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The role of chromatin loop extrusion in antibody diversification

Abstract

Cohesin mediates chromatin loop formation across the genome by extruding chromatin between convergently oriented CTCF-binding elements. Recent studies indicate that cohesin-mediated loop extrusion in developing B cells presents immunoglobulin heavy chain (Igh) variable (V), diversity (D) and joining (J) gene segments to RAG endonuclease through a process referred to as RAG chromatin scanning. RAG initiates V(D)J recombinational joining of these gene segments to generate the large number of different Igh variable region exons that are required for immune responses to diverse pathogens. Antigen-activated mature B cells also use chromatin loop extrusion to mediate the synapsis, breakage and end joining of switch regions flanking Igh constant region exons during class-switch recombination, which allows for the expression of different antibody constant region isotypes that optimize the functions of antigen-specific antibodies to eliminate pathogens. Here, we review recent advances in our understanding of chromatin loop extrusion during V(D)J recombination and class-switch recombination at the Igh locus.

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Fig. 1: Loop extrusion model.
Fig. 2: The Igh locus initiates V(D)J recombination at the V(D)J recombination centre.
Fig. 3: Discovery of RAG long-range chromatin scanning.
Fig. 4: Loop extrusion-mediated RAG scanning drives D-to-JH and proximal VH-to-DJH recombination.
Fig. 5: Long-range RAG scanning mediates VH use across the Igh locus.
Fig. 6: Potential substrate accessibility and distal RAG scanning mechanisms in the VH locus.
Fig. 7: Loop extrusion mediates physiological, deletional class-switch recombination.

