. B. (1996). Genes Cells 1, 543–553. Steen, S. B., Han, J. O., Mundy, C., Oettinger, M. A., and Roth, D. B. (1999). Mol Cell Biol 19, 3010–3017. Swanson, P. C. (2002). Mol Cell Biol 22, 7790–801. Swanson, P. C., and Desiderio, S. (1998). Immunity 9, 115–125. Taccioli, G. E., and Alt, F. W. (1995). Curr Opin Immunol 7, 436–440. Taccioli, G. E., Amatucci, A. G., Beamish, H. J., Gell, D., Xiang, X. H., Torres Arzayus, M. I., Priestley, A., Jackson, S. P., Marshak Rothstein, A., Jeggo, P. A., and Herrera, V. L. (1998). Immunity 9, 355–366. Taccioli, G. E., Cheng, H. L., Varghese, A. J., Whitmore, G., and Alt, F. W. (1994). J Biol Chem 269, 7439–7442. Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., and Jeggo, P. A. (1994). Science 265, 1442–1445. Taccioli, G. E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P. A., and Alt, F. W. (1993). Science 260, 207–210. Takata, M., Sasaki, M. S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., Yamaguchi-Iwai, Y., Shinohara, A., and Takeda, S. (1998). EMBO J 17, 5497–5508. Teo, S. H., and Jackson, S. P. (1997). EMBO J 16, 4788–4795. Thomas, J. O., and Travers, A. A. (2001). Trends Biochem Sci 26, 167–174. Thompson, C. B. (1995). Immunity 3, 531–539.
82
Sekiguchi et al.
Thompson, L. H., and Schild, D. (2001). Mutat Res 477, 131–153. Tillman, R. E., Wooley, A. L., Khor, B., Wehrly, T. D., Little, C. A., and Sleckman, B. P. (2003). J Immunol 170, 5–9. Timsit, Y., Vilbois, E., and Moras, D. (1991). Nature 354, 167–170. Tonegawa, S. (1983). Nature 302, 575–581. Tsai, C. L., Drejer, A. H., and Schatz, D. G. (2002). Genes Dev 16, 1934–1949. Tsai, C. L., and Schatz, D. G. (2003). EMBO J. •• Tsai, C. L., and Schatz, D. G. (2003). EMBO J 22, 1922–1930. Tuteja, R., and Tuteja, N. (2000). Crit Rev Biochem Mol Biol 35, 1–33. van Gent, D. C., Hiom, K., Paull, T. T., and Gellert, M. (1997). EMBO J 16, 2265–2670. van Gent, D. C., McBlane, J. F., Ramsden, D. A., Sadofsky, M. J., Hesse, J. E., and Gellert, M. (1995). Cell 81, 925–934. van Gent, D. C., Mizuuchi, K., and Gellert, M. (1996). Science 271, 1592–1594. van Gent, D. C., Ramsden, D. A., and Gellert, M. (1996). Cell 85, 107–113. Vanasse, G. J., Halbrook, J., Thomas, S., Burgess, A., Hoekstra, M. F., Disteche, C. M., and Willerford, D. M. (1999). J Clin Invest 103, 1669–1675. Villa, A., Santagata, S., Bozzi, F., Giliani, S., Frattini, A., Imberti, L., Gatta, L. B., Ochs, H. D., Schwarz, K., Notarangelo, L. D., Vezzoni, P., and Spanopoulou, E. (1998). Cell 93, 885–896. Villa, A., Sobacchi, C., Notarangelo, L. D., Bozzi, F., Abinun, M., Abrahamsen, T. G., Arkwright, P. D., Baniyash, M., Brooks, E. G., Conley, M. E., Cortes, P., Duse, M., Fasth, A., Filipovich, A. M., Infante, A. J., Jones, A., Mazzolari, E., Muller, S. M., Pasic, S., Rechavi, G., Sacco, M. G., Santagata, S., Schroeder, M. L., Seger, R., Strina,
D., Ugazio, A., Valiaho, J., Vihinen, M., Vogler, L. B., Ochs, H., Vezzoni, P., Friedrich, W., and Schwarz, K. (2001). Blood 97, 81–88. Walker, J. R., Corpina, R. A., and Goldberg, J. (2001). Nature 412, 607–614. Ward, I. M., and Chen, J. (2001). J Biol Chem 276, 47759–47762. West, R. B., and Lieber, M. R. (1998). Mol Cell Biol 18, 6408–6415. Willett, C. E., Cherry, J. J., and Steiner, L. A. (1997). Immunogenetics 45, 394–404. Wilson, T. E., Grawunder, U., and Lieber, M. R. (1997). Nature 388, 495–498. Wilson, T. E., and Lieber, M. R. (1999). J Biol Chem 274, 23599–23609. Wu, C., Bassing, C. H., Jung, D., Woodman, B. B., Foy, D., and Alt, F. W. (2003). Immunity 18, 75–85. Xu, Y., Ashley, T., Brainerd, E. E., Bronson, R. T., Meyn, M. S., and Baltimore, D. (1996). Genes Dev 10, 2411–2422. Yarnell Schultz, H., Landree, M. A., Qiu, J. X., Kale, S. B., and Roth, D. B. (2001). Mol Cell 7, 65–75. Yeo, T. C., Xia, D., Hassouneh, S., Yang, X. O., Sabath, D. E., Sperling, K., Gatti, R. A., Concannon, P., and Willerford, D. M. (2000). Mol Immunol 37, 1131–1139. Yu, K., and Lieber, M. R. (2000). Mol Cell Biol 20, 7914–7921. Yurchenko, V., Xue, Z., and Sadofsky, M. (2003). Genes Dev 17, 581–585. Zachau, H. G. (1993). Gene 135, 167–173. Zhu, C., Bogue, M. A., Lim, D. S., Hasty, P., and Roth, D. B. (1996). Cell 86, 379–389. Zhu, C., Mills, K. D., Ferguson, D. O., Lee, C., Manis, J., Fleming, J., Gao, Y., Morton, C. C., and Alt, F. W. (2002). Cell 109, 811–821. Zhu, C., and Roth, D. B. (1995). Immunity 2, 101–112.
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6 Transcription of Immunoglobulin Genes KATHRYN CALAME
RANJAN SEN
Departments of Microbiology and Biochemistry & Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York. New York, USA
Department of Biology, Brandeis University, Waltham, Massachusetts, USA
Given the abundance of mRNA encoding immunoglobulin (Ig) light and heavy chains in murine plasmacytoma lines, immunoglobulin cDNAs and genes were among the first to be cloned in the late 1970s. The transcription of immunoglobulin genes quickly attracted the attention of many laboratories because of its strict B-cell specificity and developmental stage–specific regulation. In addition, with the understanding that functional Ig genes were created by a unique process of VDJ DNA rearrangement in B lymphocytes, the location and character of transcriptional regulatory elements was of particular interest. Ig gene transcriptional regulation became more intriguing with the discovery, in 1983, of the Ig heavy chain intronic enhancer (Em) (Banerji, Olson et al., 1983; Gillies, Morrison et al., 1983; Mercola, Wang et al., 1983). This enhancer, located between the JH and Cm gene segments, provided an explanation for transcriptional activation of rearranged VH gene promoters while unrearranged VH promoters remained inactive. Furthermore, Em was the first transcriptional enhancer identified in a mammalian gene and, like the SV40 enhancer, it could activate transcription in a distance- and orientationindependent manner. Indeed, it soon became obvious that most regulatory elements for both light and heavy chain immunoglobulin genes resided in enhancers that were either located in intervening sequences between J and C gene segments or 3¢ of C gene segments, or both. The molecular mechanism(s) by which these enhancers act has been, and continues to be, a central challenge for understanding Ig gene transcription. Both previous editions of this book contained chapters that summarized our understanding of immunoglobulin gene transcriptional regulation. At this time, most of the elements and DNA binding proteins involved are probably identified
and an updated summary of this information is presented. This chapter presents general characteristics of the Ig regulatory elements and discusses areas of current research. In addition, we discuss how, in a striking example of serendipity in science, research on Ig transcriptional regulation has provided unexpected insights into other aspect of immune system biology.
Molecular Biology of B Cells
TRANSCRIPTIONAL REGULATORY ELEMENTS IN IMMUNOGLOBULIN HEAVY AND LIGHT CHAIN GENES The regulatory elements in Ig genes have been described in previous editions of this volume and in many reviews (Leanderson and Hogbom, 1991; Li, Rothman et al., 1991; Staudt and Lenardo, 1991; Eckhardt, 1992; Kadesch, 1992; Ernst and Smale, 1995; Henderson and Calame, 1995; Henderson and Calame, 1998; Magor, Ross et al., 1999; Khamlichi, Pinaud et al., 2000). Our current understanding of these elements is summarized in Figures 6.1 and 6.2 and discussed below, followed by a discussion of the proteins that bind these elements.
Ig Promoters Each functional Ig V gene segment has a transcriptional initiation site, a TATA element, and regulatory sequences comprising a promoter extending approximately 100 to 200 bp 5¢ of the leader coding sequences. Both heavy and light chain gene promoters are remarkably simple (Figures 6.1 and 6.2). The most important regulatory element in both light and heavy chain promoters is an octamer element,
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Copyright 2004, Elsevier Science (USA). All rights reserved.
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FIGURE 6.1 Summary of transcriptional regulatory elements in the immunoglobulin heavy chain locus. Boxes indicate protein binding sites that are thought to be functional. Where known, the proteins that bind these sites are shown with activators in green and repressors or inhibitors of the activators in red. The arrow indicates the transcription initiation site. Distances are not to scale. See color insert.
which is usually located within 100 bp of the transcription initiation site. Initially, it was hypothesized that this element would confer B-cell specificity to V gene promoters; however, as discussed below, the roles of different B cellspecific and non-B cell-specific octamer proteins remain unclear, leaving the questions of oct-dependent B cell specificity unresolved. A few other regulatory elements (E, mE3) have been identified in VH and Vk promoters, but in functional assays their roles are less important than the oct sites (Avitahl and Calame, 1996). Indeed, it is interesting that C/EBP family proteins, which often bind VH and Vk pro-
moters, have recently been shown to interact with octamer proteins (Hatada, Chen-Kiang et al., 2000), underscoring the role of octamer protein for V promoters. Matrix attachment regions (MARs) have also been found 5¢ of many VH promoters (Goebel, Montalbano et al., 2002). Different VH promoters have been found to have different strengths and different degrees of enhancer dependence (Buchanan, Hodgetts et al., 1995; Love, Lugo et al., 2000). In general, however, the strong enhancer-dependence of V gene promoters in vivo renders the intrinsic activity of the promoters themselves less important for understanding Ig gene
6. Transcription of Immunoglobulin Genes
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FIGURE 6.2 Summary of transcriptional regulatory elements in the immunoglobulin light chain loci. Boxes indicate protein binding sites that are thought to be functional. Where known, the proteins that bind these sites are shown with activators in green and repressors or inhibitors of the activators in red. Parentheses indicate sites where proteins are presumed to bind but have not been shown experimentally. The arrow indicates the transcription initiation site. Distances are not to scale. See color insert.
expression, although it may be important for determining the accessibility of the V gene segments to recombinase activity (Sikes, Suarez et al., 1999).
