Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

An evolutionarily conserved target motif for immunoglobulin class-switch recombination

Nature Immunology volume 5, pages 1275–1281 (2004)Cite this article

Abstract

Immunoglobulin H class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis switch region Sμ, which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis Sμ–mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID–replication protein A complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID–replication protein A complex.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: R-loop formation of XSμ versus mouse Sγ1.
Figure 2: Targeted replacement of the Sγ1a allele.
Figure 3: RT-PCR analysis of the Ighγ1 germline transcripts.
Figure 4: ELISA of IgG1 in the supernatants of splenocyte cultures.
Figure 5: Sequence analysis of Sμ to XSμ+, XSμ−, Xpf+ and Xpf− hybridoma junctions.
Figure 6: Sμ to XSμ switch junctions.
Figure 7: The AID-RPA complex deaminates the XSμ region in vitro.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Jung, D. & Alt, F.W. Unraveling V(D)J recombination; insights into gene regulation. Cell 116, 299–311 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Martin, A. & Scharff, M.D. AID and mismatch repair in antibody diversification. Nat. Rev. Immunol. 2, 605–614 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Reynaud, C.A., Aoufouchi, S., Faili, A. & Weill, J.C. What role for AID: mutator, or assembler of the immunoglobulin mutasome? Nat. Immunol. 4, 631–638 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Manis, J.P., Tian, M. & Alt, F.W. Mechanism and control of class-switch recombination. Trends Immunol. 23, 31–39 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Honjo, T., Muramatsu, M. & Fagarasan, S. AID: how does it aid antibody diversity? Immunity 20, 659–668 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Luby, T.M., Schrader, C.E., Stavnezer, J. & Selsing, E. The μ switch region tandem repeats are important, but not required, for antibody class switch recombination. J. Exp. Med. 193, 159–168 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shinkura, R. et al. The influence of transcriptional orientation on endogenous switch region function. Nat. Immunol. 4, 435–441 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Khamlichi, A.A. et al. Immunoglobulin class-switch recombination in mice devoid of any S μ tandem repeat. Blood 103, 3828–3836 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Dunnick, W., Hertz, G.Z., Scappino, L. & Gritzmacher, C. DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. 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 

  12. Doi, T., Kinoshita, K., Ikegawa, M., Muramatsu, M. & Honjo, T. De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl. Acad. Sci. USA 100, 2634–2638 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Begum, N.A. et al. De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. USA 101, 13003–13007 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Begum, N.A. et al. Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch. Science 305, 1160–1163 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Chaudhuri, J. & Alt, F.W. Class-switch recombination: interplay of transcription, DNA deamination and DNA repair. Nat. Rev. Immunol. 4, 541–552 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Petersen-Mahrt, S.K., Harris, R.S. & Neuberger, M.S. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Bransteitter, R., Pham, P., Scharff, M.D. & Goodman, M.F. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A. & Bhagwat, A.S. Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Res. 31, 2990–2994 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chaudhuri, J. et al. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Dickerson, S.K., Market, E., Besmer, E. & Papavasiliou, F.N. AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ramiro, A.R., Stavropoulos, P., Jankovic, M. & Nussenzweig, M.C. Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat. Immunol. 4, 452–456 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Yu, K., Huang, F.T. & Lieber, M.R. DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279, 6496–6500 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Di Noia, J. & Neuberger, M.S. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Rada, C. et al. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Imai, K. et al. Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat. Immunol. 4, 1023–1028 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  27. Chaudhuri, J., Khuong, C. & Alt, F.W. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Tian, M. & Alt, F.W. Transcription-induced cleavage of immunoglobulin switch regions by nucleotide excision repair nucleases in vitro. J. Biol. Chem. 275, 24163–24172 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E. & Lieber, M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Rogozin, I.B. & Diaz, M. Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. J. Immunol. 172, 3382–3384 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Betz, A.G., Neuberger, M.S. & Milstein, C. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14, 405–411 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Binz, S.K., Sheehan, A.M. & Wold, M.S. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair (Amst) 3, 1015–1024 (2004).

