Loop extrusion mediates physiological Igh locus contraction for RAG scanning

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J_H-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs^1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning³. Scanning of RAG from the DJ_H-RC-RSS to upstream convergent V_H-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)^2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the V_H locus that is proposed to provide distal V_Hs access to the DJ_H-RC^6-9. Here we report that an inversion of the entire 2.4-Mb V_H locus in mouse primary progenitor 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 the utilization of cryptic V_H-RSSs that are normally in opposite orientation and RAG scanning beyond the V_H locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the V_H locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)¹⁰, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the V_H locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.