Em The Em heavy chain intronic enhancer was the first Ig enhancer to be identified, and it has been extensively studied. It has strong, classical transcriptional enhancer activity that is promoter-, distance-, and orientation-
independent. Transgenes in which expression is dependent on Em show that the enhancer is active throughout B cell development from earliest pro B cells to plasma cells and it also has some activity in medullary thymocytes (Cook, Meyer et al., 1995). Multiple protein binding sites are present in Em, and experimental evidence indicates that those shown in Figure 6.1 are functionally important. The activity of many individual sites appears to be redundant with other sites, since mutation of individual sites usually has only a minor effect on activity whereas deletion of mul-
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tiple sites has significant impact. As detailed below, many sites in Em can be recognized by multiple proteins, some of which activate and some of which repress transcription, providing ample possibilities for subtle regulation. The “core” enhancer, which provides transcriptional activation in B cells, is flanked by two MARs that have been postulated to positively or negatively modulate its activity. As discussed below and elsewhere in this volume, a requirement for Em in VDJ recombination has been shown by gene targeting. However, there are no studies in which Em has been deleted from a rearranged heavy chain gene. This would assess its importance for transcription of a rearranged VH promoter in normal B cells, although there are cell lines that express Ig heavy chains normally from rearranged alleles that lack Em (Klein, Sablitzky et al., 1984; Wabl and Burrows, 1984).
Enhancers 3¢ of Ca Cell lines in which Em was deleted, but which continue to transcribe Ig heavy chains, provided the first indication that other enhancers might be present in the IgH locus. Indeed, it is now clear that a complex region 3¢ of Ca and more than 200 kb 3¢ of Em has enhancer activity (Figure 6.1). A recent review on this enhancer region provides a detailed summary of studies to determine its role and activity (Khamlichi, Pinaud et al., 2000). The region denoted HS1,2 is 16 kb 3¢ of Ca in the mouse IgH locus and was the first B-cell specific enhancer in this region to be identified. Subsequently, others, which are numbered based on the occurrence of DNaseI hypersensitive sites, were also identified. As indicated in Figure 6.1 the region contains several inverted repeats; HS3a and HS3b are 97% identical but in opposite orientations. Transfection studies suggested that HS1,2 had highest activity in activated B cells (Dariavach, Williams et al., 1991), and this was largely confirmed by transgene studies, although complete B-cell specificity was not observed (Arulampalam, Grant et al., 1994). HS3a and b and HS4 have weaker activity, primarily in activated B cells, and HS4 appears to be active throughout B cell development. The entire region displays locus control activity (Madisen and Groudine, 1994; Madisen, Krumm et al., 1998). Em and the 3¢ enhancers appear to synergize in a position- and distance-dependent manner (Mocikat, Kardinal et al., 1995), and the 3¢ enhancers probably function in vivo as co-enhancers. Mice with a targeted deletion of HS1,2, but retaining Em, had normal IgH transcription and selective defects in CH germline transcripts (Cogne, Lansford et al., 1994). Mice lacking the entire region 3¢ of Ca have not yet been described.
of these enhancers and has shown that they both play a role in VkJk recombination and that each has redundant and unique functions (Inlay, Alt et al., 2002). The intronic enhancer appears to be more important for secondary rearrangements that allow receptor editing (Nemazee, 2000) and for monoallelic demethylation that is required for ordered kappa rearrangement (Mostoslavsky, Singh et al., 2001). The developmental stage specificity of kappa gene rearrangement and expression was originally thought to be largely determined by the binding of NF-kB/rel family proteins in the kB site of the intronic enhancer. However, in vivo footprinting studies showed that the kB site was occupied in both pro and pre B cells; changes in occupancy of Cre, BSAP, and mB, NF-EM5 sites in the 3¢ enhancer, suggest these sites may be more important for the developmental stage-specific rearrangement and expression of kappa genes (Shaffer, Peng et al., 1997).
Lambda Enhancers Enhancers 3¢ of the constant gene segments have been found in lambda loci in mouse and human. Both the murine and human elements are illustrated in Figure 6.2 because the human element has been studied in some detail recently (Asenbauer, Combriato et al., 1999). Most protein binding sites in these enhancers are also found in other Ig enhancers, but a role for Mef proteins appears to be unique to the lambda enhancers (Satyaraj and Storb, 1998). A role for PU.1/IRF-4 in the murine lambda enhancer was evident in early studies (Pongubala, Nagulapalli et al., 1992; Eisenbeis, Singh et al., 1993) and provided a paradigm for understanding the activity of PU.1, in conjunction with other proteins, in many Ig enhancers.
PROTEINS BINDING IN IG TRANSCRIPTIONAL REGULATORY ELEMENTS Most proteins that bind to individual sites in the Ig promoters and enhancers have been identified and studied in detail. Since much of this basic information has been discussed in earlier editions of this volume and in other reviews, it has been summarized in Table 6.1 along with pertinent references. Below, we discuss some general features of the regulation and mechanism of action of proteins that bind sites in the Ig promoters and enhancers.
Kappa Intronic and 3¢ Enhancers
Ig Enhancer Activities Are Regulated by Multiple Sites and Mechanisms
In an arrangement similar to the IgH locus, the murine kappa locus has both an intronic and a 3¢ enhancer (Figure 6.2). Gene targeting has been used to compare the activities
A consistent finding has been that the Ig enhancers are complex elements and their B-cell and developmental stage specificity is not easily explained by a single B cell or devel-
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6. Transcription of Immunoglobulin Genes
TABLE 6.1 Protein
Sites
Activity
ATF/CREB
Cre
Pos.
Bright
MARs
CBF
Family bZip
Expression
Comments and references
Ubiquitous
Regulated by cAMP (De Cesare and Sassone-Corsi, 2000)
Pos.
B cells
Competes with Cux/CDP; related to HMG and Swi/Snf; may remodel chromatin (Webb, 2001)
Pos.
Ubiquitous
Associates with E2A and PU.1 (Erman, Cortes et al., 1998)
C/EBPb
E
Pos.
bZip
Wide
Induced by LPS and negatively regulated by heterodimerizing, short forms (LIP) and C/EBPg, (Lekstrom-Himes and Xanthopoulos, 1998)
Cux/CDP
MARs
Neg.
Hox
Non-B cells
Competes with Bright (Wang, Goldstein et al., 1999)
E2-A/HEB/ E2-2
mE5, mE2, kE2
Pos.
bHLH
Ubiquitous
E47 homodimera are found in B cells; negatively regulated by heterodimerizing Id proteins (Kee, Quong et al., 2000)
Ets family
mA
Pos.
Wide
Many family members; usually require association with other proteins for activity (Nikolajczyk, Sanchez et al., 1999)
Fos/Jun
AP1
Pos.
bZip
Wide
AP-1, often inducible with mitogens; negatively regulated by JunB (Shaulian and Karin, 2002)
IRF4
NF-EM5 lB
Pos.
IRF
Lymphoid, Plasma cells
Associates with PU.1 and E2-A proteins (Eisenbeis, Singh et al., 1995)
Maf/Bach2
MARE
Neg.
bZip/
Bach2 in early B cells
Heterodimer negatively regulates transcription (Muto, Hoshino et al., 1998)
Mef2
lA
Pos.
MADS
Ubiquitous
Large family, some important for myocytes (Satyaraj and Storb, 1998)
MiT
mE3, kE3
Pos.
bHLHzip
Wide
TFE3, TFEB or USF; homo- or heterodimerize; association of TFE3 with ets proteins; possible enhancer–promoter interactions (Rehli, Den Elzen et al., 1999)
NF-kB/rel
kB
Pos.
Rel
Wide
Heterodimers activate, IkB proteins regulate nuclear localization and respond to many signaling pathways (Li and Verma, 2002)
Oct1/2
Oct
Pos.
Pou/hox
Wide
Require association with the B-cell specific coactivator OCA-B (Matthias, 1998)
Pax5
BSAP
Pos/Neg.
B cells, not
Activity depends on gene context plasma cells (Nutt, Eberhard et al., 2001)
PU.1
mB, kB, lB
Pos.
Ets
B cells, Myeloid
Usually requires association with another protein such as IRF4 (Singh, Dekoter et al., 1999)
YY1
mE1, kE1
Pos./Neg
Zn finger
Ubiquitous
Recruits enzymes that modify histone acetylation; associates with many other proteins (Thomas and Seto, 1999)
ZEB
mE5
Neg.
Zn finger
Ubiquitous
Competes with E2A proteins for binding (Genetta, Ruezinsky et al., 1994)
opmental stage-specific protein. Some mechanisms contributing to the lineage and stage-specific activity of these elements are discussed below. Dimerizing Proteins with Different Partners Several families of transcriptional activators bind DNA as obligate dimers, providing the opportunity for shortened forms to act as dominant negative regulators by forming nonfunctional heterodimers. For example, the bHLH proteins encoded by E2-A, HEB, and E2–2 are negatively regulated by Id proteins, encoded by four genes (Id1–4) (Engel and Murre, 2001). The shorter HLH Id proteins lack both an activation domain and a basic region. Thus, Id/bHLH heterodimers fail to bind DNA and cannot activate transcription. Regulated expression of Id proteins is important for regulating E2-A, HEB, and E2–2 activity during B cell development (Sun, Copeland et al., 1991; Barndt and Zhuang, 1999; Becker-Herman, Lantner et al., 2002).
