Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes

Abstract

SUMMARY RAG initiates antibody V(D)J recombination in developing lymphocytes by generating “on-target” DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically-inserted RSS pairs to identify huge numbers of RAG “off-target” breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage-site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation-dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

Highly sensitive and unbiased approach for elucidating antibody repertoires

“…Because V(D)J recombination generates rearrangements with junctions at borders of V, D, and J segments, we can use primers for any of these gene segments as LAM-HTGTS bait to identify sites of RAG-generated DSBs, both in progenitor or precursor lymphocytes undergoing V(D)J recombination, as well as in mature lymphocytes to retrospectively identify V(D)J recombination events that occurred earlier in development. Notably, LAM-HTGTS using endogenous RAG-generated DSBs identified RAG-generated DJ H joins, RSS joins in excision circles, and off-target junctions in developing B-lineage cells that were not detected by prior assays (22), illustrating the high sensitivity of the assay. Based on these earlier studies, we now describe an adaptation of LAM-HTGTS as a robust repertoire-sequencing assay that we term "HTGTS-adapted repertoire sequencing" (HTGTS-Rep-seq).…”

“…LAM-HTGTS, like its predecessor HTGTS (17), employs a single primer for a DSB-associated bait sequence to perform linear amplification across bait-prey junctions to identify all prey sequences joined to the bait DSBs in an unbiased manner (16,18). We have used various types of DSBs as bait for LAM-HTGTS, including those generated by engineered nucleases and endogenous DSBs (17)(18)(19)(20)(21)(22). Because V(D)J recombination generates rearrangements with junctions at borders of V, D, and J segments, we can use primers for any of these gene segments as LAM-HTGTS bait to identify sites of RAG-generated DSBs, both in progenitor or precursor lymphocytes undergoing V(D)J recombination, as well as in mature lymphocytes to retrospectively identify V(D)J recombination events that occurred earlier in development.…”

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

“…9 and this study). Because of increased contact frequency, translocation frequency is increased further between sequences on the same cis chromosome (3,28,29). HTGTS detects only those DSBs that translocate.…”

“…Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref. 9 and this study).…”

“…We previously developed an unbiased, high-throughput, genomewide, translocation sequencing (HTGTS) approach to identify DSBs via their translocation to bait DSBs at defined genomic sites in primary progenitor and activated mature B cells, NSPCs, and human cell lines (4,9,(26)(27)(28). Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref.…”