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References

  1. Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dowen, J. M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lupianez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Narendra, V. et al. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347, 1017–1021 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hnisz, D., Day, D. S. & Young, R. A. Insulated neighborhoods: structural and functional units of mammalian gene control. Cell 167, 1188–1200 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Merkenschlager, M. & Nora, E. P. CTCF and cohesin in genome folding and transcriptional gene regulation. Annu. Rev. Genomics Hum. Genet. 17, 17–43 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Stadhouders, R., Filion, G. J. & Graf, T. Transcription factors and 3D genome conformation in cell-fate decisions. Nature 569, 345–354 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pope, B. D. et al. Topologically associating domains are stable units of replication-timing regulation. Nature 515, 402–405 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Moindrot, B. et al. 3D chromatin conformation correlates with replication timing and is conserved in resting cells. Nucleic Acids Res. 40, 9470–9948 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hu, J. et al. Chromosomal loop domains direct the recombination of antigen receptor genes. Cell 163, 947–959 (2015). This study is the first to show that RAG, upon binding a bona fide RSS, can linearly explore megabase distances within convergent CTCF site-based chromatin loop domains to identify convergently oriented cryptic RSS targets for V(D)J recombination-based cleavage and joining, thereby providing the basis for subsequent models of linear RAG chromatin scanning.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dong, J. et al. Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching. Nature 525, 134–139 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Collins, P. L. et al. DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner. Nat. Commun. 11, 3158 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Arnould, C. et al. Loop extrusion as a mechanism for formation of DNA damage repair foci. Nature 590, 660–665 (2021). This study provides evidence for a key role of cohesin-mediated loop extrusion in general double-strand break repair, which involves facilitating the formation of long double-strand break response foci that are crucial for the synapsis and joining of double-strand break ends.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ochs, F. et al. Stabilization of chromatin topology safeguards genome integrity. Nature 574, 571–574 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Dekker, J. & Mirny, L. The 3D genome as moderator of chromosomal communication. Cell 164, 1110–1121 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). This study is the first to report kilobase high-resolution Hi-C maps of the human genome, which reveal a genome-wide prevalence of convergent CTCF sites at the chromatin loop anchors, thus providing a key observation for the cohesin-mediated loop extrusion model.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nasmyth, K. Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Alipour, E. & Marko, J. F. Self-organization of domain structures by DNA-loop-extruding enzymes. Nucleic Acids Res. 40, 11202–11212 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sanborn, A. L. et al. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc. Natl Acad. Sci. USA 112, E6456–E6465 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nichols, M. H. & Corces, V. G. A CTCF code for 3D genome architecture. Cell 162, 703–705 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bouwman, B. A. & de Laat, W. Getting the genome in shape: the formation of loops, domains and compartments. Genome Biol. 16, 154 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Fudenberg, G. et al. Formation of chromosomal domains by loop extrusion. Cell Rep. 15, 2038–2049 (2016). Together with Sanborn et al. (2015), this paper provides strong evidence to support a chromatin loop extrusion process, probably driven by cohesin, as the underlying mechanism for the formation of convergent CTCF site-based contact loop domains.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jain, S., Ba, Z., Zhang, Y., Dai, H. Q. & Alt, F. W. CTCF-binding elements mediate accessibility of RAG substrates during chromatin scanning. Cell 174, 102–116 e114 (2018). This paper provides evidence that supports a model in which the Igh V(D)J recombination centre functions as a dynamic loop anchor to allow cohesin-mediated loop extrusion to present upstream chromatin for scanning by RAG, and also shows that CTCF sites promote accessibility of associated proximal VH regions by increasing their interaction with the V(D)J recombination centre during scanning.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang, Y. et al. The fundamental role of chromatin loop extrusion in physiological V(D)J recombination. Nature 573, 600–604 (2019). This paper provides evidence that loop extrusion-mediated RAG scanning is the major mechanism for physiological, deletional D-to-JH recombination and shows that mechanisms in addition to CTCF site-based loop anchors, including active transcription and nuclease-‘dead’ Cas9 protein binding, can impede loop extrusion-mediated scanning and promote RAG targeting activity.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ba, Z. et al. CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning. Nature 586, 305–310 (2020). This paper, through targeted depletion of cohesin or CTCF in v-Abl cell lines, provides evidence that cohesin drives loop extrusion-mediated RAG chromatin scanning and provides proof of principle that dampening of CTCF site-based anchors can promote loop extrusion, locus contraction and long-range RAG scanning.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hill, L. et al. Wapl repression by Pax5 promotes V gene recombination by Igh loop extrusion. Nature 584, 142–147 (2020). This study shows that PAX5-mediated suppression of Wapl transcription in pro-B cells results in global extension of chromosomal loops, which implicates loop extrusion as the long-sought mechanism for VH locus contraction and distal VH use.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dai, H. Q. et al. Loop extrusion mediates physiological Igh locus contraction for RAG scanning. Nature 590, 338–343 (2021). This work, through analyses of the use of cryptic RSSs across normal and inverted VH loci, shows linear RAG scanning across the VH locus in normal pro-B cells and also implicates WAPL downregulation in promoting loop extrusion-mediated locus contraction and long-range RAG scanning.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang, X. et al. Fundamental roles of chromatin loop extrusion in antibody class switching. Nature 575, 385–389 (2019). This paper shows that cohesin-mediated loop extrusion has a fundamental role in the physiological, deletional Igh CSR mechanism, by establishing a CSR centre that orchestrates substrate S region activation and synapsis, as well as post-cleavage deletional joining of S region double-strand breaks.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, X., Yoon, H. S., Chapdelaine-Williams, A. M., Kyritsis, N. & Alt, F. W. Physiological role of the 3′IgH CBEs super-anchor in antibody class switching. Proc. Natl Acad. Sci. USA 118 (2021). This paper shows that the 3′ Igh CTCF sites function as an insulator to focus chromatin loop extrusion-mediated transcriptional and CSR activities within the upstream CH-containing domain.