C/EBPb, an important activator of Ig promoters and enhancers, is a bZip protein that binds DNA as an obligate dimer. LIP, a shorter form of C/EBPb that lacks an activation domain, is generated by alternate translation initiation (Descombes and Schibler, 1991). A similar shorter form is also encoded by a separate gene, C/EBPg (Roman, Platero et al., 1990). Both short forms act as dominant negative inhibitors by forming DNA-binding heterodimers that cannot activate transcription. Both C/EBPb and the dominant negative short forms are regulated during B cell development, suggesting that activity of their binding sites is determined by both absolute and relative levels of these proteins. A similar situation has been described for the bHLHZip protein TFE3, wherein differential RNA splicing creates a truncated form that acts as a dominant negative in heterodimers with full-length proteins (Roman, Cohn et al., 1991). The Maf family of bZip proteins also contains both activating and short, nonfunctional forms. However, for this
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family an additional twist is present in B cells where a small, nonfunctional Maf protein heterodimerizes with a bZip protein called Bach2 that represses transcription (Muto, Hoshino et al., 1998). Bach2 has B-cell and neuron-specific expression; it is present in most B cells but absent in plasma cells and represses transcription by association with the corepressor SMRT. Different Proteins Binding to One Site Bright (B-cell-restricted regulator of IgH transcription) binds to the A/T rich sequences present in a subset of MARs, including those present in Em (Webb, Zong et al., 1999). Bright levels vary at different stages of B cell development and it is absent in plasma cells. Although it can activate transcription in some artificial settings, in vivo Bright may be important for chromatin remodeling early in B cell development. Interestingly, in non-B cells where Bright is absent, a negative regulator, Cux/CDP, binds to the Em MAR sites (Wang, Goldstein et al., 1999). Cux/CDP is absent in B cells, and it has been suggested that switching from Cux/CDP to Bright provides a MAR-mediated switch for Em activity. bHLH proteins encoded by the E2-A, HEB, and E2–2 genes bind sites in Ig enhancers (Figures 6.1 and 6.2). A “two-handed” zinc finger protein called ZEB has also been shown to bind the E5 site in Em in non-B cells (Genetta, Ruezinsky et al., 1994). ZEB is a transcriptional repressor that is ubiquitously expressed. The reason bHLH activator proteins overcome ZEB repression in B cells is intriguing, but incompletely understood at present. Octamer Proteins The striking conservation of oct sites in both heavy chain and light chain V gene promoters and the presence of oct sites in Em and heavy chain 3¢ enhancers, suggested that oct binding proteins might be critical for B-cell specificity of Ig transcription. However, despite extensive studies, the roles of oct sites and the proteins that bind them remains murky (Matthias 1998; Bertolino, Tiedt et al., 2000). Two octbinding proteins are present in B cells: Oct-1, which is ubiquitously expressed, and Oct-2, restricted to lymphoid and central nervous sytems cells. Both proteins interact with a B-cell specific co-activator, OCA-B, suggesting a way in which oct-dependent activation could be B-cell specific. However, B cell development and IgM secretion are normal in mice lacking OCA-B, whereas expression of secondary isotypes and the entry of B cells into peripheral pools is defective (Kim, Qin et al., 1996; Nielsen, Georgiev et al., 1996). Thus, at a minimum, OCA-B does not confer nonredundant B-cell-specific regulation on Ig transcription in vivo. It is also possible that another B-cell specific coactivator, like OCA-B, may exist. Oct sites are most important functional elements in V gene promoters and altered speci-
ficity mutants (Shah, Bertolino et al., 1997), and knock-out mice (Schubart, Massa et al., 2001) suggest that Oct-1 is more important in this context than Oct-2. Lineage and Stage Specificity of Ig Enhancers via Regulation of PU.1 and Pax5 PU.1 is an ets family protein, expressed in hematopoietic cells, which appears to be important for the B-cell specificity of Em (Nelsen, Tian et al., 1993; Shaffer, Peng et al., 1997). PU.1 also binds to kappa and lambda enhancers (Figure 6.2) and in the kappa 3¢ enhancer occupation of the PU.1 site, detected by in vivo footprinting, correlates with pre B cell, but not pro-B cell activity of the enhancer (Shaffer, Peng et al., 1997). Pax5, also called B cell lineage specific activator protein (BSAP), has a B-cell specific expression pattern but is not present in plasma cells (Nutt, Eberhard et al., 2001). Pax5, along with YY1, is a transcriptional regulator that can either activate or repress transcription, depending on the gene context of its binding site. In the enhancers HS1,2 and HS 4 3¢ of Ca, and in the 3¢ kappa enhancer, Pax5 appears to repress enhancer activity. Thus, it is likely that the decreased expression of Pax5 in plasma cells is important for the high activity of these enhancers in terminally differentiated B cells.
Cooperative Interactions Are Important for the Activity of Many DNA Binding Proteins That Regulate Ig Transcription Our current understanding of transcriptional activators that bind in promoter regions near the start of transcription is that they recruit, either directly or via co-activators and/or chromatin remodeling machines, components of the basal transcription machinery to form a stable transcription initiation complex. Ig promoters, however, are very simple and most transcriptional regulatory elements in Ig genes reside in enhancers (Figure 6.1 and 6.2). The molecular mechanism(s) by which these elements activate transcription from distances of several kilobases remains an intriguing puzzle. The question is further complicated by the complexity of most Ig enhancers. Many protein binding sites exist and complicated patterns of both functional redundancy and functional cooperativity have been observed in transfection studies. Ets Family Proteins and Their Partners PU.1 is an ets family protein that preferentially binds mB or lB sites in Em, the 3¢ kappa enhancer and the lambda enhancers (Figure 6.1 and 6.2). In the lB and 3¢ kappa enhancer sites, PU.1 associates with a lymphoid-restricted
6. Transcription of Immunoglobulin Genes
IRF family protein, IRF4, and activates transcription (Eisenbeis, Singh et al., 1995). Further study indicates that PU.1 in this context may play an architectural role in recruiting IRF4, which actively promotes transcription (Pongubala and Atchison, 1997). In Em, a tripartite region containing mA, mE3, and mB is sufficient to activate transcription in B cells (Nelsen, Tian et al., 1993; Nikolajczyk, Cortes et al., 1997), and the spacing of these three sites is important for their activity, due at least in part to the ability of PU.1 to bend DNA (Nikolajczyk, Nelsen et al., 1996). The Mi-T bHLHZip protein TFE3 cooperates with PU.1 and Ets-1 to activate transcription dependent on this region (Tian, Erman et al., 1999). Similar to the situation in light chain enhancers, the transactivation domain of PU.1 is not important for cooperative transcriptional activation (Erman and Sen, 1996). Other Enhancer-Binding Proteins and Cooperativity Many other examples of cooperative interactions among Ig transcription factors have been reported. IRF4 interacts with the E2-A proteins E12 and E47 to activate transcription from the 3¢ kappa enhancer (Nagulapalli and Atchison, 1998). C/EBPb associates with Oct1 and Oct2 in solution and forms a ternary complex on Ig heavy chain and kappa promoters, implying functional cooperativity (Hatada, Chen-Kiang et al., 2000). Functional synergy has been shown for the bHLHZip protein TFE3, binding in Em at the mE3 site with both Ets-1 and bHLH protein E47 (Nikolajczyk, Dang et al., 1999).
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transgene expression in single cells revealed that, in fact, enhancers do not increase the rate of transcription initiation, but instead increase the probability that transcription will initiate at a given promoter without affecting the rate of transcription initiation (Walters, Fiering et al., 1995; Fiering, Whitelaw et al., 2000). For enhancers in Ig genes it is important to remember that in addition to activating the transcription of V gene promoters, they are also required for VDJ recombination. Although it is not clear if the same DNA-binding proteins and the same mechanism(s) are involved in their effects on transcription and their effects on DNA recombination, it is reasonable to assume some or most may be in common. Tracking How do enhancers increase the probability of transcription initiation when they are often located significant distances from their target promoters? One model suggests some form of a tracking mechanism, by which activator proteins are recruited to enhancers and then track along the DNA until they encounter a promoter, at which point they act to facilitate transcription initiation. This idea is consistent with, but not proven by, the finding that some DNA elements (called insulators) can block enhancer activity when placed between an enhancer and a promoter (Felsenfeld, Boyes et al., 1996). However, neither the mechanism of insulators nor enhancers are known, and they may or may not involve protein tracking on DNA. Looping
AREAS OF CURRENT RESEARCH Mechanism(s) of Enhancer Action In all the Ig loci, transcriptional enhancers located several kilobases 3¢ to the V gene promoters activate the promoters and are critical for regulated gene expression. In spite of much effort, we still do not understand the molecular mechanism(s) by which enhancers in the Ig loci, or in any mammalian gene, actually work. Indeed, because there is little consensus on what an enhancer does, we discuss below some of the possibilities that have been considered. It is likely that enhancers associated with different loci will incorporate one or more of these possible mechanisms depending on the regulatory requirements of the locus. Probability of Transcription Initiation Enhancers increase the amount of transcription initiation at target promoters, and it was originally believed that they did this by increasing the rate of transcription initiation at each promoter. However, analysis of enhancer-dependent
Alternatively, looping models show that proteins bound at enhancers directly associate with proteins bound at the promoter to facilitate transcription initiation. Since in vivo enhancer-dependent transcriptional activation occurs in the context of chromatin, this “looping” of DNA may actually involve or depend on the remodeling of chromatin. Consistent with this idea, in Escherichia coli enhancer activity requires DNA supercoiling (Liu, Bondarenko et al., 2001), and in in vitro transcription reactions using mammalian genes, enhancer-dependent transcription requires a chromatinized template (Barton and Emerson, 1994). Past studies on Ig genes have addressed the issue of enhancer–promoter interactions using transfection assays, and enhancers have robust activity in these assays. However, it is important to remember the limitations of such systems, especially since chromatin structure and nuclear sublocalization may not be faithfully recapitulated in these systems. Several proteins have binding sites in both VH promoters and Em and, when isolated protein binding sites were tested for their ability to activate transcription from a distance, TFE3, but not C/EBPb or octamer proteins were able to mediate activation (Artandi, Cooper et al., 1994). This finding sug-
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gested that the self-association of bHLHZip dimers might be important for enhancer–promoter interactions. Similarly, more recent studies have shown that Bright, which binds to MARs associated with both VH promoters and Em, can selfassociate and might mediate long-range enhancer–promoter interactions in vivo (Webb, Zong et al., 1999). Enhancer–promoter interactions may also involve associations between different proteins bound at the enhancer and the promoter, although to date there is no direct evidence for this. However, distant enhancers cannot activate transcription in the absence of at least one proximal transcriptional activator and a recent study has explored this observation in the context of an Ig core promoter. The data show that a truncated octamer protein, containing only the POU domain, is sufficient to mediate activation through a distant enhancer (Bertolino, Tiedt et al., 2000). The POU domain binds DNA and recruits TBP to the TATA box. The ability of this minimal protein to mediate enhancer activity suggests that initial recruitment of TFIIB is both independent of the enhancer and required for enhancer activity. Subsequently, RNAPolII/mediator complexes recruited to the distant enhancer, may, via looping, mediate the assembly of the complete transcription initiation complex. The nature of protein–protein associations involved in the enhancer– promoter interaction are not elucidated in this paper, but the data open the possibility of associations between enhancer–bound proteins with TFIIB as well as activators bound at the promoter. Chromatin Structure Another model for enhancer activity, which is not mutually exclusive with either tracking or looping mechanisms, is that enhancers block gene silencing by preventing the localization of a gene to centromeric heterochromatin (Francastel, Walters et al., 1999). Many proteins that bind in Ig enhancers are “classical” transcription factors that often bind in the promoter elements of other genes, and few features distinguish proteins that act proximally versus those that act distally. However, two Ig enhancer binding proteins have a unique activity that may have important implications for enhancer activity. The bHLH E2-A proteins and the ets family protein PU.1, when overexpressed in T cells, are capable of inducing Em activity, evidenced by sterile mu
transcripts (Choi, Shen et al., 1996; Nikolajczyk, Sanchez et al., 1999). This has been interpreted as indicating that these proteins are capable of binding to nucleosomal DNA and activating silent chromatin, although overexpression experiments may not replicate in vivo conditions and could be misleading. In the context of models in which enhancers act by changing chromatin structure or by facilitating the localization of the gene to particular regions within the nucleus and thus affecting chromatin structure, it is important to consider the activity of the matrix attachment regions (MARs) that are associated with many Ig enhancers. In Em, the MARs appear to be important for extending chromatin activity for transcription over long distances (Jenuwein, Forrester et al., 1997), and their activity includes blocking DNA methylation and extending the domains of histone acetylation (Forrester, Fernandez et al., 1999; Fernandez, Winkler et al., 2001). Interestingly, the MARs do not appear to be required for Em to activate VDJ recombination (Sakai, Bottaro et al., 1999), suggesting differences in the mechanism of action of Em in transcription and VDJ recombination. Certainly chromatin structure and subnuclear localization are likely to be important for enhancer activity. However, the activity of enhancer elements in transient transfection assays, in which the chromatin structure of the transfected DNA only partially resembles endogenous chromatin and in which nuclear localization is unlikely to recapitulate that of endogenous loci, suggest that these features may not be entirely responsible for enhancer activity. The challenge for future experiments will be to develop assays in which enhancer activity can be systematically dissected in a context wherein the genes are in their physiological context with respect to location on the chromosome, subnuclear localization of the chromosome, and chromatin structure. Activation of the IgH Locus for Rearrangement and Transcription In its germline (unrearranged) state, the immunoglobulin heavy chain gene locus spans approximately 2.5 to 3 Mb close to the telomere of the short arm of murine chromosome 12 (Chevillard, Ozaki et al., 2002) (Figure 6.3). Approximately 1.5 to 2 Mb of this comprises multiple VH gene segments, the sixteen DH gene segments are spread
FIGURE 6.3 Schematic representation of the IgH locus including VH gene segments and all heavy chain isotypes, showing some approximate distances, not drawn to scale, on the top. Although some VHJ558 genes are interspersed with other families, the VHJ558 family is the most DH-distal and VH 7183 family is the most DH-proximal VH gene family. DFL16.1 and Dq52 are the 5¢- and 3¢- most DH gene segments, respectively.
