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.

RAG endonuclease initiates IgH V(D)J recombination in pro-B cells by binding a J H -recombination signal sequence (RSS) within a recombination center (RC) and then linearly scanning upstream chromatin, presented by cohesin-mediated loop extrusion, for convergent D-RSSs 1 , 2 . Utilization of convergently-oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning 3 . RAG scanning from the DJ H -RC-RSS to upstream convergent V H -RSSs is impeded by D-proximal CTCF-binding elements (CBEs) 2 – 5 . Primary pro-B cells undergo a mechanistically-undefined V H locus contraction proposed to provide distal V H s access to the DJ H -RC 6 – 9 . Here, we report that a 2.4 mega-base V H locus inversion in primary pro-B cells abrogates rearrangement of both V H -RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both utilization of cryptic V H -locus RSSs normally in opposite orientation and RAG scanning beyond the V H locus through multiple convergent-CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary pro-B cells promotes loop extrusion-mediated RAG V H locus-scanning. We further found that expression of Wapl 10 , a cohesin-unloading factor, is low in primary pro-B cells versus v-Abl -transformed pro-B lines that lack contraction and RAG-scanning of the V H locus. Correspondingly, Wapl depletion in v-Abl -tranformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.