  32. Alt, F. W., Zhang, Y., Meng, F. L., Guo, C. & Schwer, B. Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell 152, 417–429 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Methot, S. P. & Di Noia, J. M. Molecular mechanisms of somatic hypermutation and class switch recombination. Adv. Immunol. 133, 37–87 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Ong, C. T. & Corces, V. G. CTCF: an architectural protein bridging genome topology and function. Nat. Rev. Genet. 15, 234–246 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Degner, S. C., Wong, T. P., Jankevicius, G. & Feeney, A. J. Cutting edge: developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development. J. Immunol. 182, 44–48 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Proudhon, C., Hao, B., Raviram, R., Chaumeil, J. & Skok, J. A. Long-range regulation of V(D)J recombination. Adv. Immunol. 128, 123–182 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. MacPherson, M. J. & Sadowski, P. D. The CTCF insulator protein forms an unusual DNA structure. BMC Mol. Biol. 11, 101 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nakahashi, H. et al. A genome-wide map of CTCF multivalency redefines the CTCF code. Cell Rep. 3, 1678–1689 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Vietri Rudan, M. et al. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10, 1297–1309 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. de Wit, E. et al. CTCF binding polarity determines chromatin looping. Mol. Cell 60, 676–684 (2015).

    Article  PubMed  CAS  Google Scholar 

  41. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zuin, J. et al. A cohesin-independent role for NIPBL at promoters provides insights in CdLS. PLoS Genet. 10, e1004153 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Dorsett, D. & Merkenschlager, M. Cohesin at active genes: a unifying theme for cohesin and gene expression from model organisms to humans. Curr. Opin. Cell Biol. 25, 327–333 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li, Y. et al. The structural basis for cohesin–CTCF-anchored loops. Nature 578, 472–476 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Nishana, M. et al. Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation. Genome Biol. 21, 108 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pugacheva, E. M. et al. CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention. Proc. Natl Acad. Sci. USA 117, 2020–2031 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nora, E. P. et al. Molecular basis of CTCF binding polarity in genome folding. Nat. Commun. 11, 5612 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Mirny, L. & Dekker, J. Mechanisms of chromosome folding and nuclear organization: their interplay and open questions. Cold Spring Harb. Perspect. Biol. https://doi.org/10.1101/cshperspect.a040147 (2021).

    Article  Google Scholar 

  49. Hansen, A. S., Pustova, I., Cattoglio, C., Tjian, R. & Darzacq, X. CTCF and cohesin regulate chromatin loop stability with distinct dynamics. eLife 6, e25776 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Hansen, A. S., Cattoglio, C., Darzacq, X. & Tjian, R. Recent evidence that TADs and chromatin loops are dynamic structures. Nucleus 9, 20–32 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Rowley, M. J. & Corces, V. G. Organizational principles of 3D genome architecture. Nat. Rev. Genet. 19, 789–800 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Davidson, I. F. & Peters, J. M. Genome folding through loop extrusion by SMC complexes. Nat. Rev. Mol. Cell Biol. 22, 445–464 (2021).

    Article  CAS  PubMed  Google Scholar 

  53. Lin, S. G., Ba, Z., Alt, F. W. & Zhang, Y. RAG chromatin scanning during V(D)J recombination and chromatin loop extrusion are related processes. Adv. Immunol. 139, 93–135 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. Kraft, K. et al. Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations. Nat. Cell Biol. 21, 305–310 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Hsieh, T. S. et al. Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Mol. Cell 78, 539–553.e8 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Thiecke, M. J. et al. Cohesin-dependent and -independent mechanisms mediate chromosomal contacts between promoters and enhancers. Cell Rep. 32, 107929 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Nora, E. P. et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169, 930–944.e22 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wutz, G. et al. Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins. EMBO J. 36, 3573–3599 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Gassler, J. et al. A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture. EMBO J. 36, 3600–3618 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Rao, S. S. P. et al. Cohesin loss eliminates all loop domains. Cell 171, 305–320.e24 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Schwarzer, W. et al. Two independent modes of chromatin organization revealed by cohesin removal. Nature 551, 51–56 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Vian, L. et al. The energetics and physiological impact of cohesin extrusion. Cell 173, 1165–1178.e20 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Gandhi, R., Gillespie, P. J. & Hirano, T. Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase. Curr. Biol. 16, 2406–2417 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kueng, S. et al. Wapl controls the dynamic association of cohesin with chromatin. Cell 127, 955–967 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. Haarhuis, J. H. I. et al. The cohesin release factor WAPL restricts chromatin loop extension. Cell 169, 693–707.e14 (2017). Together with Wutz et al. (2017) and Gassler et al. (2017), this study shows that depletion of WAPL, the cohesin unloading factor, increases the cohesin retention time on chromatin and leads to extension of chromatin loop sizes genome wide.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Davidson, I. F. et al. DNA loop extrusion by human cohesin. Science 366, 1338–1345 (2019).