6. Transcription of Immunoglobulin Genes
over 40 kb, and the JH/Cm/Cd region extends another 10 to 15 kb. This part of the locus is activated during antigenindependent B cell differentiation in the bone marrow. The first gamma isotypes lie approximately 50 kb 3¢ of Cd, followed by the other isotypes spread over 100 kb, which culminate in Ca. Activation of the IgH locus for rearrangement and expression has been and continues to be studied extensively, serving as a paradigm for understanding the activation of all Ig loci. Gene Rearrangement and Transcription Transcription of unrearranged (germline) Ig gene segments precedes both VDJ recombination and class switch recombination (CSR), suggesting that transcription and/or transcriptional control elements play a role in regulating these two critical DNA rearrangements. Deletion of Em inhibits VDJ recombination, with a greater effect on VDJ than DJ recombination (Serwe and Sablitzky, 1993). Surprisingly, only the core of Em is necessary, and the MARs are dispensable (Sakai, Bottaro et al., 1999). In the kappa locus, both the intronic and 3¢ enhancers are important for VJ recombination (Inlay, Alt et al., 2002). Both Em (Sakai, Bottaro et al., 1999) and the enhancers 3¢ of Ca (Cogne, Lansford et al., 1994) appear to be important for CSR. Understanding how these transcriptional control elements function to control DNA recombination is an area of intensely active study. Since the molecular mechanisms responsible for VDJ recombination and CSR are discussed in detail elsewhere in this volume, we will not detail these studies here. However, models for enhancers’ role in these DNA rearrangements include: 1) altering chromatin structure to make gene segments more accessible to recombinase machinery, 2) activating transcription which, either via the process itself or via the mRNA produced, is required for the process of recombination, or 3) recruiting proteins directly involved in recombination. Activation of the DH-Cm Region Acetylation of lysine residues at the N-termini of histones H3 and H4 has recently emerged as a marker of activated regions of the genome (Workman and Kingston, 1998). Genes that are transcriptionally active, or those that are poised to be transcribed, are associated with acetylated histones and can be assayed by immunoprecipitating DNA/protein complexes using antimodified histone antibodies and scoring for the gene of interest by the polymerase chain reaction (PCR). Analysis of the unrearranged IgH locus by this assay shows that the locus is activated in discrete, independently regulated steps during B cell differentiation. The first domain of hyperacetylation is approximately 90 to 100 kb and includes all the DH gene segments, the JH gene segments, and the Cm exons. It is
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likely that DH to JH recombination is initiated within this domain (Chowdhury and Sen, 2001). Because the VH regions are hypoacetylated at this stage, and therefore inactive by the criteria described above, these observations also provide a simple explanation for DH to JH recombination preceding VH to DJH recombination. There are several additional features of this domain. First, only two closely located DNase 1 hypersensitive sites have been identified within this region (Chowdhury and Sen, 2001). One marks the IgH m intron enhancer and the second a region, close to the 3¢ most DH gene segment, Dq52. The latter probably marks a sterile promoter. The region between the two hypersensitive sites contains the four JH gene segments and is immunoprecipitated more efficiently with antiacetylated histone antibodies than flanking sequences. These observations suggest that the JH cluster is contained within a microdomain of increased histone acetylation, which may play a role in targeting the V(D)J recombinase to this part of the locus. Interestingly, the JH gene segments are located asymmetrically within the 90 kb domain; the 3¢ end of the domain ends abruptly within 10 kb 3¢ of the JHs between the Cm and Cd exons, whereas the domain extends at least 60 kb 5¢ to include even the most distal DH gene segment, DFL16.1. It is possible that the short 3¢ extension minimizes abortive scanning of the genome 3¢ to JHs by the recombinase, where no other recombinogenic gene segments exist. The nature of the domain boundary between Cm and Cd is unclear. The few boundary elements and insulators that are known are marked by DNase1 hypersensitive sites. However, no hypersensitive site exists between Cm and Cd, suggesting that this boundary may be generated by a different mechanism. The Em has been shown to activate V(D)J recombination in engineered substrates. Yet deletion of the enhancer has no effect on DH to JH recombination (Sakai, Bottaro et al., 1999), although VH to DJH recombination is severely diminished. The identification of a second Dq52 hypersensitive site suggests that this element may provide the requisite recombinational enhancer activity, in the absence of Em, to allow DH to JH recombination. This region has also been deleted from the genome (Nitschke, Kestler et al., 2001). Unlike Em, however, this mutation permits both DH to JH as well as VH to DJH recombination. Thus, while each element may substitute for the other to activate DH to JH recombination, Em is uniquely essential for the second step of IgH gene assembly. All DH gene segments are not marked by a proximal hypersensitive site. No such sites were found in 10 kb spanning DFL16.1 and DSP2.2 gene segments. Because these gene segments recombine efficiently, it is unlikely that a closely associated hypersensitive site is required for recombination. However, it cannot be ruled out that there are other regulatory sequences within the DH region that contribute to activating the 90 kb domain.
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VH Gene Activation Histone acetylation and other studies suggest that the VH locus contains at least two independently regulated domains (Chowdhury and Sen, 2001; Johnson, Angelin-Duclos et al., 2003). The largest region comprises the telomere-proximal VHJ558 and VH3609 gene families that are activated by interleukin-7 (IL-7) in adult pro-B cells. Both H3 and H4 are acetylated; however, the acetylation is limited to about 1 kb surrounding each gene segment and does not extend into the intergenic regions (Johnson, Angelin-Duclos et al., 2003). These genes make up more than half of all VH genes. The DH/Cm-proximal VH genes, including 7183, S107, VGAM, V10, and SM7 families, are not highly acetylated in adult pro-B cells prior to DJH recombination, but become hyperacetylated in cells that contain DJH joins (Chowdhury and Sen, 2001). Although the connection is correlative, this observation raises the interesting possibility that DJH recombination itself may trigger the next step of IgH recombination. An obvious, but untested, mechanism could be that DJH joining brings the 3¢ VH genes closer to, and therefore under control of, Cm-proximal regulatory sequences such as Em (the Dq52 element being deleted by any DJH recombination other than Dq52 itself). This region of low acetylation extends approximately halfway into the VH locus and includes some DH-proximal J558 genes. A characteristic feature of IgH gene assembly, which is most obvious during fetal development, is that 3¢ VH genes, such as VH7183 family members, recombine preferentially in early B cell ontogeny (Yancopoulos, Desiderio et al., 1984; Jeong and Teale, 1989; Malynn, Yancopoulos et al., 1990; ten Boekel, Melchers et al., 1997). In the fetal liver, these genes, as well as the more DH-distal VH genes, are associated with acetylated H4 following culture in IL-7, but prior to DJH recombination (Johnson, Angelin-Duclos et al., 2003). These observations highlight a basic difference in the mechanisms that activate VH genes in the fetus versus the adult. Interestingly, the absence of Pax 5 also causes a complete loss of VH to DJH recombination in the fetal liver, but only a loss of JH-distal VH gene recombination in the adult (Nutt, 1997; Hesslein, Pflugh et al., 2003). This also makes the case for differences in VH gene regulation in fetal and adult ontogeny. However, histone acetylation of proximal and distal VH genes is similar in Pax 5-/- and normal mice, suggesting that Pax 5 may regulate VH to DJH recombination at a step other than altering the state of histone acetylation (Hesslein, Pflugh et al., 2003). Differential activation of segments of the VH locus provides insight into the basis for ordered rearrangements in adult developing B cells. Two factors may contribute to the overall outcome. First, proximal VH genes may be activated early in response to DJH recombination, as suggested above. In addition, the differential IL-7 sensitivity of developing pro-B cells may delay activation of the large cluster of VHJ558 genes. Early pro-B cells express low levels of the
IL-7 receptor a chain and are generally less responsive to IL-7 (Marshall et al., 1998). As a result of weak IL-7 signaling, the distal VHJ558 genes may not be effectively activated early to compete with the proximal genes for recombination. The net result is that proximal VH genes recombine early and the distal genes recombine later. Thus, preferential rearrangement of VH7183 family is the result of independent control of different parts of the VH locus and the complex pattern of IL-7 sensitivity of developing B cells. However, much work remains to fully understand the mechanisms and signals that differentially control histone acetylation and VH gene rearrangements. Nuclear Sublocalization Developmentally regulated changes in IgH locus chromatin structure are accompanied by alterations in the nuclear organization of the locus. Three kinds of changes have been noted. In non-B lineage cells, such as thymocytes or ES cells, both IgH alleles are located close to the nuclear periphery (Kosak, Skok et al., 2002). In pro-B cells IgH alleles were found to be more centrally located in the nucleus and away from centromeric DNA regardless of the rearrangement status. Centromeric heterochromatin has been implicated in keeping genes turned off, and these observations are consistent with both alleles being simultaneously active for transcription and recombination. The state of the locus is not permanent, however, because one allele co-localizes with centromeric DNA in mature splenic B cells activated to enter the cell cycle (Skok, Brown et al., 2001). Singh and colleagues also made the intriguing observation that in-situ hybridization signals from two ends of the VH locus were closer together in T cell nuclei (where it is peripherally located) compared to pro-B cell nuclei; they suggested that the decreased compaction in pro-B cells may reflect some aspect of recombination control or may facilitate VH to DJH rearrangements (Kosak, Skok et al., 2002). Third, a correlation has been noted between replication pattern of the IgH locus and its nuclear location (Zhou, Ermakova et al., 2002). Pre- and pro-B cell lines that replicate IgH early in the S phase localize this locus centrally in the nucleus, whereas mature B and non-B cells that follow a triphasic replication pattern localize the IgH locus to the nuclear periphery. Matrix Attachment Regions The core of the m heavy chain gene enhancer is flanked by matrix attachment regions (MARs). These A/T-rich sequences have been implicated in positive and negative regulation of Ig expression. Evidence for negative regulation stems from the observation that the tissue-range in which the m enhancer is active in transfection assays is increased if the flanking MARs are missing (Weinberger, Jat et al., 1988; Scheuermann and Chen, 1989). Conversely,
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expression of a functionally rearranged IgH transgene is significantly increased when MARs are included in the normal location flanking the enhancer (Forrester et al., 1994). Based on these studies, Grosschedl and colleagues have proposed that MARs help to propagate enhancer effects over long distances (Jenuwein et al., 1997; Fernandez et al., 2001). Recently, the IgH intron MARs have also been individually, or jointly, deleted from the genome. However, MAR deletion does not alter VDJ recombination or IgH gene expression from the altered allele (Sakai et al., 1999). The apparent discrepancy between transgenic and endogenous locus studies is best reconciled by considering that other MARs within the locus may compensate for the loss of the intronic MARs. If so, an interesting corollary is that position within the locus is not important for MAR function. Another MAR binding protein, SATB1, was identified based on its ability to bind A/T-rich sequnces that unwound easily upon torsional stress (also referred to as base unpairing regions, BURs) (Dickinson, Joh et al., 1992). SATB1 DNA binding in vitro is significantly diminished if the propensity to unwind DNA is weakened by appropriately placed mutations. Since many MARs contain BURs, the possible role of MAR binding proteins in stabilizing alternate DNA conformations should not be overlooked. In addition, both BRIGHT and SATB1 have features that underscore their importance in chromatin structure. For example, the BRIGHT DNA binding domain is similar to that found in SWI1 (a component of the chromatin remodeling complex SWI/SNF) and SATB1 is complexed to histone de-acetylases and nuclear co-repressors in cells. Genetic deletion of SATB1 inhibits T cell development and alters the structure of the interleukin-2 receptor alpha chain gene (Yasui, Miyano et al., 2002). Understanding how classical enhancers and MARs coordinately regulate gene expression remains a challenge for the future.
