Loop extrusion mediates physiological Igh locus contraction for RAG scanning

Author(s): Hai-Qiang Dai [sup.1] [sup.2] , Hongli Hu [sup.1] [sup.2] , Jiangman Lou [sup.1] [sup.2] , Adam Yongxin Ye [sup.1] [sup.2] , Zhaoqing Ba [sup.1] [sup.2] , Xuefei Zhang [sup.1] [...]
RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J.sub.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.sup.1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning.sup.3. Scanning of RAG from the DJ.sub.H-RC-RSS to upstream convergent V.sub.H-RSSs is impeded by D-proximal CTCF-binding elements (CBEs).sup.2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the V.sub.H locus that is proposed to provide distal V.sub.Hs access to the DJ.sub.H-RC.sup.6-9. Here we report that an inversion of the entire 2.4-Mb V.sub.H locus in mouse primary progenitor B cells abrogates rearrangement of both V.sub.H-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic V.sub.H-RSSs that are normally in opposite orientation and RAG scanning beyond the V.sub.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.sub.H locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL).sup.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.sub.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.