Loop Extrusion Mediates Physiological IgH Locus Contraction For RAG Scanning

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 scanning3. 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 Wapl10, 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.