Loop extrusion mediates physiological Igh locus contraction for RAG scanning.

Abstract

RAG endonuclease initiates IgH V(D)J recombination in pro-B cells by binding a JH-recombination signal sequence (RSS) within a recombination center (RC) and then linearly scanning upstream chromatin, presented by cohesin-mediated loop extrusion, for convergent D-RSSs1,2. Utilization of convergently-oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. RAG scanning from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2–5. Primary pro-B cells undergo a mechanistically-undefined VH locus contraction proposed to provide distal VHs access to the DJH-RC6–9. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both utilization of cryptic VH-locus RSSs normally in opposite orientation and RAG scanning beyond the VH 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 VH 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 VH locus. Correspondingly, Wapl depletion in v-Abl-tranformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.