DISCOVERIES RESULTING FROM THE STUDY OF IG GENE TRANSCRIPTION Since studies on Ig gene transcriptional regulation were initiated early, when little was understood regarding mammalian gene regulation, and since Em was the first mammalian transcriptional enhancer to be identified, studies in this field have not only illuminated our understanding of Ig gene regulation, but have also established paradigms for understanding general mechanisms of transcriptional regulation. In addition, several regulatory proteins first identified and studied for their roles in Ig gene regulation upon further study have been found to play critical roles in the immune system unrelated to their regulation of Ig genes. Two of the most important “additional” discoveries are discussed below.
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Certain Ig Transcriptional Regulators Are Critical for Early Stages of Hematopoietic Cell Development The ets Family Protein PU.1 in Early Hematopoiesis Gene targeting studies originally revealed a requirement for PU.1 in the development of both myeloid and lymphoid lineages (Scott et al., 1994). Interestingly, in in vitro reconstitution studies, graded levels of PU.1 differentially regulate macrophage versus B cell differentiation, with higher levels being associated with macrophage development (DeKoter and Singh, 2000). PU.1 also appears important for mast cell and dendritic cell development (Singh et al., 1999; Anderson et al., 2000).
Pax5 and Commitment to the B Cell Lineage and B Cell Development Pax5 is an interesting transcription factor that can either activate or repress transcription, depending on gene context (Wallin et al., 1998). In hematopoietic cells, its expression is limited to the B lymphoid lineage. Cells lacking Pax5 are not committed to the B lineage (Nutt et al., 1999) and recently Pax5 has been shown to inhibit the Notch pathway, required for T cell commitment (Souabni et al., 2002). Thus, Pax5 has a unique role in lineage commitment. Roles for Pax5 also have been demonstrated during early B cell development and in the germinal center (Nutt et al., 2001). Like some other transcription factors, Pax5 is shut down during plasma cell differentiation, thus relieving the repression of genes such as J chain and XBP-1 that are expressed in Ig secreting cells (Schebesta et al., 2002).
E2-A Proteins in Early B Cell Development B cell development is arrested at the pro-B stage in mice lacking the E2A gene (Barndt and Zhuang, 1999; Kee et al., 2000). Furthermore, E12, encoded by E2-A, induces early B cell factor (EBF) (Kee and Murre, 1998), and together the EBF and E2-A gene products synergize in early B cell development (O’Riordan and Grosschedl, 1999).
NF-kB/rel Proteins Are Important in Many Immune and Inflammatory Processes NF-kB/rel proteins were first discovered because of their binding to the kB site in the Igk intronic enhancer. However, it soon became obvious that this family of transcriptional regulators plays an important role in many other immune cells, as well as in B cells. NF-kB/rel proteins exist in inactive forms in the cytoplasm of most cells and, in response to a wide variety of signals, they rapidly become activated and enter the nucleus to affect expression of target genes.
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These proteins are important in a wide range of diseases ranging from inflammation to cancer (Yamamoto and Gaynor, 2001; Li and Verma, 2002). They are critical for inflammatory responses and have an anti-apoptotic role in both normal and malignant cells. They are critical for normal splenic architecture, B cell survival and B cell-dependent immune responses (Caamano, Rizzo et al., 1998; Franzoso, Carlson et al., 1998), dendritic cell survival and differentiation (Ouaaz, Arron et al., 2002), and T cell survival following activation (Wang, Guttridge et al., 1999). The family of Rel homology domain (RHD)-containing proteins consists of p105/NFkB1 (the precursor to p50), p110/NFk2 (the precursor to p52), p65/RelA, c-Rel, and RelB. Most family members can homo- or heterodimerize to produce transcription factors that recognize the DNA sequence GGG(A/T)4CCC, often referred to as a kB element (for recent more comprehensive reviews see Chen and Ghosh, 1999; Li and Verma, 2002). Of the various proteins, the term NF-kB usually refers to the p50/p65 heterodimer, which is usually the most abundant form of the factor detected by electrophoretic mobility shift assays. The approximately 300-amino acid RHD is sufficient for dimerization, DNA binding, nuclear localization, and association with a family of regulatory proteins called inhibitors of NFkB (IkB) (Ghosh and Karin, 2002). X-ray crystallographic structures of several Rel proteins reveals a novel DNA binding motif utilizing loops that protrude from more defined secondary structures (Chen, Huang et al., 1998; Chen, Ghosh et al., 1998; Huxford, Huang et al., 1998; Jacobs and Harrison, 1998; Huang, Chen et al., 2001). Cocrystals of Rel/IkBa complexes show extensive contacts between the subunits and conformational alteration of the N-terminus of the RHD that contains most of the DNA binding residues. Structural similarity of the RHDs is reflected in their recognizing closely related DNA sequences, such that it is virtually impossible to ascertain the functional Rel protein from the sequence of the kB-like element in a gene. However, mice deficient in Rel genes show different phenotypes, indicating that even very similar Rel genes serve distinct functions in-vivo (Beg, Sha et al., 1995; Sha, Liou et al., 1995; Weih, Carrasco et al., 1995; Kontgen, Grumont et al., 1995). Other than p50-/p65-mice, analyses of double-deficient Rel mice are ongoing (Grumont, Rourke et al., 1998; Grossmann, Metcalf et al., 1999; Gugasyan, Grumont et al., 2000; Grumont, 1998; Pohl, Gugasyan et al., 2002). Regulation by Subcellular Localization via IkB Proteins In most cells, Rel/IkB interactions were proposed to sequester the complex in the cytoplasm by hiding the nuclear localization sequence (NLS) of the Rel protein. That the mechanism was more complex was first revealed by the
crystallographic structure of the p65/IkBa complex in which the p65 NLS was not obscured by IkBa. Furthermore, because IkB proteins did not interact with p50 or p52, heterodimers containing these subunits would be expected to have at least one available NLS for nuclear import. Recent studies show that cytoplasmic localization by IkB proteins is a dynamic process. In particular, IkBa contains a very strong nuclear export sequence (NES) located in the Nterminus of the protein (Johnson, Van Antwerp et al., 1999; Huang, Kudo et al., 2000; Tam, Lee et al., 2000). This NES interacts with the nuclear export receptor CRM1, which directs IkBa and any associated proteins out of the nucleus. That IkBa-associated proteins are in constant flux is best visualized by treating cells with the drug leptomycin B (LMB), which blocks CRM1-dependent export. In LMBtreated cells, IkBa and associated Rel proteins accumulate in the nucleus. Similar results are observed in yeast with exogenously introduced p65 and IkBa proteins in a strain with a hypomorphic mutation in the yeast crm1 gene (Tam, Lee et al., 2000). Finally, increased nuclear distribution of Rel/IkBa complexes is observed when the IkBa NES is mutated (Johnson, Van Antwerp et al., 1999; Huang, Kudo et al., 2000; Tam, Lee et al., 2000). Taken together, the combined genetic and pharmacological experiments suggest that Rel/IkBa complexes are continuously shuttling between the nucleus and the cytoplasm. The net cytosolic location observed in earlier studies is therefore the result of nuclear export dominating over nuclear import; an imbalance in the import–export equilibrium, such as that created by LMB, results in the net subcellular redistribution of the complexes. This mechanism of cytoplasmic localization also provides a ready explanation for the availability of functional NLSs in Rel/IkBa complexes. In contrast to IkBa, IkBb and IkBe do not contain strong NESs and also interact more closely with Rel proteins in the vicinity of the NLS (Malek, Chen et al., 2001; Tam and Sen, 2001). Thus, these molecules probably truly sequester Rel proteins in the cytoplasm, as envisaged earlier for all IkBs. One of the benefits of the dynamic mechanism may be that the same properties of IkBa that mediate cytosolic localization in unactivated cells can also be used to restore cells to a resting state after termination of an activating signal. NF-kB induction by diverse stimuli leads to IkBa gene transcription and new protein synthesis. The newly synthesized IkBa can enter the nucleus (Chiao, Miyamoto et al., 1994; Arenzana-Seisdedos, Thompson et al., 1995) either by passive diffusion or aided by a nonclassical NLS (Sachdev, Hoffmann et al., 1998), disrupt Rel/DNA complexes and export Rel/IkBa complexes out to the cytoplasm to await retriggering by another signal. Indeed, continued signals result in cyclical Rel protein expression in the nucleus due to the dynamics of retrieval and reinduction in the cytoplasm (Hoffmann, Levchenko et al., 2002). It is unclear whether transit of IkBa-containing complexes through the nucleus
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serves additional biological function. An untested intriguing possibility remains that nuclear kinases or phosphatases may participate in NF-kB regulation, for example in response to nuclear inducing signals such as DNA double strand breaks. In this regard, it is noteworthy that DNA-dependent protein kinase (DNA-PK) has been shown to phosphorylate IkBa in vitro (Liu, Kwak et al., 1998), and NF-kB activation is diminished in ATM-deficient mouse embryo fibroblasts (Li, Banin et al., 2001). Signaling to Activate NF-kB/rel Proteins Only B lymphocytes contain nuclear Rel proteins that bind DNA (that is, are not complexed to IkBs) prior to any activating signal. This activity consists largely of p50/c-Rel heterodimers and lower levels of p50–p65 heterodimers (Liou, Sha et al., 1994; Miyamoto, Schmitt et al., 1994). Increased IkBa turnover has been proposed as the basis for constitutive nuclear NF-kB in B cells (Miyamoto, Chiao et al., 1994). However, the mechanism of IkBa turnover remains unclear. Miyamoto and colleagues have shown that constitutive IkBa degradation is insensitive to proteasome inhibitors and may be mediated by calpainlike proteases (Fields, Seufzer et al., 2000; Shen, Channavajhala et al., 2001). Further studies are required to identify the features of IkBa that target it for increased basal turnover in B cells. The dominance of nuclear p50 and c-Rel heterodimers has been proposed to be due to inefficient export of these complexes from the nucleus (Tam, Wang et al., 2001). This model is based on two observations: that p65/RelA contains an NES in its C-terminal domain, and that c-Rel/IkBa complexes are only found in B cells. Enhanced IkBa degradation in B cells thus creates nuclear pools of both p65 and c-Rel containing homo- and heterodimers. However, the p65 NES leads to more efficient export of p65-containing complexes, with the result that c-Rel containing complexes, accumulate in the nucleus. The central feature of the NF-kB family is its inducible activation to a nuclear DNA binding form by multiple signals. This occurs by signal-induced phosphorylation of IkB proteins at two conserved serine residues within the Nterminal domain. This domain is sometimes also referred to as the signal receptor domain. Phosphorylation of IkBs marks them for proteasome-mediated degradation. Released from the inhibitory influence of IkB proteins, DNA-binding Rel dimers translocate to the nucleus to activate gene expression. IkB phosphorylation is mediated by a heterotrimeric IkB kinase (IKK) complex that consists of IKKa, b, and g (Karin and Ben-Neriah, 2000; Karin and Delhase, 2000; Ghosh and Karin, 2002). IKKa and b are catalytic subunits that homo- or heterodimerize via leucine zipper–containing dimerization domains. Either kinase can phosphorylate IkBa in vitro at the appropriate residues, though IKKb usually appears to be the more active kinase.
