Loop Extrusion Mediates Physiological Locus Contraction for V(D)J Recombination

Abstract

AbstractImmunoglobulin heavy chain locus (Igh) VH, D, and JHgene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation1,2. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJHjoining via a mechanistically-undefined VH-locus contraction process3-7. Here, we report that a 2.4 mega-base VHlocus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contractionper seis maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription ofWapl8, a cohesin-unloading factor, versus levels inv-Ablpro-B lines that lack marked locus contraction or distal VHrearrangements2,9-11. Correspondingly, Wapl depletion inv-Abllines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJHjoining both are regulated via circumvention of CBE scanning impediments.