AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching (original) (raw)

Nature volume 414, pages 660–665 (2001)Cite this article

Abstract

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction1. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair2,3,4, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in _H2AX_-/- mice. Localization of Nbs1 and γ-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sµ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

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References

  1. 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 Google Scholar
  2. Carney, J. P. et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477–486 (1998).
    Article CAS Google Scholar
  3. Rogakou, E. P., Boon, C., Redon, C. & Bonner, W. M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell. Biol. 146, 905–916 (1999).
    Article CAS Google Scholar
  4. Downs, J. A., Lowndes, N. F. & Jackson, S. P. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001–1004 (2000).
    Article ADS CAS Google Scholar
  5. 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 Google Scholar
  6. Paull, T. T. et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).
    Article CAS Google Scholar
  7. Li, M. J. et al. Rad51 expression and localization in B cells carrying out class switch recombination. Proc. Natl Acad. Sci. USA 93, 10222–10227 (1996).
    Article ADS CAS Google Scholar
  8. Scully, R. et al. Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90, 425–435 (1997).
    Article CAS Google Scholar
  9. Zhu, X. D., Kuster, B., Mann, M., Petrini, J. H. & Lange, T. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nature Genet. 25, 347–352 (2000).
    Article CAS Google Scholar
  10. Chen, H. T. et al. Response to RAG-mediated V(D)J Cleavage by NBS1 and γ-H2AX. Science 290, 1962–1964 (2000).
    Article ADS CAS 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 ADS CAS Google Scholar
  12. 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 Google Scholar
  13. Bross, L. et al. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597 (2000).
    Article CAS Google Scholar
  14. Casellas, R. et al. Ku80 is required for immunoglobulin isotype switching. EMBO J. 17, 2404–2411 (1998).
    Article CAS Google Scholar
  15. Manis, J. P. et al. Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081–2089 (1998).
    Article CAS Google Scholar
  16. Rolink, A., Melchers, F. & Andersson, J. The SCID but not the RAG-2 gene product is required for Sµ-S_ε_ heavy chain class switching. Immunity 5, 319–330 (1996).
    Article CAS Google Scholar
  17. Stavnezer-Nordgren, J. & Sirlin, S. Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5, 95–102 (1986).
    Article CAS Google Scholar
  18. Yancopoulos, G. D. et al. Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching. EMBO J. 5, 3259–3266 (1986).
    Article CAS Google Scholar
  19. Barnes, W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl Acad. Sci. USA 91, 2216–2220 (1994).
    Article ADS CAS Google Scholar
  20. Dunnick, W., Hertz, G. Z., Scappino, L. & Gritzmacher, C. DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372 (1993).
    Article CAS Google Scholar
  21. Kinoshita, K. & Honjo, T. Linking class-switch recombination with somatic hypermutation. Nature Rev. Mol. Cell Biol. 2, 493–503 (2001).
    Article CAS Google Scholar
  22. Winter, E., Krawinkel, U. & Radbruch, A. Directed Ig class switch recombination in activated murine B cells. EMBO J. 6, 1663–1671 (1987).
    Article CAS Google Scholar
  23. Rogozin, I. B. & Kolchanov, N. A. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochim. Biophys. Acta 1171, 11–18 (1992).
    Article CAS Google Scholar
  24. Pasqualucci, L. et al. BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci. Proc. Natl Acad. Sci. USA 95, 11816–11821 (1998).
    Article ADS CAS Google Scholar
  25. Shen, H. M., Peters, A., Baron, B., Zhu, X. & Storb, U. Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes. Science 280, 1750–1752 (1998).
    Article ADS CAS Google Scholar
  26. Dunnick, W., Wilson, M. & Stavnezer, J. Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch. Mol. Cell. Biol. 9, 1850–1856 (1989).
    CAS PubMed PubMed Central Google Scholar
  27. Bemark, M. et al. Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity. J. Exp. Med. 192, 1509–1514 (2000).
    Article CAS Google Scholar
  28. Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S. & Neuberger, M. S. Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926 (2001).
    Article ADS CAS Google Scholar
  29. Brenner, S. & Milstein, C. Origin of antibody variation. Nature 211, 242–243 (1966).
    Article ADS CAS Google Scholar
  30. Casellas, R. et al. Contribution of receptor editing to the antibody repertoire. Science 291, 1541–1544 (2001).
    Article ADS CAS Google Scholar

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Acknowledgements

We thank F. Roschenthaler for Igκ probes; S. Sharrow for help with FACS; S. Ganesan for Brca1 antibodies; and A. Singer, E. Max, M. Gellert, R. Hodes and E. Besmer for comments on the manuscript and discussions. This work was supported in part by grants from the National Institutes of Health and the Leukemia Society to M.C.N. M.C.N. is a Howard Hughes Medical Institute investigator. The first two authors (S.P. and R.C.) contributed equally to this work.

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Author notes

  1. Duane R. Pilch, Christophe Redon, William M. Bonner, Michel C. Nussenzweig and André Nussenzweig: These authors contributed equally to this work

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Simone Petersen, Hua Tang Chen, Arkady Celeste & André Nussenzweig
  2. Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Michael J. Difilippantonio & Thomas Ried
  3. Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Duane R. Pilch, Christophe Redon & William M. Bonner
  4. Laboratory of Molecular Immunology, The Rockefeller University, and Howard Hughes Medical Institute, New York, 10021, New York, USA
    Rafael Casellas, Bernardo Reina-San-Martin, Patrick C. Wilson, Leif Hanitsch & Michel C. Nussenzweig
  5. Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
    Masamichi Muramatsu & Tasuku Honjo

Authors

  1. Simone Petersen
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  2. Rafael Casellas
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  3. Bernardo Reina-San-Martin
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  4. Hua Tang Chen
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  5. Michael J. Difilippantonio
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  6. Patrick C. Wilson
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  7. Leif Hanitsch
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  8. Arkady Celeste
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  9. Masamichi Muramatsu
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  10. Duane R. Pilch
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  11. Christophe Redon
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  12. Thomas Ried
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  13. William M. Bonner
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  14. Tasuku Honjo
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  15. Michel C. Nussenzweig
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  16. André Nussenzweig
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Corresponding author

Correspondence toAndré Nussenzweig.

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Supplementary information

Figure 1. Inactivation of mouse H2AX.

(JPG 18.97 KB)

a, Genomic structure of the wild-type H2AX locus and targeting vector. A genomic clone covering the H2AX locus was isolated from a 129/Sv mouse lambda phage library (Stratagene). The targeting construct was designed as a null mutation that replaces a segment of H2AX between the unique Asc1 and Xho1 site in the coding sequence by a neomycin resistance cassette. b, Southern blot of XhoI-digested tail DNA from wildtype (+/+), heterozygous (+/-) and homozygous (-/-) H2AX-targeted mice hybridized with a probe indicated in a. The wild-type and mutant fragments are 8.8 and 10.6 kb respectively.

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Petersen, S., Casellas, R., Reina-San-Martin, B. et al. AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching.Nature 414, 660–665 (2001). https://doi.org/10.1038/414660a

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