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Indeed, a complex consisting of catalytically inactive IKKb with an active IKKa fails to activate NF-kB in response to most pro-inflammatory stimuli; in contrast, a complex of catalytically inactive IKKa with normal IKKb is functional under most conditions. Recently it has been shown that IKKa may play an essential role in NF-kB activation mediated by the cytokines RANKL and Blys in B cells (Cao, Bonizzi et al., 2001; Schiemann, Gommerman et al., 2001; Thompson, Bixler et al., 2001). No catalytic activity has been attributed to IKKg; rather, it is believed to serve as a scaffold that targets IKKa/b to the right substrates. However, IKKg is essential for NF-kB induction (Makris, Godfrey et al., 2000; Schmidt-Supprian, Bloch et al., 2000) and small peptides that disrupt IKKg interactions with IKKa/b inhibit NF-kB activation (May, D’Acquisto et al., 2000). Mutations in IKKg have also been implicated in human immunodeficiencies (Smahi, Courtois et al., 2000; Courtois, Smahi et al., 2001; Jain, Ma et al., 2001). Although the central importance of the IKK complex is well established, it is less clear how diverse stimuli converge at IKK. Catalytic activity of IKKa and b is induced by phosphorylation of two conserved serine residues in an activation loop present in each protein. Consequently, several “upstream” kinases have been implicated in IKK activation, although the physiological relevance of many of these remains to be established. Gene knock-out studies have verified the importance of two other kinases for NF-kB activation. The kinase RIP lies in the TNFR1 signaling pathway, and protein kinase C theta is necessary for NF-kB induction via the T cell receptor (Kelliher, Grimm et al., 1998; Sun, Arendt et al., 2000). Interestingly, catalytically inactive RIP can restore NF-kB activation, suggesting that it may play the role of an adapter (Hsu, Huang et al., 1996). Similar function has been attributed to another kinase, PKR, which is required for NF-kB induction by double-stranded RNA (Bonnet, Weil et al., 2000). Recently the CARDdomain–containing proteins Bcl10 and CARD11 have been shown to be essential for NF-kB induction by B- and T-cell antigen receptors (Gaide, Favier et al., 2002; Pomerantz, Denny et al., 2002; Wang, You et al., 2002). The connection of these (nonkinase) proteins to PKC theta or the IKK complex remains to be determined. The cytoplasm is likely to be the site of IkB phosphorylation since the IKK resides here. However, phospho-IkBs must be recognized by the bTrCP/SCF complex (Yaron, Hatzubai et al., 1998), which ubiquitinates IkB at a lysine residue also located in the N-terminal signal receptor domain (Spencer, Jiang et al., 1999; Winston, Strack et al., 1999). Poly-ubiquitinated IkB then is a target for the proteasome. In an intriguing twist to the compartmentalization problem of components involved in NF-kB activation, bTrCP/SCF has been shown to be predominantly nuclear (Davis, Hatzubai et al., 2002). If IkB phosphorylation only occurs in the cytoplasm, these observations suggest that
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phosphorylated IkB must translocate to the nucleus to find bTrCP/SCF. This is feasible for IkBa complexes because they shuttle through the nucleus, but more difficult to imagine for other IkBs. Other possibilities are that there may be a small but functionally relevant amount of bTrCP/SCF in the cytoplasm, or that this protein may also shuttle through the nucleus by a presently undefined pathway. Overall, all the emerging evidence serves to underscore the dynamic state of NF-kB regulation in resting as well as activated cells.
CONCLUSION The transcriptional regulation of immunoglobulin genes has been actively studied for more than twenty years. As reviewed in this chapter, we now have a good understanding of most transcriptional regulatory elements in these genes and appreciate the key roles of various enhancers. Families of DNA-binding transcriptional regulators, binding to individual sites in the promoter and enhancer elements, have also been identified and their mechanisms of action defined at least in vitro. In addition, as described above, these studies have illuminated transcription factors that play important roles in the early development of hematopoietic and lymphoid cells and in many other aspects of immune function. However, important questions remain. The most pressing is that the mechanism(s) by which enhancers activate transcription is still unknown. In the Ig loci, these enhancers also play a role in allowing DNA rearrangements; how this occurs and how it may relate to transcriptional activation also remains unknown. Related questions involve the relationship(s) between transcription, DNA rearrangement, and DNA replication, as well as the role of subnuclear localization in determining the activity of a gene. Recent advances in studying chromatin structure and how it may be regulated by histone modification and remodeling machines, chromatin immunoprecipitations to monitor the association in vivo of particular proteins with DNA sequences, and the ability to track the subnuclear localization and replication times of particular genes may help us finally unravel the remaining secrets of immunoglobulin gene regulation.
References Anderson, K. L., Perkin, H., et al. (2000). Transcription factor PU.1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells. J Immunol 164(4), 1855–1861. Arenzana-Seisdedos, F., Thompson, J., et al. (1995). Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B. Mol Cell Biol 15(5), 2689–2696. Artandi, S. E., Cooper, C., et al. (1994). The basic helix-loop-helix-zipper domain of TFE3 mediates enhancer-promoter interaction. Mol Cell Biol 14(12), 7704–7716.
Arulampalam, V., Grant, P. A., et al. (1994). Lipopolysaccharide-dependent transactivation of the temporally regulated immunoglobulin heavy chain 3¢ enhancer. Eur J Immunol 24(7), 1671–1677. Asenbauer, H., Combriato, G., et al. (1999). The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription. Eur J Immunol 29(2), 713–724. Avitahl, N., and Calame, K. (1996). A 125 bp region of the Ig VH1 promoter is sufficient to confer lymphocyte-specific expression in transgenic mice. Int Immunol 8(9), 1359–1366. Banerji, J., Olson, L., et al. (1983). A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33(3), 729–740. Barndt, R. J., and Zhuang, Y. (1999). Controlling lymphopoiesis with a combinatorial E-protein code. Cold Spring Harb Symp Quant Biol 64, 45–50. Barton, M. C., and Emerson, B. M. (1994). Regulated expression of the beta-globin gene locus in synthetic nuclei. Genes Dev 8(20), 2453–2465. Becker-Herman, S., Lantner, F., et al. (2002). Id2 negatively regulates B cell differentiation in the spleen. J Immunol 168(11), 5507–5513. Beg, A. A., Sha, W. C., et al. (1995). Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376(6536), 167–170. Bertolino, E., Tiedt, R., et al. (2000). Role of octamer transcription factors and their coactivators in the lymphoid system. In Transcription factors: Normal and malignant development of blood cells. K. Ravid and J. Licht, eds. New York: Wiley, 2000. Bonnet, M. C., Weil, R., et al. (2000). PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex. Mol Cell Biol 20(13), 4532–4542. Buchanan, K. L., Hodgetts, S. I., et al. (1995). Differential transcription efficiency of two Ig VH promoters in vitro. J Immunol 155(9), 4270–4277. Caamano, J. H., Rizzo, C. A., et al. (1998). Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cellmediated immune responses. J Exp Med 187(2), 185–196. Cao, Y., Bonizzi, G., et al. (2001). IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 107(6), 763–775. Chen, F. E., and Ghoshm, G. (1999). Regulation of DNA binding by Rel/NF-kappaB transcription factors: structural views. Oncogene 18(49), 6845–6852. Chen, F. E., Huang, D. B., et al. (1998). Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature 391(6665), 410–413. Chen, Y. Q., Ghosh, S., et al. (1998). A novel DNA recognition mode by the NF-kappa B p65 homodimer. Nat Struct Biol 5(1), 67–73. Chevillard, C., Ozaki, J., et al. (2002). A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus. J Immunol 168(11), 5659–66. Chiao, P. J., Miyamoto, S., et al. (1994). Autoregulation of I kappa B alpha activity. Proc Natl Acad Sci U S A 91(1), 28–32. Choi, J. K., Shen, C. P., et al. (1996). E47 activates the Ig-heavy chain and TdT loci in non-B cells. EMBO J 15(18), 5014–5021. Chowdhury, D., and Sen, R. (2001). Stepwise activation of the immunoglobulin mu heavy chain gene locus. EMBO J 20(22), 6394–6403. Cogne, M., Lansford, R., et al. (1994). A class switch control region at the 3¢ end of the immunoglobulin heavy chain locus. Cell 77(5), 737–747. Cook, G. P., Meyer, K. B., et al. (1995). Regulated activity of the IgH intron enhancer (E mu) in the T lymphocyte lineage. Int Immunol 7(1), 89–95. Courtois, G., Smahi, A., et al. (2001). NEMO/IKK gamma: Linking NFkappa B to human disease. Trends Mol Med 7(10), 427–430. Dariavach, P., Williams, G. T., et al. (1991). The mouse IgH 3’-enhancer. Eur J Immunol 21(6), 1499–1504.
