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Antisense intergenic transcription in V(D)J recombination

Nature Immunology volume 5, pages 630–637 (2004)Cite this article

Abstract

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJH to VDJH recombination and terminates concomitantly with VDJH recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate VH-to-DJH recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

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Figure 1: Organization of the Igh locus.
Figure 2: RT-PCR of germline transcription at the Igh locus.
Figure 3: RNA FISH analysis of J558 antisense and VDJH sense transcription during B cell development.
Figure 4: Semiquantitative RT-PCR of J558 germline transcription in wild-type fractions A, B and C.
Figure 5: Comparison of J558 antisense with μ0 and VDJH sense transcription.

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References

  1. Chevillard, C., Ozaki, J., Herring, C.D. & Riblet, R. A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus. J. Immunol. 168, 5659–5666 (2002).

    Article  CAS  Google Scholar 

  2. Krangel, M.S. Gene segment selection in V(D)J recombination: accessibility and beyond. Nat. Immunol. 4, 624–630 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Corcoran, A.E., Riddell, A., Krooshoop, D. & Venkitaraman, A.R. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391, 904–907 (1998).

    Article  CAS  Google Scholar 

  5. Angelin-Duclos, C. & Calame, K. Evidence that immunoglobulin VH-DJ recombination does not require germ line transcription of the recombining variable gene segment. Mol. Cell. Biol. 18, 6253–6264 (1998).

    Article  CAS  Google Scholar 

  6. Haines, B.B. & Brodeur, P.H. Accessibility changes across the mouse Igh-V locus during B cell development. Eur. J. Immunol. 28, 4228–4235 (1998).

    Article  CAS  Google Scholar 

  7. Nelson, K.J., Haimovich, J. & Perry, R.P. Characterization of productive and sterile transcripts from the immunoglobulin heavy-chain locus: processing of micron and μS mRNA. Mol. Cell. Biol. 3, 1317–1332 (1983).

    Article  CAS  Google Scholar 

  8. Schlissel, M.S., Corcoran, L.M. & Baltimore, D. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J. Exp. Med. 173, 711–720 (1991).

    Article  CAS  Google Scholar 

  9. Duber, S., Engel, H., Rolink, A., Kretschmer, K. & Weiss, S. Germline transcripts of immunoglobulin light chain variable regions are structurally diverse and differentially expressed. Mol. Immunol. 40, 509–516 (2003).

    Article  CAS  Google Scholar 

  10. Goldman, J.P., Spencer, D.M. & Raulet, D.H. Ordered rearrangement of variable region genes of the T cell receptor γ locus correlates with transcription of the unrearranged genes. J. Exp. Med. 177, 729–739 (1993).

    Article  CAS  Google Scholar 

  11. Sleckman, B.P., Gorman, J.R. & Alt, F.W. Accessibility control of antigen-receptor variable-region gene assembly: role of cis-acting elements. Annu. Rev. Immunol. 14, 459–481 (1996).

    Article  CAS  Google Scholar 

  12. Stanhope-Baker, P., Hudson, K.M., Shaffer, A.L., Constantinescu, A. & Schlissel, M.S. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro . Cell 85, 887–897. (1996).

    Article  CAS  Google Scholar 

  13. Mostoslavsky, R. et al. κ chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev. 12, 1801–1811 (1998).

    Article  CAS  Google Scholar 

  14. McBlane, F. & Boyes, J. Stimulation of V(D)J recombination by histone acetylation. Curr. Biol. 10, 483–486 (2000).

    Article  CAS  Google Scholar 

  15. McMurry, M.T. & Krangel, M.S. A role for histone acetylation in the developmental regulation of VDJ recombination. Science 287, 495–498 (2000).

    Article  CAS  Google Scholar 

  16. Chowdhury, D. & Sen, R. Stepwise activation of the immunoglobulin μ heavy chain gene locus. EMBO J. 20, 6394–6403 (2001).

    Article  CAS  Google Scholar 

  17. Johnson, K., Angelin-Duclos, C., Park, S. & Calame, K.L. 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 (2003).

    Article  CAS  Google Scholar 

  18. Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D. & Oettinger, M.A. Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4. Proc. Natl. Acad. Sci. USA 100, 11577–11582 (2003).

    Article  CAS  Google Scholar 

  19. Maes, J. et al. Chromatin remodeling at the Ig loci prior to V(D)J recombination. J. Immunol. 167, 866–874 (2001).

    Article  CAS  Google Scholar 

  20. Golding, A., Chandler, S., Ballestar, E., Wolffe, A.P. & Schlissel, M.S. Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase. EMBO J. 18, 3712–3723 (1999).

    Article  CAS  Google Scholar 

  21. Kwon, J., Morshead, K.B., Guyon, J.R., Kingston, R.E. & Oettinger, M.A. Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA. Mol. Cell 6, 1037–1048 (2000).

    Article  CAS  Google Scholar 

  22. Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. & Fraser, P. Intergenic transcription and developmental remodeling of chromatin subdomains in the human β-globin locus. Mol. Cell 5, 377–386 (2000).

    Article  CAS  Google Scholar 

  23. Li, Y.S., Hayakawa, K. & Hardy, R.R. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178, 951–960 (1993).

    Article  CAS  Google Scholar 

  24. Allman, D., Li, J. & Hardy, R.R. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J. Exp. Med. 189, 735–740 (1999).

