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AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line

Nature Immunology volume 3, pages 815–821 (2002)Cite this article

Abstract

Immunoglobulin (Ig) gene hypermutation can be induced in the BL2 Burkitt's lymphoma cell line by IgM cross-linking and coculture with normal or transformed T helper clones. We describe here a T cell–independent in vitro induction assay, by which hypermutation is induced in BL2 cells through simultaneous aggregation of three surface receptors: IgM, CD19 and CD21. The mutations arise as a post-transcriptional event within 90 min. They are stably introduced in the G1 phase of the cell cycle, occurring in one of the two variable gene DNA strands, and eventually become fixed by replication in one of the daughter cells. Inactivation of AID (activation-induced cytidine deaminase) by homologous recombination in BL2 cells completely inhibits the process, thus validating this induction procedure as a model for the in vivo mechanism.

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Figure 1: Stable induction of V gene mutations in the BL2 cell line in the G1 phase of the cell cycle.
Figure 2: Occurrence of somatic mutation in one DNA strand in the G1 phase of the cell cycle.
Figure 3: Inactivation, restoration and overexpression of AICDA in the BL2 cell line.
Figure 4: Detection of DNA breaks in the normal and AID−/− BL2 cell line before and after stimulation with the three-antibody procedure.

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References

  1. Weigert, M.G., Cesari, I.M., Yonkovich, S.J. & Cohn, M. Variability in the λ light chain sequences of mouse antibody. Nature 228, 1045–1047 (1970).

    Article  CAS  PubMed  Google Scholar 

  2. Wagner, S.D. & Neuberger, M.S. Somatic hypermutation of immunoglobulin genes. Annu. Rev. Immunol 14, 441–457 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. Reynaud, C.A. & Weill, J.C. Postrearrangement diversification processes in gut-associated lymphoid tissues. Curr. Top. Microbiol. Immunol. 212, 7–15 (1996).

    CAS  PubMed  Google Scholar 

  4. Weller, S. et al. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proc. Natl. Acad. Sci. USA 98, 1166–1170 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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 

  6. Revy, P. et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Arakawa, H., Hauschild, J. & Buerstedde, J.M. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295, 1301–1306 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Harris, R.S., Sale, J.E., Petersen-Mahrt, S.K. & Neuberger, M.S. AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr. Biol. 12, 435–438 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Martin, A. et al. Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas. Nature 415, 802–806 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Sale, J.E. & Neuberger, M.S. TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line. Immunity 9, 859–869 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Papavasiliou, F.N. & Schatz, D.G. Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes. Nature 408, 216–221 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Bross, L. et al. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Kong, Q. & Maizels, N. DNA breaks in hypermutating immunoglobulin genes: evidence for a break-and-repair pathway of somatic hypermutation. Genetics 158, 369–378 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Peters, A. & Storb, U. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4, 57–65 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Goyenechea, B. et al. Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers. EMBO J. 16, 3987–3994 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Denepoux, S. et al. Induction of somatic mutation in a human B cell line in vitro. Immunity 6, 35–46 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Zan, H. et al. Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation. J. Immunol. 162, 3437–3447 (1999).

    CAS  PubMed  Google Scholar 

  18. Rogozin, I.B. & Kolchanov, N.A. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequence on mutagenesis. Biochim. Biophys. Acta 1171, 11–18 (1992).

    Article  CAS  PubMed  Google Scholar 

  19. Mittrucker, H.W., Muller-Fleckenstein, I., Fleckenstein, B. & Fleischer, B. Herpes virus saimiri-transformed human T lymphocytes: normal functional phenotype and preserved T cell receptor signalling. Int. Immunol. 5, 985–990 (1993).

    Article  CAS  PubMed  Google Scholar 

  20. Poltoratsky, V. et al. Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation. Proc. Natl. Acad. Sci. USA 98, 7976–7981 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lazebnik, Y., Kaufmann, S., Desnoyers, S., Poirier, G. & Earnshaw, W. Cleavage of poly(ADP-ribose)polymerase by a proteinase with properties like ICE. Nature 371, 346–347 (1994).

    Article  CAS  PubMed  Google Scholar 

  22. Pasqualucci, L. et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412, 341–346 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Muto, T., Muramatsu, M., Taniwaki, M., Kinoshita, K. & Honjo, T. Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene. Genomics 68, 85–88 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Levy et al. Defect in IgV gene somatic hypermutation in Common Variable Immuno-Deficiency Syndrome. Proc. Natl. Acad. Sci. USA 95, 13135–13140 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Denepoux, S., Fournier, N., Peronne, C., Banchereau, J. & Lebecque, S. T cells can induce somatic mutation in B cell receptor-engaged BL2 Burkitt's lymphoma cells independently of CD40-CD40 ligand interactions. J. Immunol. 164, 1306–1313 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Fearon, D.T. & Carroll, M.C. Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex. Annu. Rev. Immunol. 18, 393–422 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Zhang, W. et al. Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line. Int. Immunol. 13, 1175–1184 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Rada, C., Jarvis, J.M. & Milstein, C. AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization. Proc. Natl. Acad. Sci. USA 99, 7003–7008 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bertocci, B. et al. Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process. Immunity 9, 257–265 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Papavasiliou, F.N. & Schatz, D.G. The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process. J. Exp. Med. 195, 1193–1198 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bross, L., Muramatsu, M., Kinoshita, K., Honjo, T. & Jacobs . DNA double-strand breaks: prior to but not sufficient in targeting hypermutation. J. Exp. Med. 195, 1187–1192 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zeng, X. et al. DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nature Immunol. 2, 537–541 (2001).