6. Transcription of Immunoglobulin Genes Davis, M., Hatzubai, A., et al. (2002). Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U. Genes Dev 16(4), 439–451. De Cesare, D., and Sassone-Corsi, P. (2000). Transcriptional regulation by cyclic AMP-responsive factors. Prog Nucleic Acid Res Mol Biol 64, 343–369. DeKoter, R. P., and Singh, H. (2000). Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288(5470), 1439–1441. Descombes, P., and Schibler, U. (1991). A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67, 569–579. Dickinson, L. A., Joh, T., et al. (1992). A tissue-specific MAR/SAR DNAbinding protein with unusual binding site recognition. Cell 70(4), 631–645. Eckhardt, L. A. (1992). Immunoglobulin gene expression only in the right cells at the right time. FASEB J 6(8), 2553–2560. Eisenbeis, C. F., Singh, H., et al. (1993). PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2–4 enhancer. Mol Cell Biol 13(10), 6452–6461. Eisenbeis, C. F., Singh, H., et al. (1995). Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev 9(11), 1377–1387. Engel, I., and Murre, C. (2001). The function of E- and Id proteins in lymphocyte development. Nat Rev Immunol 1(3), 193–199. Erman, B., Cortes, M., et al. (1998). ETS-core binding factor: A common composite motif in antigen receptor gene enhancers. Mol Cell Biol 18(3), 1322–1330. Erman, B., and Sen, R. (1996). Context dependent transactivation domains activate the immunoglobulin mu heavy chain gene enhancer. EMBO J 15(17), 4565–4575. Ernst, P., and Smale, S. T. (1995). Combinatorial regulation of transcription II: The immunoglobulin mu heavy chain gene. Immunity 2(5), 427–438. Felsenfeld, G., Boyes, J., et al. (1996). Chromatin structure and gene expression. Proc Natl Acad Sci U S A 93(18), 9384–9388. Fernandez, L. A., Winkler, M., et al. (2001). Matrix attachment regiondependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy. Mol Cell Biol 21(1), 196–208. Fields, E. R., Seufzer, B. J., et al. (2000). A switch in distinct I kappa B alpha degradation mechanisms mediates constitutive NF-kappa B activation in mature B cells. J Immunol 164(9), 4762–4767. Fiering, S., Whitelaw, E., et al. (2000). To be or not to be active: The stochastic nature of enhancer action. Bioessays 22(4), 381–387. Forrester, W. C., Fernandez, L. A., et al. (1999). Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer-promoter interactions. Genes Dev 13(22), 3003–3014. Forrester, W. C., van Genderen, C., et al. (1994). Dependence of enhancermediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions. Science 265(5176), 1221–1225. Francastel, C., Walters, M. C., et al. (1999). A functional enhancer suppresses silencing of a transgene and prevents its localization close to centrometric heterochromatin. Cell 99(3), 259–269. Franzoso, G., Carlson, L., et al. (1998). Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J Exp Med 187(2), 147–159. Gaide, O., Favier, B., et al. (2002). CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation. Nat Immunol 3(9), 836–843. Genetta, T., Ruezinsky, D., et al. (1994). Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell
97
specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol 14(9), 6153–6163. Ghosh, S., and Karin, M. (2002). Missing pieces in the NF-kappaB puzzle. Cell 109 Suppl, S81–96. Gillies, S. D., Morrison, S. L., et al. (1983). A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33(3), 717–728. Goebel, P., Montalbano, A., et al. (2002). High frequency of matrix attachment regions and cut-like protein x/CCAAT-displacement protein and B cell regulator of IgH transcription binding sites flanking Ig V region genes. J Immunol 169(5), 2477–2487. Grossmann, M., Metcalf, D., et al. (1999). The combined absence of the transcription factors Rel and RelA leads to multiple hemopoietic cell defects. Proc Natl Acad Sci U S A 96(21), 11848–11853. Grumont, R. J., Rourke, I. J., et al. (1998). B lymphocytes differentially use the Rel and nuclear factor kappaB1 (NF-kappaB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells. J Exp Med 187(5), 663–674. Gugasyan, R., Grumont, R., et al. (2000). Rel/NF-kappaB transcription factors: key mediators of B-cell activation. Immunol Rev 176, 134– 140. Hatada, E. N., Chen-Kiang, S., et al. (2000). Interaction and functional interference of C/EBPbeta with octamer factors in immunoglobulin gene transcription. Eur J Immunol 30(1), 174–184. Henderson, A., and Calame, K. (1998). Transcriptional regulation during B cell development. Annu Rev Immunol 16, 163–200. Henderson, A. J., and Calame, K. L. (1995). Lessons in transcriptional regulation learned from studies on immunoglobulin genes. Crit Rev Eukaryot Gene Expr 5(3–4), 255–280. Hesslein, D. G., Pflugh, D. L., et al. (2003). Pax5 is required for recombination of transcribed, acetylated, 5¢ IgH V gene segments. Genes Dev 17(1). Hoffmann, A., Levchenko, A., et al. (2002). The IkappaB-NF-kappaB signaling module: Temporal control and selective gene activation. Science 298(5596), 1241–1245. Hsu, H., Huang, J., et al. (1996). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4(4), 387–396. Huang, D. B., Chen., Y. Q., et al. (2001). X-ray crystal structure of protooncogene product c-Rel bound to the CD28 response element of IL-2. Structure (Camb) 9(8), 669–678. Huang, T. T., Kudo, N., et al. (2000). A nuclear export signal in the Nterminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc Natl Acad Sci U S A 97(3), 1014–1019. Huxford, T., Huang, D. B., et al. (1998). The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 95(6), 759–770. Inlay, M., Alt., F. W., et al. (2002). Essential roles of the kappa light chain intronic enhancer and 3¢ enhancer in kappa rearrangement and demethylation. Nat Immunol 3(5), 463–468. Jacobs, M. D., and Harrison, S. C. (1998). Structure of an IkappaBalpha/NF-kappaB complex. Cell 95(6), 749–758. Jain, A., Ma, C. A., et al. (2001). Specific missense mutations in NEMO result in hyper-IgM syndrome with hypohydrotic ectodermal dysplasia. Nat Immunol 2(3), 223–228. Jenuwein, T., Forrester, W., et al. (1997). Extension of chromatin accessibility by nuclear matrix attachment regions. Nature 385, 269–272. Jenuwein, T., Forrester, W. C., et al. (1997). Extension of chromatin accessibility by nuclear matrix attachment regions. Nature 385(6613), 269–272. Jeong, H. D., and Teale, J. M. (1989). VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets. J Immunol 143(8), 2752–2760.
98
Calame and Genes
Johnson, C., Van Antwerp, D., et al. (1999). An N-terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IkappaBalpha. EMBO J 18(23), 6682–6693. Johnson, K., Angelin-Duclos, C., et al. (2003). Changes in histone acetylation are associated with differences in accessibility of VH gene segments to V-DJ recombination during B-cell ontogeny and development. Mol Cell Biol. In press. Kadesch, T. (1992). Helix-loop-helix proteins in the regulation of immunoglobulin gene transcription. Immunol Today 13(1), 316. Karin, M., and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: The control of NF-[kappa]B activity. Annu Rev Immunol 18, 621–663. Karin, M., and Delhase, N. (2000). The I kappa B kinase (IKK) and NFkappa B: key elements of proinflammatory signalling. Semin Immunol 12(1), 85–98. Kee, B. L., and Murre, C. (1998). Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12. J Exp Med 188(4), 699–713. Kee, B. L., Quong, M. W., et al. (2000). E2A proteins: Essential regulators at multiple stages of B-cell development. Immunol Rev 175, 138–149. Kelliher, M. A., Grimm, S., et al. (1998). The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8(3), 297–303. Khamlichi, A. A., Pinaud, E., et al. (2000). The 3¢ IgH regulatory region: a complex structure in a search for a function. Adv Immunol 75, 317–345. Kim, U., Qin, X. F., et al. (1996). The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes. Nature 383(6600), 542–547. Klein, S., Sablitzky, F., et al. (1984). Deletion of the IgH enhancer does not reduce immunoglobulin heavy chain production of a hybridoma IgD class switch variant. EMBO J 3(11), 2473–2476. Kontgen, F., Grumont, R. J., et al. (1995). Mice lacking the c-rel protooncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev 9(16), 1965–1977. Kosak, S. T., Skok, J. A., et al. (2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296(5565), 158–162. Leanderson, T., and Hogbom, E. (1991). Regulation of immunoglobulin transcription. Exp Clin Immunogenet 8(2), 75–84. Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998). Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273(44), 28545–28548. Li, N., Banin, S., et al. (2001). ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks. J Biol Chem 276(12), 8898–8903. Li, Q., and Verma, I. M. (2002). NF-kappaB regulation in the immune system. Nat Rev Immunol 2(10), 725–734. Li, S. C., Rothman, P., et al. (1991). Control of immunoglobulin heavy chain constant region gene expression. Adv Exp Med Biol 292, 245–251. Liou, H. C., Sha, W. C., et al. (1994). Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation. Mol Cell Biol 14(8), 5349–5359. Liu, L., Kwak, Y. T., et al. (1998). DNA-dependent protein kinase phosphorylation of IkappaB alpha and IkappaB beta regulates NF-kappaB DNA binding properties. Mol Cell Biol 18(7), 4221–4234. Liu, Y., Bondarenko, V., et al. (2001). DNA supercoiling allows enhancer action over a large distance. Proc Natl Acad Sci U S A 98(26), 14883–14888. Love, V. A., Lugo, G., et al. (2000). Individual V(H) promoters vary in strength, but the frequency of rearrangement of those V(H) genes does not correlate with promoter strength nor enhancer-independence. Mol Immunol 37(12), 29–39. Madisen, L., and Groudine, M. (1994). Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc
expression in plasmacytoma and Burkitt’s lymphoma cells. Genes Dev 8(18), 2212–2226. Madisen, L., Krumm, A., et al. (1998). The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes. Mol Cell Biol 18(11), 6281–6292. Magor, B. G., Ross, D. A., et al. (1999). Transcriptional enhancers and the evolution of the IgH locus. Immunol Today 20(1), 13–17. Makris, C., Godfrey, V. L., et al. (2000). Female mice heterozygous for IKK gamma/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 5(6), 969–979. Malek, S., Chen, Y., et al. (2001). IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells. J Biol Chem 276(48), 45225–45235. Malynn, B. A., Yancopoulos, G. D., et al. (1990). Biased expression of JHproximal VH genes occurs in the newly generated repertoire of neonatal and adult mice. J Exp Med 171(3), 843–859. Marshall, A. J., Fleming, H. E., et al. (1998). Modulation of the IL-7 doseresponse threshold during pro-B cell differentiation is dependent on preB cell receptor expression. J Immunol 161(11), 6038–6045. Matthias, P. (1998). Lymphoid-specific transcription mediated by the conserved octamer site: Who is doing what? Semin Immunol 10(2), 155–163. May, M. J., D’Acquisto, F., et al. (2000). Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science 289(5484), 1550–1554. Mercola, M., Wang, X. F., et al. (1983). Transcriptional enhancer elements in the mouse immunoglobulin heavy chain locus. Science 221(4611), 663–665. Miyamoto, S., Chiao, P. J., et al. (1994). Enhanced I kappa B alpha degradation is responsible for constitutive NF-kappa B activity in mature murine B-cell lines. Mol Cell Biol 14(5), 3276–3282. Miyamoto, S., Schmitt, M. J., et al. (1994). Qualitative changes in the subunit composition of kappa B-binding complexes during murine Bcell differentiation. Proc Natl Acad Sci U S A 91(11), 5056–5060. Mocikat, R., Kardinal, C., et al. (1995). Differential interactions between the immunoglobulin heavy chain mu intron and 3¢ enhancer. Eur J Immunol 25(11), 3195–3198. Mostoslavsky, R., Singh, N., et al. (2001). Asynchronous replication and allelic exclusion in the immune system. Nature 414(6860), 221–225. Muto, A., Hoshino, H., et al. (1998). Identification of Bach2 as a B-cellspecific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3¢ enhancer. EMBO J 17(19), 5734–5743. Nagulapalli, S., and Atchison, M. L. (1998). Transcription factor Pip can enhance DNA binding by E47, leading to transcriptional synergy involving multiple protein domains. Mol Cell Biol 18(8), 4639– 4650. Nelsen, B., Tian, G., et al. (1993). Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins. Science 261(5117), 82–86. Nemazee, D. (2000). Receptor editing in B cells. Adv Immunol 74, 89–126. Nielsen, P. J., Georgiev, O., et al. (1996). B lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1. Eur J Immunol 26(12), 3214–3218. Nikolajczyk, B. S., Cortes, M., et al. (1997). Combinatorial determinants of tissue-specific transcription in B cells and macrophages. Mol Cell Biol 17(7), 3527–3535. Nikolajczyk, B. S., Dang, W., et al. (1999). Mechanisms of mu enhancer regulation in B lymphocytes. Cold Spring Harb Symp Quant Biol 64, 99–107. Nikolajczyk, B. S., Nelsen, B., et al. (1996). Precise alignment of sites required for mu enhancer activation in B cells. Mol Cell Biol 16(8), 4544–4554.
