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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Gene Expression Omnibus
HTGTS sequencing data has been deposited in the GEO database under the accession number GSE71005.
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.
Extended data figures
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.