Letter | Published:

Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching

Nature volume 525, pages 134139 (03 September 2015) | Download Citation

Abstract

During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons1,2. In mice, six additional sets of constant region exons (CHs) lie 100–200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons2. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region2,3; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors3. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing4 into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.

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Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

HTGTS sequencing data has been deposited in the GEO database under the accession number GSE71005.

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Acknowledgements

We thank K. Yu for providing the CH12F3-RMCE (1F7) cell line and exchange cassette plasmid. This work was supported by National Institute of Health grants AI077595 to F.W.A., CA133781 to J.M., AI112602 to D.F.R., and AI037526 and AI072529 to M.C.N. S.V. was supported by NIH training grant T32HL066987. F.W.A. and M.C.N. are investigators of the Howard Hughes Medical Institute. J.H. is supported by a Robertson Foundation/Cancer Research Institute Irvington Fellowship. F.M. is a Lymphoma Research Foundation postdoctoral fellow and was a Cancer Research Institute postdoctoral fellow.

Author information

Author notes

    • Tingting Zhang
    • , Yu Zhang
    • , Yu-Jui Ho
    •  & Monica Gostissa

    Present addresses: Eli Lilly and Company, Alexandria Center for Life Sciences, 450 East 29th Street, New York, New York 10016, USA (T.Z.); National Institute of Biological Sciences, Beijing 102206, Beijing, China (Y.Z.); Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Cold Spring Harbor, New York 11724, USA (Y.-J.H.); 121Bio, 700 Main Street, Cambridge, Massachusetts 02139, USA (M.G.).

    • Junchao Dong
    • , Rohit A. Panchakshari
    •  & Tingting Zhang

    These authors contributed equally to this work.

Affiliations

  1. Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Junchao Dong
    • , Rohit A. Panchakshari
    • , Tingting Zhang
    • , Yu Zhang
    • , Jiazhi Hu
    • , Robin M. Meyers
    • , Yu-Jui Ho
    • , Zhou Du
    • , Feilong Meng
    • , Monica Gostissa
    •  & Frederick W. Alt
  2. Boston Children’s Hospital and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Sabrina A. Volpi
    •  & John P. Manis
  3. Howard Hughes Medical Institute, Laboratory of Molecular Immunology, The Rockefeller University, New York, New York 10065, USA

    • Davide F. Robbiani
    •  & Michel C. Nussenzweig

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Contributions

J.D., R.P., T.Z., J.P.M. and F.W.A. designed the study; J.D., R.P., T.Z., J.H. and S.V. performed experiments; Y.H. and R.M. designed bioinformatics pipelines; R.M., J.D., R.P. and Z.D. performed computational analyses of sequencing data; J.D., R.P. and F.W.A. wrote the paper. Other authors provided reagents, designed or performed certain experiments, and helped revise the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to John P. Manis or Frederick W. Alt.

Extended data

Extended data figures

  1. 1.

    Deletional CSR in in vitro activated B cells by DC-PCR; I-Sce1 DSBs within the Igh constant region locus in activated B cells join with orientation-independence.

  2. 2.

    Genome-wide translocation junctions lack orientation bias; statistical analyses for experimental replicates orientation-biased joining between I-SceI break in place of Sα and AID-initiated Sμ breaks in CH12F3 cells.

  3. 3.

    Joining between I-SceI break at Sμ and AID-initiated S-region breaks in lipopolysaccharide (LPS)-activated ΔSμ2×I/ΔSγ12×I B cells and clustered I-Sce1 breaks in ΔSμ2×I/ΔSγ128×I B cells in place of Sγ1; inverted Sμ in CH12F3 cells support robust IgA CSR.

  4. 4.

    Orientation-biased joining between AID-initiated rSμ and downstream AID-initiated S-region breaks in anti-CD40/IL4-activated and LPS-activated Sμ-truncated B cells.

  5. 5.

    Orientation-biased joining between rSμ and AID-induced Sα DSBs in CSR-activated CH12F3 cells.

  6. 6.

    Level of junctions to downstream S regions in wild-type and DSBR-deficient 5′Sμ HTGTS libraries correlate with CSR levels; 5′Sμ break site undergoes variable degrees of resection from stimulated DSBR-deficient B cells.

  7. 7.

    Orientation-biased joining between rSμ and AID-induced Sγ and Sε DSBs in wild-type, ATM-deficient, and 53BP1-deficient B cells.

  8. 8.

    Orientation-biased joining of I-SceI DSBs at Sγ1 to AID-induced S-region breaks in various DSBR-deficient backgrounds.

  9. 9.

    Inhibition of resection in 53BP1-deficient B cells by an ATM inhibitor (ATMi) does not rescue directional CSR joining to Sγ1.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Text, full legends for Extended Data Figures 1-9 and Extended Data Table 1, and 2 Supplementary Tables, which show the DNA oligos and Plotting coordinates used in this study.

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DOI

https://doi.org/10.1038/nature14970

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