CTCF-binding elements mediate control of V(D)J recombination


Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the VH and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VH gene segments and promoting rearrangement of distal VH segments. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination by suppressing VH joining to D segments not joined to JH segments, and VH to DJH joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal VH-to-DJH recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.

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Figure 1: Mutation of IGCR1 CBEs impairs B-cell development.
Figure 2: IGCR1 mutations alter V H usage, germline transcription and rearrangement order.
Figure 3: IGCR1/CBE mutations lead to V H (D)J H and V H D rearrangements in thymocytes.
Figure 4: IGCR1 is required to allow feedback regulation of proximal V H -to-DJ H recombination.
Figure 5: IGCR1 mediates long-distance Igh chromosomal loops.


  1. 1

    Schatz, D. G. Antigen receptor genes and the evolution of a recombinase. Semin. Immunol. 16, 245–256 (2004)

    CAS  Article  Google Scholar 

  2. 2

    Zhang, Y. et al. The role of mechanistic factors in promoting chromosomal translocations found in lymphoid and other cancers. Adv. Immunol. 106, 93–133 (2010)

    CAS  Article  Google Scholar 

  3. 3

    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)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Perlot, T. & Alt, F. W. Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv. Immunol. 99, 1–32 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Alt, F. W. et al. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 3, 1209–1219 (1984)

    CAS  Article  Google Scholar 

  6. 6

    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)

    CAS  Article  Google Scholar 

  7. 7

    Bates, J. G., Cado, D., Nolla, H. & Schlissel, M. S. Chromosomal position of a V H gene segment determines its activation and inactivation as a substrate for V(D)J recombination. J. Exp. Med. 204, 3247–3256 (2007)

    CAS  Article  Google Scholar 

  8. 8

    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)

    CAS  Article  Google Scholar 

  9. 9

    Reth, M. G., Ammirati, P., Jackson, S. & Alt, F. W. Regulated progression of a cultured pre-B-cell line to the B-cell stage. Nature 317, 353–355 (1985)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Melchers, F. et al. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175, 33–46 (2000)

    CAS  Article  Google Scholar 

  11. 11

    Malynn, B. A., Yancopoulos, G. D., Barth, J. E., Bona, C. A. & Alt, F. W. Biased expression of JH-proximal VH genes occurs in the newly generated repertoire of neonatal and adult mice. J. Exp. Med. 171, 843–859 (1990)

    CAS  Article  Google Scholar 

  12. 12

    Decker, D. J., Boyle, N. E. & Klinman, N. R. Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains. J. Immunol. 147, 1406–1411 (1991)

    CAS  PubMed  Google Scholar 

  13. 13

    ten Boekel, E., Melchers, F. & Rolink, A. G. Changes in the VH gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7, 357–368 (1997)

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    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)

    CAS  Article  Google Scholar 

  16. 16

    Corcoran, A. E. The epigenetic role of non-coding RNA transcription and nuclear organization in immunoglobulin repertoire generation. Semin. Immunol. 22, 353–361 (2010)

    CAS  Article  Google Scholar 

  17. 17

    Feeney, A. Epigenetic regulation of V(D)J recombination. Semin. Immunol. 22, 311–312 (2010)

    Article  Google Scholar 

  18. 18

    Subrahmanyam, R. & Sen, R. RAGs’ eye view of the immunoglobulin heavy chain gene locus. Semin. Immunol. 22, 337–345 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Jhunjhunwala, S., van Zelm, M. C., Peak, M. M. & Murre, C. Chromatin architecture and the generation of antigen receptor diversity. Cell 138, 435–448 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Abarrategui, I. & Krangel, M. S. Germline transcription: a key regulator of accessibility and recombination. Adv. Exp. Med. Biol. 650, 93–102 (2009)

    CAS  Article  Google Scholar 

  21. 21

    Bergman, Y. & Cedar, H. Epigenetic control of recombination in the immune system. Semin. Immunol. 22, 323–329 (2010)

    CAS  Article  Google Scholar 

  22. 22

    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)

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Roldán, E. et al. Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nature Immunol. 6, 31–41 (2004)

    Article  Google Scholar 

  25. 25

    Pinaud, E. et al. The IgH locus 3′ regulatory region: pulling the strings from behind. Adv. Immunol. 110, 27–70 (2011)

    CAS  Article  Google Scholar 

  26. 26

    Sakai, E., Bottaro, A., Davidson, L., Sleckman, B. P. & Alt, F. W. 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. USA 96, 1526–1531 (1999)

    ADS  CAS  Article  Google Scholar 

  27. 27

    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)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Afshar, R., Pierce, S., Bolland, D. J., Corcoran, A. & Oltz, E. M. Regulation of IgH gene assembly: role of the intronic enhancer and 5′DQ52 region in targeting DHJH recombination. J. Immunol. 176, 2439–2447 (2006)

    CAS  Article  Google Scholar 

  29. 29

    Featherstone, K., Wood, A. L., Bowen, A. J. & Corcoran, A. E. The mouse immunoglobulin heavy chain V-D intergenic sequence contains insulators that may regulate ordered V(D)J recombination. J. Biol. Chem. 285, 9327–9338 (2010)