    Article  CAS  Google Scholar 

  33. Petersen, S. et al. AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nagaoka, H., Muramatsu, M., Yamamura, N., Kinoshita, K. & Honjo, T. Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Sμ region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation. J. Exp. Med. 195, 529–534 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zeng, X., Negrete, G.A., Kasmer, C., Yang, W.W. & Gearhart, P.J. Absence of DNA polymerase {eta} reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions. J Exp. Med. 199, 917–924 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Du Pasquier, L. The immune system of invertebrates and vertebrates. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 129, 1–15 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Mussmann, R., Courtet, M., Schwager, J. & Du Pasquier, L. Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals. Eur. J. Immunol. 27, 2610–2619 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Kitao, H., Arakawa, H., Yamagishi, H. & Shimizu, A. Chicken immunoglobulin μ-chain gene: germline organization and tandem repeats characteristic of class switch recombination. Immunol. Lett. 52, 99–104 (1996).

    Article  CAS  PubMed  Google Scholar 

  39. Cogne, M. et al. A class switch control region at the 3′ end of the immunoglobulin heavy chain locus. Cell 77, 737–747 (1994).

    Article  PubMed  Google Scholar 

  40. Seidl, K.J. et al. An expressed neor cassette provides required functions of the 1γ2b exon for class switching. Int. Immunol. 10, 1683–1692 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Sternberg, N. & Hoess, R. The molecular genetics of bacteriophage P1. Annu. Rev. Genet. 17, 123–154 (1983).

    Article  CAS  PubMed  Google Scholar 

  42. Tashiro, J., Kinoshita, K. & Honjo, T. Palindromic but not G-rich sequences are targets of class switch recombination. Int. Immunol. 13, 495–505 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Bardwell, P.D. et al. Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nat. Immunol. 5, 224–229 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Petersen-Mahrt, S.K. & Neuberger, M.S. In vitro deamination of cytosine to uracil in single-stranded DNA by apolipoprotein B editing complex catalytic subunit 1 (APOBEC1). J. Biol. Chem. 278, 19583–19586 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Flajnik, M.F. Comparative analyses of immunoglobulin genes: surprises and portents. Nat. Rev. Immunol. 2, 688–698 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Pasqualucci, L., Neri, A., Baldini, L., Dalla-Favera, R. & Migliazza, A. BCL-6 mutations are associated with immunoglobulin variable heavy chain mutations in B-cell chronic lymphocytic leukemia. Cancer Res. 60, 5644–5648 (2000).

    CAS  PubMed  Google Scholar 

  47. Kuppers, R. & Dalla-Favera, R. Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene 20, 5580–5594 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Ramiro, A.R. et al. AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118, 431–438 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Tian, M., Shinkura, R., Shinkura, N. & Alt, F.W. Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF. Mol. Cell Biol. 24, 1200–1205 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 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 

Download references

Acknowledgements

We thank Y. Fujiwara, T. Borjeson and A. Williams for mouse work, and J. Manis, R. Shinkura, C. Giallourakis, E. Pinaud, J. Wang and S. Ranganath for discussions. Supported by National Institutes of Health (AI31541 to F.W.A. and AI07512 to M.T.), National Cancer Institute of Canada (A.A.Z.) and Howard Hughes Medical Institute (F.W.A.).

Author information

Author notes

  1. Ali A Zarrin and Ming Tian: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, The Children's Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard University Medical School, Boston, 02115, Massachusetts, USA

    Ali A Zarrin, Frederick W Alt, Jayanta Chaudhuri, Nicole Stokes, Dhruv Kaushal & Ming Tian

  2. Institute of Zoology, University of Basel, Basel, Switzerland

    Louis Du Pasquier

Authors
  1. Ali A Zarrin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frederick W Alt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jayanta Chaudhuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicole Stokes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dhruv Kaushal
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Louis Du Pasquier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ming Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frederick W Alt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Correlation of AGCT density and CSR breakpoint frequency. (PDF 137 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zarrin, A., Alt, F., Chaudhuri, J. et al. An evolutionarily conserved target motif for immunoglobulin class-switch recombination. Nat Immunol 5, 1275–1281 (2004). https://doi.org/10.1038/ni1137

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1137

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing