Recombination activating gene 1/2 endonuclease (RAG) initiates V(D)J recombination in developing B and T lymphocytes via “on-target” activity at bona fide recombination signal sequences (RSSs) to generate diverse antibodies. “Off-target” RAG activity is thought to be rare, but can cause oncogenic interstitial deletions or chromosomal translocations that contribute to immature B and T lymphocyte cancers. Given that potential RAG off-target sites occur frequently in the genome, it has been a mystery how this potentially dangerous activity is suppressed. We generated mice with a pair of bona fide V(D)J RSSs (“V(D)J cassette”) in the first intron of c-myc on mouse chromosome 15. When introduced into an ATM-deficient background, the c-myc V(D)J cassette-containing mice recurrently develop B cell lymphomas with oncogenic c-myc translocations/amplifications involving the Igh locus on chromosome 12. These amplifications are generated by breakage-fusion-bridge (“BFB”) cycles from dicentric (15;12) chromosomes formed through fusion of RAG-generated Igh double-stranded breaks (DSBs) to sequences downstream of c-myc. Notably, the translocations downstream of c-myc occurred exclusively on V(D)J cassette-containing alleles, but did not involve the cassette. Based on this finding, we proposed that the V(D)J cassette mediates DSBs downstream of c-myc by activating RAG cutting at linked cryptic RSSs. We developed a linear amplification-mediated (“LAM”) version of our high-throughput genome-wide translocation sequencing approach (“LAM-HTGTS”) to robustly identify “prey” endogenous DNA DSBs that join to introduced or endogenous “bait” DSBs. We used LAM-HTGTS to reveal genome-wide DSBs that joined to RAG-generated bait DSBs. We found that translocations between RAG-induced DSBs in the c-myc V(D)J cassette to Igh occur only in the absence of ATM. Strikingly, however, we discovered a large number of deletions from the c-myc cassette to sequences just upstream of c-myc (1 kb) and up to 1.7 Mb downstream of c-myc in WT and ATM-deficient cells. Moreover, insertion of a pair of bona fide RSSs into 12 random sites genome-wide induced large numbers of RAG-initiated DSBs leading to cassette involved deletions and inversions spreading upstream and downstream of a given cassette from 200 kb to over 2 Mb, respectively. In each case, regions of RAG off-target activity fell into a well-defined topologically associated domain (TAD) into which the cassette integrated. Such domains generally were flanked by convergent CTCF binding sites (CBEs), such as those our lab implicated in Igh locus V(D)J recombination control, but sometimes also involved sub-domains of a CTCF-defined TAD. We showed these TAD-associated DSBs to enrich at “cryptic RSSs” determined by conserved “CAC” motifs. We also captured these DSBs via LAM-HTGTS from Cas9/gRNA DSBs within a TAD and proved them to be RAG-generated. We found dramatic changes of on-target profiles and expansion of off-target RAG-generated Igh locus DSBs when CBEs acting as boundary of DH-JH recombination domain were deleted. Based on these overall findings, we propose that antigen receptor loci exploit fixed CBE-based TADs to restrict RAG activity within them, but also evolved “regulated” sub-domains to further focus and re-focus RAG activity developmentally. We also used LAM-HTGTS to identify 89 RAG off-targets that participate in chromosomal translocations and show RAG off-targets occur in pairs in highly accessible regions such as enhancers and promoters within a TAD. Beyond providing a mechanisms for the generation of recurrent B cell lymphomas in ATM-deficient mice with a V(D)J cassette in c-myc, our findings help explain the origin of certain recurrent interstitial deletions and translocations in tumors that arise from human developing lymphocytes.
Citation Format: Jiazhi Hu, Yu Zhang, Lijuan Zhao, Richard Frock, Zhou Du, Robin M. Meyers, Feilong Meng, David G. Schatz, Frederick W. Alt. Topologically associated domains genome-wide restrict the off-target activity of recombination activating gene 1/2 endonuclease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A180.