    CAS  Article  Google Scholar 

  30. 30

    Giallourakis, C. C. et al. Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination. Proc. Natl Acad. Sci. USA 107, 22207–22212 (2010)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Lin, Y. C. et al. A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nature Immunol. 11, 635–643 (2010)

    CAS  Article  Google Scholar 

  32. 32

    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)

    CAS  Article  Google Scholar 

  33. 33

    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)

    CAS  Article  Google Scholar 

  34. 34

    Williams, A. & Flavell, R. A. The role of CTCF in regulating nuclear organization. J. Exp. Med. 205, 747–750 (2008)

    CAS  Article  Google Scholar 

  35. 35

    Phillips, J. E. & Corces, V. G. CTCF: master weaver of the genome. Cell 137, 1194–1211 (2009)

    Article  Google Scholar 

  36. 36

    Bulger, M. & Groudine, M. Enhancers: the abundance and function of regulatory sequences beyond promoters. Dev. Biol. 339, 250–257 (2010)

    CAS  Article  Google Scholar 

  37. 37

    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)

    ADS  CAS  Article  Google Scholar 

  38. 38

    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)

    CAS  Article  Google Scholar 

  39. 39

    Hirano, S. L. et al. Identity of IGHV-7183.1 (V81x) coding and recombination signal sequences among wild-derived mice. Immunogenetics 53, 54–58 (2001)

    CAS  Article  Google Scholar 

  40. 40

    Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621 (2001)

    CAS  Article  Google Scholar 

  41. 41

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

    CAS  Article  Google Scholar 

  42. 42

    Su, I. H. et al. Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nature Immunol. 4, 124–131 (2003)

    CAS  Article  Google Scholar 

  43. 43

    Liu, H. et al. Yin Yang 1 is a critical regulator of B-cell development. Genes Dev. 21, 1179–1189 (2007)

    CAS  Article  Google Scholar 

  44. 44

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

    CAS  Article  Google Scholar 

  45. 45

    Degner-Leisso, S. C. & Feeney, A. J. Epigenetic and 3-dimensional regulation of V(D)J rearrangement of immunoglobulin genes. Semin. Immunol. 22, 346–352 (2010)

    CAS  Article  Google Scholar 

  46. 46

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

    CAS  Article  Google Scholar 

  47. 47

    Kyrchanova, O., Chetverina, D., Maksimenko, O., Kullyev, A. & Georgiev, P. Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements. Nucleic Acids Res. 36, 7019–7028 (2008)

    CAS  Article  Google Scholar 

  48. 48

    Dudley, D. D. et al. Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice. J. Exp. Med. 198, 1439–1450 (2003)

    CAS  Article  Google Scholar 

  49. 49

    Hagège, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nature Protocols 2, 1722–1733 (2007)

    Article  Google Scholar 

  50. 50

    Chen, J. et al. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus. EMBO J. 12, 4635–4645 (1993)

    CAS  Article  Google Scholar 

  51. 51

    Barreto, V. & Cumano, A. Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice. J. Immunol. 164, 893–899 (2000)

    CAS  Article  Google Scholar 

  52. 52

    Sonoda, E. et al. B cell development under the condition of allelic inclusion. Immunity 6, 225–233 (1997)

    CAS  Article  Google Scholar 

  53. 53

    McManus, S. et al. The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells. EMBO J. 30, 2388–2404 (2011)

    CAS  Article  Google Scholar 

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We thank Y. Fujiwara and P.-Y. Huang for generating chimaeric mice. This work was supported by NIH grants RO1 AI20047 (to F.W.A.), RO1 HL48702 and AI40227 (to M.S.S.), CA054198-20 (to C.M.) and K08 AI070839 (to C.C.G.). M.B. was supported by the Austrian GEN-AU initiative and Boehringer Ingelheim. C.G. is supported by an Irvington Institute Postdoctoral Fellowship from the Cancer Research Institute. C.V. is supported by a Marie Curie Fellowship. C.B. is supported by an EMBO fellowship. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

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C.G., H.S.Y., A.F., C.C.G. and F.W.A. conceived, designed and/or peformed most experiments, interpreted most results, and wrote the manuscript. With respect to other authors, S.J. performed experiments for Figs 2, 3, 4 and Supplementary Fig. 11 and contributed to interpretation of the data; A.E. and M.B. contributed the work in Fig. 2c and Supplementary 7a–c. C.V., J.G.B. and M.S.S. contributed the work in Supplementary Fig. 10; and C.B. and C.M. performed FISH experiments on IGCR1−/− cells that helped frame aspects of the discussion and models; all of these authors contributed to polishing the manuscript. All other authors provided technical assistance with various experiments or data analysis.

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Correspondence to Cosmas C. Giallourakis or Frederick W. Alt.

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Guo, C., Yoon, H., Franklin, A. et al. CTCF-binding elements mediate control of V(D)J recombination. Nature 477, 424–430 (2011). https://doi.org/10.1038/nature10495

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