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 scanning(3). 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(H)s access to the DJ(H)-RC6-9. 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. Long-distance V(D)J recombination is facilitated by contraction of the Igh locus and linear RAG scanning along chromatin, both driven by cohesin-mediated loop extrusion, which allows recombination of widely separated gene segments to occur.

Automated summary

RAG endonuclease initiates gene rearrangement in B cells by binding to specific DNA sequences and this process is influenced by chromatin structure. In progenitor B cells, locus contraction promotes gene rearrangement and loop extrusion also drives this process.