6. Transcription of Immunoglobulin Genes Nikolajczyk, B. S., Sanchez, J. A., et al. (1999). ETS protein-dependent accessibility changes at the immunoglobulin mu heavy chain enhancer. Immunity 11(1), 11–20. Nitschke, L., Kestler, J., et al. (2001). Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage. J Immunol 166(4), 2540– 2552. Nutt, S. L., Eberhard, D., et al. (2001). Pax5 determines the identity of B cells from the beginning to the end of B-lymphopoiesis. Int Rev Immunol 20(1), 65–82. Nutt, S. L., Heavey, B., et al. (1999). Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401(6753), 556–562. Nutt S. L, Rolink, U. P., Rolink A, Busslinger M. (1997). Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoies and reduced V-to-DJ recombination at the IgH locus. Genes Dev 11(4), 476–491. O’Riordan, M., and Grosschedl, R. (1999). Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 11(1), 21–31. Ouaaz, F., Arron, J., et al. (2002). Dendritic cell development and survival require distinct NF-kappaB subunits. Immunity 16(2), 257– 270. Pohl, T., Gugasyan, R., et al. (2002). The combined absence of NF-kappa B1 and c-Rel reveals that overlapping roles for these transcription factors in the B cell lineage are restricted to the activation and function of mature cells. Proc Natl Acad Sci U S A 99(7), 4514– 4519. Pomerantz, J. L., Denny, E. M., et al. (2002). CARD11 mediates factorspecific activation of NF-kappaB by the T cell receptor complex. EMBO J 21(19), 5184–5194. Pongubala, J. M., and Atchison, M. L. (1997). PU.1 can participate in an active enhancer complex without its transcriptional activation domain. Proc Natl Acad Sci U S A 94(1), 127–132. Pongubala, J. M., Nagulapalli, S., et al. (1992). PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3¢ enhancer activity. Mol Cell Biol 12(1), 368–378. Rehli, M., Den Elzen, N., et al. (1999). Cloning and characterization of the murine genes for bHLH-ZIP transcription factors TFEC and TFEB reveal a common gene organization for all MiT subfamily members. Genomics 56(1), 111–120. Roman, C., Cohn, L., et al. (1991). A dominant negative form of transcription activator mTFE3 created by differential splicing. Science 254, 94–97. Roman, C., Platero, J. S., et al. (1990). Ig/EBP-1: A ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev 4, 1404–1415. Sachdev, S., Hoffmann, A., et al. (1998). Nuclear localization of IkappaB alpha is mediated by the second ankyrin repeat: the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences. Mol Cell Biol 18(5), 2524–2534. Sakai, E., Bottaro, A., et al. (1999). The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination. Int Immunol 11(10), 1709–1713. Sakai, E., Bottaro, A., et al. (1999). Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions. Proc Natl Acad Sci U S A 96(4), 1526–1531. Satyaraj, E., and Storb, U. (1998). Mef2 proteins, required for muscle differentiation, bind an essential site in the Ig lambda enhancer. J Immunol 161(9), 4795–4802. Schebesta, M., Heavey, B., et al. (2002). Transcriptional control of B-cell development. Curr Opin Immunol 14(2), 216–223. Scheuermann, R. H., and Chen, U. (1989). A developmental-specific factor binds to suppressor sites flanking the immunoglobulin heavy-chain enhancer. Genes Dev 3(8), 1255–1266.
99
Schiemann, B., Gommerman, J. L., et al. (2001). An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293(5537), 2111–2114. Schmidt-Supprian, M., Bloch, W., et al. (2000). NEMO/IKK gammadeficient mice model incontinentia pigmenti. Mol Cell 5(6), 981–992. Schubart, K., Massa, S., et al. (2001). B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1. Nat Immunol 2(1), 69–74. Scott, E. W., Simon, M. C., et al. (1994). Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265(5178), 1573–1577. Serwe, M., and Sablitzky, F. (1993). V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer. EMBO J 12(6), 2321–2327. Sha, W. C., Liou, H. C., et al. (1995). Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80(2), 321–330. Shaffer, A. L., Peng, A., et al. (1997). In vivo occupancy of the kappa light chain enhancers in primary pro- and pre-B cells: a model for kappa locus activation. Immunity 6(2), 131–143. Shah, P. C., Bertolino, E., et al. (1997). Using altered specificity Oct-1 and Oct-2 mutants to analyze the regulation of immunoglobulin gene transcription. EMBO J 16(23), 7105–7117. Shaulian, E., and Karin, M. (2002). AP-1 as a regulator of cell life and death. Nat Cell Biol 4(5), E131–E136. Shen, J., Channavajhala, P., et al. (2001). Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha. J Immunol 167(9), 4919–4925. Sikes, M. L., Suarez, C. C., et al. (1999). Regulation of V(D)J recombination by transcriptional promoters. Mol Cell Biol 19(4), 2773–2781. Singh, H., DeKoter, R. P., et al. (1999). PU.1, a shared transcriptional regulator of lymphoid and myeloid cell fates. Cold Spring Harb Symp Quant Biol 64, 13–20. Skok, J. A., Brown, K. E., et al. (2001). Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes. Nat Immunol 2(9), 848– 854. Smahi, A., Courtois, G., et al. (2000). Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium. Nature 405(6785), 466–472. Souabni, A., Cobaleda, C., et al. (2002). Pax5 promotes B lymphopoiesis and blocks T cell development by repressing notch1. Immunity 17(6), 781–793. Spencer, E., Jiang, J., et al. (1999). Signal-induced ubiquitination of IkappaBalpha by the F-box protein Slimb/beta-TrCP. Genes Dev 13(3), 284–294. Staudt, L. M., and Lenardo, M. J. (1991). Immunoglobulin gene transcription. Annu Rev Immunol 9, 373–398. Sun, X. H., Copeland, N. G., et al. (1991). Id proteins Id1 and Id2 selectively inhibit DNA binding by class of helix-loop-helix proteins. Mol Cell Biol 11, 5603. Sun, Z., Arendt, C. W., et al. (2000). PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes. Nature 404(6776), 402–407. Tam, W. F., Lee, L. H., et al. (2000). Cytoplasmic sequestration of rel proteins by IkappaBalpha requires CRM1-dependent nuclear export. Mol Cell Biol 20(6), 2269–2284. Tam, W. F., and Sen, R. (2001). IkappaB family members function by different mechanisms. J Biol Chem 276(11), 7701–7704. Tam, W. F., Wang, W., et al. (2001). Cell-specific association and shuttling of IkappaBalpha provides a mechanism for nuclear NF-kappaB in B lymphocytes. Mol Cell Biol 21(14), 4837–4846. ten Boekel, E., Melchers, F., et al. (1997). Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7(3), 357–368.
100
Calame and Genes
Thomas, M. J., and Seto, E. (1999). Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Gene 236(2), 197–208. Thompson, J. S., Bixler, S. A., et al. (2001). BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293(5537), 2108–2111. Tian, G., Erman, B., et al. (1999). Transcriptional activation by ETS and leucine zipper-containing basic helix-loop-helix proteins. Mol Cell Biol 19(4), 2946–2957. Wabl, M. R., and Burrows, P. D. (1984). Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis. Proc Natl Acad Sci U S A 81(8), 2452–2455. Wallin, J. J., Gackstetter, E. R., et al. (1998). Dependence of BSAP repressor and activator functions on BSAP concentration. Science 279(5358), 1961–1964. Walters, M. C., Fiering, S., et al. (1995). Enhancers increase the probability but not the level of gene expression. Proc Natl Acad Sci U S A 92(15), 7125–7129. Wang, C. Y., Guttridge, D. C., et al. (1999). NF-kappaB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol 19(9), 5923–5929. Wang, D., You, Y., et al. (2002). A requirement for CARMA1 in TCRinduced NF-kappa B activation. Nat Immunol 3(9), 830–835. Wang, Z., Goldstein, A., et al. (1999). Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator. Mol Cell Biol 19(1), 284–295. Webb, C., Zong, R. T., et al. (1999). Differential regulation of immunoglobulin gene transcription via nuclear matrix-associated regions. Cold Spring Harb Symp Quant Biol 64, 109–118.
Webb, C. F. (2001). The transcription factor, Bright, and immunoglobulin heavy chain expression. Immunol Res 24(2), 149–161. Weih, F., Carrasco, D., et al. (1995). Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family. Cell 80(2), 331– 340. Weinberger, J., Jat, P. S., et al. (1988). Localization of a repressive sequence contributing to B-cell specificity in the immunoglobulin heavy-chain enhancer. Mol Cell Biol 8(2), 988–992. Winston, J. T., Strack, P., et al. (1999). The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro. Genes Dev 13(3), 270–283. Workman, J. L., and Kingston, R. E. (1998). Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu Rev Biochem 67, 545–579. Yamamoto, Y., and Gaynor, R. B. (2001). Role of the NF-kappaB pathway in the pathogenesis of human disease states. Curr Mol Med 1(3), 287–296. Yancopoulos, G. D., Desiderio, S. V., et al. (1984). Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature 311(5988), 727–733. Yaron, A., Hatzubai, A., et al. (1998). Identification of the receptor component of the IkappaBalpha-ubiquitin ligase. Nature 396(6711), 590–594. Yasui, D., Miyano, M., et al. (2002). SATB1 targets chromatin remodelling to regulate genes over long distances. Nature 419(6907), 641– 645. Zhou, J., Ermakova, O. V., et al. (2002). Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells. Mol Cell Biol 22(13), 4876–4889.
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7 Early B Cell Development to a Mature, Antigen-Sensitive Cell FRITZ MELCHERS
PAUL KINCADE
Department of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
The development of immunoglobulin (Ig)-producing B lymphocytes proceeds, like the development of any other cell lineage within a multicellular organism, through a series of developmental steps. These steps can be defined by cellular s