    Article  CAS  PubMed  Google Scholar 

  67. Kim, Y., Shi, Z., Zhang, H., Finkelstein, I. J. & Yu, H. Human cohesin compacts DNA by loop extrusion. Science 366, 1345–1349 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Golfier, S., Quail, T., Kimura, H. & Brugues, J. Cohesin and condensin extrude DNA loops in a cell cycle-dependent manner. eLife 9, e53885 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Ribeiro de Almeida, C., Hendriks, R. W. & Stadhouders, R. Dynamic control of long-range genomic interactions at the immunoglobulin κ light-chain locus. Adv. Immunol. 128, 183–271 (2015).

    Article  PubMed  CAS  Google Scholar 

  70. Schatz, D. G. & Swanson, P. C. V(D)J recombination: mechanisms of initiation. Annu. Rev. Genet. 45, 167–202 (2011).

    Article  CAS  PubMed  Google Scholar 

  71. Teng, G. & Schatz, D. G. Regulation and evolution of the RAG recombinase. Adv. Immunol. 128, 1–39 (2015).

    Article  CAS  PubMed  Google Scholar 

  72. Jung, D., Giallourakis, C., Mostoslavsky, R. & Alt, F. W. Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24, 541–570 (2006).

    Article  CAS  PubMed  Google Scholar 

  73. Kim, M. S., Lapkouski, M., Yang, W. & Gellert, M. Crystal structure of the V(D)J recombinase RAG1–RAG2. Nature 518, 507–511 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Ru, H. et al. Molecular mechanism of V(D)J recombination from synaptic RAG1–RAG2 complex structures. Cell 163, 1138–1152 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Kim, M. S. et al. Cracking the DNA code for V(D)J recombination. Mol. Cell 70, 358–370.e4 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Ru, H. et al. DNA melting initiates the RAG catalytic pathway. Nat. Struct. Mol. Biol. 25, 732–742 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Grundy, G. J. et al. Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex. Mol. Cell 35, 217–227 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Wang, X. S., Lee, B. J. & Zha, S. The recent advances in non-homologous end-joining through the lens of lymphocyte development. DNA Repair. 94, 102874 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Zhao, B., Rothenberg, E., Ramsden, D. A. & Lieber, M. R. The molecular basis and disease relevance of non-homologous DNA end joining. Nat. Rev. Mol. Cell Biol. 21, 765–781 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Ji, Y. et al. The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141, 419–431 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Teng, G. et al. RAG represents a widespread threat to the lymphocyte genome. Cell 162, 751–765 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Guo, C. et al. CTCF-binding elements mediate control of V(D)J recombination. Nature 477, 424–430 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Lin, S. G., Guo, C., Su, A., Zhang, Y. & Alt, F. W. CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination. Proc. Natl Acad. Sci. USA 112, 1815–1820 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Garrett, F. E. et al. Chromatin architecture near a potential 3′ end of the igh locus involves modular regulation of histone modifications during B-cell development and in vivo occupancy at CTCF sites. Mol. Cell Biol. 25, 1511–1525 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Degner, S. C. et al. CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells. Proc. Natl Acad. Sci. USA 108, 9566–9571 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Benner, C., Isoda, T. & Murre, C. New roles for DNA cytosine modification, eRNA, anchors, and superanchors in developing B cell progenitors. Proc. Natl Acad. Sci. USA 112, 12776–12781 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Aiden, E. L. & Casellas, R. Somatic rearrangement in B cells: it’s (mostly) nuclear physics. Cell 162, 708–711 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Kosak, S. T. et al. Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296, 158–162 (2002).

    Article  CAS  PubMed  Google Scholar 

  89. Fuxa, M. et al. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes. Dev. 18, 411–422 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Sayegh, C. E., Jhunjhunwala, S., Riblet, R. & Murre, C. Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells. Genes Dev. 19, 322–327 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Roldan, E. et al. Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6, 31–41 (2005).

