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IgH class switching and translocations use a robust non-classical end-joining pathway

Nature volume 449, pages 478–482 (2007)Cite this article

Abstract

Immunoglobulin variable region exons are assembled in developing B cells by V(D)J recombination. Once mature, these cells undergo class-switch recombination (CSR) when activated by antigen. CSR changes the heavy chain constant region exons (Ch) expressed with a given variable region exon from Cμ to a downstream Ch (for example, Cγ, Cε or Cα), thereby switching expression from IgM to IgG, IgE or IgA. Both V(D)J recombination and CSR involve the introduction of DNA double-strand breaks and their repair by means of end joining1,2. For CSR, double-strand breaks are introduced into switch regions that flank Cμ and a downstream Ch, followed by fusion of the broken switch regions1. In mammalian cells, the ‘classical’ non-homologous end joining (C-NHEJ) pathway repairs both general DNA double-strand breaks and programmed double-strand breaks generated by V(D)J recombination2,3. C-NHEJ, as observed during V(D)J recombination, joins ends that lack homology to form ‘direct’ joins, and also joins ends with several base-pair homologies to form microhomology joins3,4. CSR joins also display direct and microhomology joins, and CSR has been suggested to use C-NHEJ5,6,7,8. Xrcc4 and DNA ligase IV (Lig4), which cooperatively catalyse the ligation step of C-NHEJ, are the most specific C-NHEJ factors; they are absolutely required for V(D)J recombination and have no known functions other than C-NHEJ2. Here we assess whether C-NHEJ is also critical for CSR by assaying CSR in Xrcc4- or Lig4-deficient mouse B cells. C-NHEJ indeed catalyses CSR joins, because C-NHEJ-deficient B cells had decreased CSR and substantial levels of IgH locus (immunoglobulin heavy chain, encoded by Igh) chromosomal breaks. However, an alternative end-joining pathway, which is markedly biased towards microhomology joins, supports CSR at unexpectedly robust levels in C-NHEJ-deficient B cells. In the absence of C-NHEJ, this alternative end-joining pathway also frequently joins Igh locus breaks to other chromosomes to generate translocations.

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Figure 1: Proliferation and ionizing radiation sensitivity of Xrcc4-deficient B cells.
Figure 2: CSR in Xrcc4-deficient B cells.
Figure 3: Frequent general and Igh -locus-specific chromosomal breaks and translocations in Xrcc4-deficient B cells.
Figure 4: Structure of Xrcc4-deficient CSR junctions.

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Acknowledgements

We thank D. Schatz for the gift of the Lig4 antibody, M. Alimzhanov, S. Casola and J.-B. Telliez for technical assistance and suggestions, and members of the Alt laboratory for discussions. This work was supported by NIH grants (to F.W.A.), an NIH postdoctoral training grant (to C.T.Y.) and a Long-Term Fellowship of the European Molecular Biology Organization (to S.F.). F.W.A. is an investigator of the Howard Hughes Medical Institute.

Author Contributions F.W.A. and C.T.Y. planned the studies and analysed and interpreted the data. C.T.Y. generated the reagents and performed or oversaw all the experiments described. E.K.S. along with C.T.Y. purified and stimulated XP-T/HL B cells for the studies described. C.B. with C.T.Y. generated and analysed the LP-T/HL mice. E.K.S. generated and analysed (with C.T.Y.) most of the switch junctions and performed all statistical analysis. C.T.Y., T.R.H., S.F. and S.G. analysed metaphases for telomere-FISH and IgH FISH. C.T.Y., T.R.H., E.K.S. and C.B. performed ELISPOT analysis with technical assistance and reagents from A.A.Z. C.B. with E.K.S. isolated and analysed hybridoma sequence junctions; J.P.M. with M.G. provided expertise in B-cell stimulation, fluorescence-activated cell sorting and analysis of sequence junctions; and K.R. provided CD21-cre and HL mice and helped interpret data. F.W.A. and C.T.Y. wrote the paper.

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Authors and Affiliations

  1. Howard Hughes Medical Institute,,

    Catherine T. Yan, Cristian Boboila, Ellen Kris Souza, Sonia Franco, Thomas R. Hickernell, Michael Murphy, Sunil Gumaste, Ali A. Zarrin & Frederick W. Alt

  2. The Children’s Hospital,,

    Catherine T. Yan, Cristian Boboila, Ellen Kris Souza, Sonia Franco, Thomas R. Hickernell, Michael Murphy, Sunil Gumaste, Mark Geyer, Ali A. Zarrin, John P. Manis & Frederick W. Alt

  3. Immune Disease Institute,,

    Catherine T. Yan, Cristian Boboila, Ellen Kris Souza, Sonia Franco, Thomas R. Hickernell, Michael Murphy, Sunil Gumaste, Ali A. Zarrin, Klaus Rajewsky & Frederick W. Alt

  4. Department of Genetics,,

    Catherine T. Yan, Cristian Boboila, Ellen Kris Souza, Sonia Franco, Thomas R. Hickernell, Michael Murphy, Sunil Gumaste, Ali A. Zarrin & Frederick W. Alt

  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA,

    John P. Manis & Klaus Rajewsky

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Correspondence to Frederick W. Alt.

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Yan, C., Boboila, C., Souza, E. et al. IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482 (2007). https://doi.org/10.1038/nature06020

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