    Article  CAS  Google Scholar 

  33. Zan, H. et al. The translesion DNA polymerase ζ plays a major role in Ig and bcl-6 somatic hypermutation. Immunity 14, 643–653 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Diaz, M., Verkoczy, L.K., Flajnik, M.F. & Klinman, N.R. Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase ζ. J. Immunol. 167, 327–335 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Diaz, M. & Casali, P. Somatic immunoglobulin hypermutation. Curr. Opin. Immunol. 14, 235–240 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bachl, J. & Wabl, M. An immunoglobulin mutator that targets G. C base pairs. Proc. Natl. Acad. Sci. USA 93, 851–855 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rada, C., Ehrenstein, M.R., Neuberger, M.S. & Milstein, C. Somatic hypermutation in MSH2-deficient mice is more focused on intrinsic hot spots suggesting hypermutation targeting to be a two stage process. Immunity 9, 135–141 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Weill, J.-C. et al. Ig gene hypermutation: a mechanism is due. Adv. Immunol. 80, 183–202 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Baba, T.W., Giroir, B.P. & Humphries, E.H. Cell lines derived from avian lymphomas exhibit two distinct phenotypes. Virology 144, 139–151 (1985).

    Article  CAS  PubMed  Google Scholar 

  40. Aoufouchi, S. et al. Two novel human and mouse DNA polymerases of the polX family. Nucleic Acids Res. 28, 3684–3693 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Aoufouchi, S. Monoclonal antibodies A4. 3.3; A6.4.12; B3.3.9; B15.4.13 anti-poly(ADP-ribose) polymerase. Hybridoma 16, 583 (1997).

  42. Schlissel, M.S. Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination. Mol. Cell. Biol. 18, 2029–2037 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Mueller, P.R. & Wold, B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786 (1989).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S. Lebecque for his advice on the BL2 cell line; B. Fleckenstein for the CB15 cell line; M. Braun for identifying the nonfunctional heavy chain allele of the BL2 cell line; A. Dahan for advice with the LM-PCR assay; A. Alcaïs for help with statistical analysis; and C. Hivroz for testing signal transduction pathways during the induction assay. Supported by the Fondation Princesse Grace de Monaco and the Ligue Nationale Française contre le Cancer (Equipe labellisée). A. F. and S. A. were supported during part of this work by the Fondation de France (Fondation contre la Leucémie).

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Author notes

  1. Jean-Claude Weill and Claude-Agnès Reynaud: These authors share senior authorship.

Authors and Affiliations

  1. INSERM U373, Faculté de Médecine Necker-Enfants Malades, Paris, France

    Ahmad Faili, Said Aoufouchi, Quentin Guéranger, Carole Zober, Barbara Bertocci, Jean-Claude Weill & Claude-Agnès Reynaud

  2. EFS, Site Henri Mondor, Créteil, France

    Anne Léon

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Supplementary information

Web Fig. 1.

Monitoring apoptosis, which occurs during induction of somatic hypermutation in the BL2 cell line through PARP proteolysis. Cells were induced to undergo V gene hypermutation (see Methods). At the indicated time points after induction, cell lysates were analyzed by immunoblotting with anti-PARP antibodies and HRP-coupled secondary antibodies. Appearance of the 86 kD cleavage product of the PARP protein monitors apoptosis occurring in BL2 cells. (JPG 5 kb)

Web Fig. 2.

Distribution of mutations obtained by the three antibodies stimulation procedure along the V4-39-JH5 gene of the BL2 cell line. The distribution of mutations listed in Table 2 is represented along the V4-39-JH5 sequence, with open triangles for deletions and closed triangles for insertions. The position of CDRs (complementarity-determining regions) is indicated. (JPG 60 kb)

Web Fig. 3.

Control of RNA transcription inhibition by actinomycin D during induction of somatic mutation in the BL2 cell line. Quantification of the unstable c-Myc transcripts versus actin transcripts was performed at the following time points: time 0, before incubation of BL2 with actinomycin D; 2 h, after 2 h incubation with actinomycin D at which time cells were induced for somatic mutation; 4 h 30, at the end of the induction procedure at which time mutations are analyzed (1 h at 4 °C with the antibody cocktail, followed by 90 min incubation at 37 °C). Mutation data are shown in Table 4. (JPG 4 kb)

Web Table 1 (PDF 16 kb)

Web Methods (PDF 24 kb)

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Faili, A., Aoufouchi, S., Guéranger, Q. et al. AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line. Nat Immunol 3, 815–821 (2002). https://doi.org/10.1038/ni826

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