    Article  CAS  PubMed  Google Scholar 

  92. Jhunjhunwala, S. et al. The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Cell 133, 265–279 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Medvedovic, J. et al. Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire. Immunity 39, 229–244 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Rother, M. B. et al. Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci. Nucleic Acids Res. 44, 175–186 (2016).

    Article  PubMed  Google Scholar 

  95. Montefiori, L. et al. Extremely long-range chromatin loops link topological domains to facilitate a diverse antibody repertoire. Cell Rep. 14, 896–906 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Bossen, C., Mansson, R. & Murre, C. Chromatin topology and the regulation of antigen receptor assembly. Annu. Rev. Immunol. 30, 337–356 (2012).

    Article  CAS  PubMed  Google Scholar 

  97. Ebert, A., Hill, L. & Busslinger, M. Spatial regulation of V-(D)J recombination at antigen receptor loci. Adv. Immunol. 128, 93–121 (2015).

    Article  CAS  PubMed  Google Scholar 

  98. Rogers, C. H., Mielczarek, O. & Corcoran, A. E. Dynamic 3D locus organization and its drivers underpin immunoglobulin recombination. Front. Immunol. 11, 633705 (2020).

    Article  CAS  PubMed  Google Scholar 

  99. Lucas, J. S., Zhang, Y., Dudko, O. K. & Murre, C. 3D trajectories adopted by coding and regulatory DNA elements: first-passage times for genomic interactions. Cell 158, 339–352 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Ranganath, S. et al. Productive coupling of accessible Vβ14 segments and DJβ complexes determines the frequency of Vβ14 rearrangement. J. Immunol. 180, 2339–2346 (2008).

    Article  CAS  PubMed  Google Scholar 

  101. Helmink, B. A. & Sleckman, B. P. The response to and repair of RAG-mediated DNA double-strand breaks. Annu. Rev. Immunol. 30, 175–202 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Nussenzweig, A. & Nussenzweig, M. C. Origin of chromosomal translocations in lymphoid cancer. Cell 141, 27–38 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Tepsuporn, S., Hu, J., Gostissa, M. & Alt, F. W. Mechanisms that can promote peripheral B-cell lymphoma in ATM-deficient mice. Cancer Immunol. Res. 2, 857–866 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Bredemeyer, A. L. et al. ATM stabilizes DNA double-strand-break complexes during V(D)J recombination. Nature 442, 466–470 (2006).

    Article  CAS  PubMed  Google Scholar 

  105. Wood, C. & Tonegawa, S. 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. USA 80, 3030–3034 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Yancopoulos, G. D. et al. Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature 311, 727–733 (1984).

    Article  CAS  PubMed  Google Scholar 

  107. Gauss, G. H. & Lieber, M. R. The basis for the mechanistic bias for deletional over inversional V(D)J recombination. Genes Dev. 6, 1553–1561 (1992).

    Article  CAS  PubMed  Google Scholar 

  108. Choi, N. M. et al. Deep sequencing of the murine IgH repertoire reveals complex regulation of nonrandom V gene rearrangement frequencies. J. Immunol. 191, 2393–2402 (2013).

    Article  CAS  PubMed  Google Scholar 

  109. Bolland, D. J. et al. Two mutually exclusive local chromatin states drive efficient V(D)J recombination. Cell Rep. 15, 2475–2487 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Khanna, N., Zhang, Y., Lucas, J. S., Dudko, O. K. & Murre, C. Chromosome dynamics near the sol-gel phase transition dictate the timing of remote genomic interactions. Nat. Commun. 10, 2771 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  111. Ebert, A. et al. The distal VH gene cluster of the Igh locus contains distinct regulatory elements with Pax5 transcription factor-dependent activity in pro-B cells. Immunity 34, 175–187 (2011).

    Article  CAS  PubMed  Google Scholar 

  112. Verma-Gaur, J. et al. Noncoding transcription within the Igh distal VH region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells. Proc. Natl Acad. Sci. USA 109, 17004–17009 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Hesslein, D. G. et al. Pax5 is required for recombination of transcribed, acetylated, 5′ IgH V gene segments. Genes Dev. 17, 37–42 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Yancopoulos, G. D. & Alt, F. W. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell 40, 271–281 (1985).

    Article  CAS  PubMed  Google Scholar 

  115. Yancopoulos, G. D., Blackwell, T. K., Suh, H., Hood, L. & Alt, F. W. Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. Cell 44, 251–259 (1986).

    Article  CAS  PubMed  Google Scholar 

  116. Bolland, D. J. et al. Antisense intergenic transcription in V(D)J recombination. Nat. Immunol. 5, 630–637 (2004).

    Article  CAS  PubMed  Google Scholar 

  117. Bolland, D. J. et al. Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Eμ. Mol. Cell Biol. 27, 5523–5533 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Beilinson, H. A. et al. The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination. J. Exp. Med. 218, e20210250 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Qiu, X. et al. Sequential enhancer sequestration dysregulates recombination center formation at the IgH locus. Mol. Cell 70, 21–33 e26 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Chakraborty, T. et al. Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus. Mol. Cell 27, 842–850 (2007).

    Article  CAS  PubMed  Google Scholar 

  121. Hwang, J. K., Alt, F. W. & Yeap, L. S. Related mechanisms of antibody somatic hypermutation and class switch recombination. Microbiol. Spectr. 3, MDNA3-0037-2014 (2015).

    PubMed  Google Scholar 

  122. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).

    Article  CAS  PubMed  Google Scholar 

  123. Yeap, L. S. & Meng, F. L. Cis- and trans-factors affecting AID targeting and mutagenic outcomes in antibody diversification. Adv. Immunol. 141, 51–103 (2019).

    Article  CAS  PubMed  Google Scholar 

  124. Cascalho, M., Wong, J., Steinberg, C. & Wabl, M. Mismatch repair co-opted by hypermutation. Science 279, 1207–1210 (1998).

    Article  CAS  PubMed  Google Scholar 

  125. Bottaro, A. et al. Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the μ locus. Int. Immunol. 10, 799–806 (1998).

    Article  CAS  PubMed  Google Scholar 

  126. Sakai, E., Bottaro, A. & Alt, F. W. The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination. Int. Immunol. 11, 1709–1713 (1999).

    Article  CAS  PubMed  Google Scholar 

  127. Perlot, T., Alt, F. W., Bassing, C. H., Suh, H. & Pinaud, E. Elucidation of IgH intronic enhancer functions via germ-line deletion. Proc. Natl Acad. Sci. USA 102, 14362–14367 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Li, F., Yan, Y., Pieretti, J., Feldman, D. A. & Eckhardt, L. A. Comparison of identical and functional Igh alleles reveals a nonessential role for Eμ in somatic hypermutation and class-switch recombination. J. Immunol. 185, 6049–6057 (2010).

    Article  CAS  PubMed  Google Scholar 

  129. Vincent-Fabert, C. et al. Genomic deletion of the whole IgH 3′ regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes. Blood 116, 1895–1898 (2010).

    Article  CAS  PubMed  Google Scholar 

  130. Saintamand, A. et al. Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3′ regulatory region. Nat. Commun. 7, 10730 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Pinaud, E. et al. Localization of the 3′ IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15, 187–199 (2001).

    Article  CAS  PubMed  Google Scholar 

  132. Wuerffel, R. et al. S–S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase. Immunity 27, 711–722 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Seidl, K. J. et al. Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus. Proc. Natl Acad. Sci. USA 96, 3000–3005 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Harriman, W., Volk, H., Defranoux, N. & Wabl, M. Immunoglobulin class switch recombination. Annu. Rev. Immunol. 11, 361–384 (1993).

    Article  CAS  PubMed  Google Scholar 

  135. Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).

    Article  CAS  PubMed  Google Scholar 

  137. Wei, P. C. et al. Long neural genes harbor recurrent DNA break clusters in neural stem/progenitor Cells. Cell 164, 644–655 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Dudley, D. D. et al. Internal IgH class switch region deletions are position-independent and enhanced by AID expression. Proc. Natl Acad. Sci. USA 99, 9984–9989 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Zhao, Y., Rabbani, H., Shimizu, A. & Hammarstrom, L. Mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted α gene upstream of a condensed υ gene. Immunology 101, 348–353 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Lundqvist, M. L., Middleton, D. L., Hazard, S. & Warr, G. W. The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes. J. Biol. Chem. 276, 46729–46736 (2001).

    Article  CAS  PubMed  Google Scholar 

  141. Xiong, H., Dolpady, J., Wabl, M., Curotto de Lafaille, M. A. & Lafaille, J. J. Sequential class switching is required for the generation of high affinity IgE antibodies. J. Exp. Med. 209, 353–364 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Mandler, R., Finkelman, F. D., Levine, A. D. & Snapper, C. M. IL-4 induction of IgE class switching by lipopolysaccharide-activated murine B cells occurs predominantly through sequential switching. J. Immunol. 150, 407–418 (1993).

    CAS  PubMed  Google Scholar 

  143. Zhang, T. et al. Downstream class switching leads to IgE antibody production by B lymphocytes lacking IgM switch regions. Proc. Natl Acad. Sci. USA 107, 3040–3045 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Yu, K. An insulator that regulates chromatin extrusion and class switch recombination. Proc. Natl Acad. Sci. USA 118, e2026399118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Bassing, C. H. & Alt, F. W. The cellular response to general and programmed DNA double strand breaks. DNA Repair. 3, 781–796 (2004).

    Article  CAS  PubMed  Google Scholar 

  146. Liu, Y. et al. Very fast CRISPR on demand. Science 368, 1265–1269 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Li, K., Bronk, G., Kondev, J. & Haber, J. E. Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break. Proc. Natl Acad. Sci. USA 117, 21354–21363 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Mirny, L. A. Cells use loop extrusion to weave and tie the genome. Nature 590, 554–555 (2021).

    Article  CAS  PubMed  Google Scholar 

  149. Desiderio, S. Temporal and spatial regulatory functions of the V(D)J recombinase. Semin. Immunol. 22, 362–369 (2010).

    Article  CAS  PubMed  Google Scholar 

  150. Majumder, K., Bassing, C. H. & Oltz, E. M. Regulation of Tcrb gene assembly by genetic, epigenetic, and topological mechanisms. Adv. Immunol. 128, 273–306 (2015).

    Article  CAS  PubMed  Google Scholar 

  151. Carico, Z. & Krangel, M. S. Chromatin dynamics and the development of the TCRα and TCRδ repertoires. Adv. Immunol. 128, 307–361 (2015).

    Article  CAS  PubMed  Google Scholar 

  152. Fischer, C. et al. Conservation of the T-cell receptor α/δ linkage in the teleost fish Tetraodon nigroviridis. Genomics 79, 241–248 (2002).

    Article  CAS  PubMed  Google Scholar 

  153. Fitzsimmons, S. P., Bernstein, R. M., Max, E. E., Skok, J. A. & Shapiro, M. A. Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Igκ locus. J. Immunol. 179, 5264–5273 (2007).

    Article  CAS  PubMed  Google Scholar 

  154. Ribeiro de Almeida, C. et al. The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus. Immunity 35, 501–513 (2011).

    Article  CAS  PubMed  Google Scholar 

  155. Stadhouders, R. et al. Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions. PLoS Biol. 12, e1001791 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  156. Lin, Y. C. et al. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat. Immunol. 13, 1196–1204 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Xiang, Y., Park, S. K. & Garrard, W. T. Vκ gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Vκ–Jκ intervening region. J. Immunol. 190, 1819–1826 (2013).

    Article  CAS  PubMed  Google Scholar 

  158. Liu, Z. et al. A recombination silencer that specifies heterochromatin positioning and ikaros association in the immunoglobulin κ locus. Immunity 24, 405–415 (2006).