    Article  CAS  Google Scholar 

  25. Rolink, A. et al. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J. Exp. Med. 183, 187–194 (1996).

    Article  CAS  Google Scholar 

  26. Spanopoulou, E. et al. Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8, 1030–1042 (1994).

    Article  CAS  Google Scholar 

  27. Lennon, G.G. & Perry, R.P. Cμ-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5′-nontranslatable exon. Nature 318, 475–478 (1985).

    Article  CAS  Google Scholar 

  28. de Krom, M., van de Corput, M., von Lindern, M., Grosveld, F. & Strouboulis, J. Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited. Mol. Cell 9, 1319–1326 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Li, Y.S., Wasserman, R., Hayakawa, K. & Hardy, R.R. Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5, 527–535 (1996).

    Article  CAS  Google Scholar 

  31. Ehlich, A., Martin, V., Muller, W. & Rajewsky, K. Analysis of the B-cell progenitor compartment at the level of single cells. Curr. Biol. 4, 573–583 (1994).

    Article  CAS  Google Scholar 

  32. Mostoslavsky, R. et al. Asynchronous replication and allelic exclusion in the immune system. Nature 414, 221–225 (2001).

    Article  CAS  Google Scholar 

  33. Skok, J.A. et al. Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes. Nat. Immunol. 2, 848–854 (2001).

    Article  CAS  Google Scholar 

  34. Trimborn, T., Gribnau, J., Grosveld, F. & Fraser, P. Mechanisms of developmental control of transcription in the murine α- and β-globin loci. Genes Dev. 13, 112–124 (1999).

    Article  CAS  Google Scholar 

  35. Allshire, R. Molecular biology. RNAi and heterochromatin—a hushed-up affair. Science 297, 1818–1819 (2002).

    Article  CAS  Google Scholar 

  36. Vanhee-Brossollet, C. & Vaquero, C. Do natural antisense transcripts make sense in eukaryotes? Gene 211, 1–9 (1998).

    Article  CAS  Google Scholar 

  37. Sleutels, F., Zwart, R. & Barlow, D.P. The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415, 810–813 (2002).

    Article  CAS  Google Scholar 

  38. Hebbes, T.R., Clayton, A.L., Thorne, A.W. & Crane-Robinson, C. Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken β-globin chromosomal domain. EMBO J. 13, 1823–1830 (1994).

    Article  CAS  Google Scholar 

  39. Orphanides, G. & Reinberg, D. RNA polymerase II elongation through chromatin. Nature 407, 471–475 (2000).

    Article  CAS  Google Scholar 

  40. Nambu, Y. et al. Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302, 2137–2140 (2003).

    Article  CAS  Google Scholar 

  41. Schlissel, M.S. & Morrow, T. Ig heavy chain protein controls B cell development by regulating germ-line transcription and retargeting V(D)J recombination. J. Immunol. 153, 1645–1657 (1994).

    CAS  PubMed  Google Scholar 

  42. Chowdhury, D. & Sen, R. Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 18, 229–241 (2003).

    Article  CAS  Google Scholar 

  43. Tripathi, R., Jackson, A. & Krangel, M.S. A change in the structure of Vβ chromatin associated with TCRβ allelic exclusion. J. Immunol. 168, 2316–2324 (2002).

    Article  CAS  Google Scholar 

  44. Stevenson, D.S. & Jarvis, P. Chromatin silencing: RNA in the driving seat. Curr. Biol. 13, R13–R15 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Urbanek, P., Wang, Z.Q., Fetka, I., Wagner, E.F. & Busslinger, M. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79, 901–912 (1994).

    Article  CAS  Google Scholar 

  47. 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  Google Scholar 

  48. Shendure, J. & Church, G.M. Computational discovery of sense-antisense transcription in the human and mouse genomes. Genome Biol. 3, 0044.1–0044.14 (2002).

    Article  Google Scholar 

  49. Kiyosawa, H., Yamanaka, I., Osato, N., Kondo, S. & Hayashizaki, Y. Antisense transcripts with FANTOM2 clone set and their implications for gene regulation. Genome Res. 13, 1324–1334 (2003).

    Article  CAS  Google Scholar 

  50. Kirch, S.A., Rathbun, G.A. & Oettinger, M.A. Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly. EMBO J. 17, 4881–4886 (1998).

    Article  CAS  Google Scholar 

  51. Gribnau, J. et al. Chromatin interaction mechanism of transcriptional control in vivo . EMBO J. 17, 6020–6027 (1998).

    Article  CAS  Google Scholar 

  52. Chakalova, L., Carter, D. & Fraser, P. RNA fluorescence in situ hybridization tagging and recovery of associated proteins to analyze in vivo chromatin interactions. Methods Enzymol. 375, 479–493 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank W. Reik, G. Butcher, J. Skok, J. Pell for critically reviewing this manuscript, and C. Osborne for technical help with RNA FISH. Supported by the Association for International Cancer Research (D.B.), Medical Research Council (A.E.C.) and Biotechnology and Biological Sciences Research Council (A.E.C. laboratory).

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Authors and Affiliations

  1. Laboratory of Chromatin and Gene Expression, The Babraham Institute, Cambridge, CB2 4AT, UK

    Daniel J Bolland, Andrew L Wood, Colette M Johnston, Sam F Bunting, Lyubomira Chakalova, Peter J Fraser & Anne E Corcoran

  2. Flow Cytometry Facility, The Babraham Institute, Cambridge, CB2 4AT, UK

    Geoff Morgan

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Correspondence to Anne E Corcoran.

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Bolland, D., Wood, A., Johnston, C. et al. Antisense intergenic transcription in V(D)J recombination. Nat Immunol 5, 630–637 (2004). https://doi.org/10.1038/ni1068

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