    Article  PubMed  CAS  Google Scholar 

  159. Xiang, Y., Zhou, X., Hewitt, S. L., Skok, J. A. & Garrard, W. T. A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location. J. Immunol. 186, 5356–5366 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants (R01AI020047 and R01AI077595 to F.W.A.; R01AI155775 to Y.Z.). H.-Q.D. was a fellow of the Cancer Research Institute (CRI) of New York. H.H. is a fellow of CRI. F.W.A. is an investigator of the Howard Hughes Medical Institute.

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Glossary

CTCF-binding elements

(Referred to here as CTCF sites). DNA sequence motifs recognized by the structural protein CTCF (CCCTC-binding factor) that mostly are characterized by a highly conserved 20-bp core consensus motif, but a subset of which are also flanked by upstream and/or downstream regulatory motifs.

Cohesin ring protein complex

A highly conserved ring-shaped multi-protein complex composed of three core subunits, SMC1, SMC3 and RAD21, that associate with SA1 or SA2 in somatic cells. The cohesin complex has ATPase activity and has essential functions in chromosome segregation and chromatin loop formation, as well as in various fundamental processes including transcription and DNA repair.

Convergent orientation

Referring to two DNA sequence motifs of opposite orientation that point towards each other in cis.

V(D)J recombination

A programmed somatic recombination process initiated by RAG1–RAG2 endonuclease in early developing lymphocytes that assembles variable (V), diversity (D) and joining (J) gene segments into exons that encode the variable regions of antibody and T cell receptor chains.

Class-switch recombination

(CSR). A programmed somatic recombination process initiated by activation-induced cytidine deaminase (AID) in antigen-activated mature B cells that replaces the upstream IgM-encoding Cμ exon with a different downstream constant region exon to change the class of antibody expressed. This allows for the antigen-specific effector functions of an antibody with a given variable region specificity to be optimized.

Classical non-homologous end joining

(C-NHEJ). A major cellular repair pathway for DNA double-strand breaks that joins DNA ends without requiring a homologous template. This pathway is commonly referred to as ‘classical’ to distinguish it from less robust, alternative end-joining pathways that become more obvious in the absence of the classical pathway.

V(D)J recombination centre

In the immunoglobulin heavy chain (Igh) locus, this is the site where RAG1–RAG2 endonuclease binds active chromatin harbouring a strong enhancer to generate a hub for the capture of substrate variable (V), diversity (D) and joining (J) segments to allow for their cleavage and joining to create V(D)J variable region exons. Similar recombination centres occur in other antigen receptor loci that undergo V(D)J recombination.

HTGTS-V(D)J-seq

This assay maps RAG-initiated V(D)J recombination events across the genome and is adapted from linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS). HTGTS-V(D)J-seq has a unique ability to detect thousands of low-level independent cryptic joining events within a large population of cells, that when viewed together provide ‘tracks’ of RAG activity within a domain. These tracks clearly show the chromatin regions that are explored by recombination centre-bound RAG, the directionality of the exploration process and the sequences RAG does or does not ‘see’ during exploration.

Phase separation

A process that forms condensates of loci with similar chromatin states through homotypic attraction of bridging proteins.

LAM-HTGTS-3C-seq

A high-resolution chromatin conformation capture (3C)-based genomic interaction assay that uses linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) for its downstream steps. This method detects sequences within chromatin loop domains that interact with a sequence of interest at high resolution.

Switch region

(S region). A 1–12 kb repetitive DNA sequence that precedes each set of immunoglobulin heavy chain (Igh) constant region exons. S regions undergo DNA double-strand breaks mediated by activation-induced cytidine deaminase (AID) that are then joined to effect IgH class-switch recombination (CSR).

Class-switch recombination centre

(CSR centre). The CSR centre is an immunoglobulin heavy chain (Igh) locus site at which activated B cells juxtapose distant cis-regulatory elements with donor and acceptor DNA switch regions (S regions), which are then cleaved and joined to carry out the process of antibody isotype switching.

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Zhang, Y., Zhang, X., Dai, HQ. et al. The role of chromatin loop extrusion in antibody diversification. Nat Rev Immunol 22, 550–566 (2022). https://doi.org/10.1038/s41577-022-00679-3

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