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

Immunoglobulin heavy chain locus () V, D, and J gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a J-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 DJ-RC formation. In primary pro-B cells, 100s of upstream V-associated RSSs, embedded in convergent orientation to the DJ-RC-RSS, gain proximity to the DJ-RC for V-to-DJ joining via a mechanistically-undefined V-locus contraction process. Here, we report that a 2.4 mega-base V locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent V-RSSs and cryptic RSSs, even though locus contraction is maintained. Moreover, this inversion activated rearrangement of both cryptic V-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 of , a cohesin-unloading factor, versus levels in pro-B lines that lack marked locus contraction or distal V rearrangements. Correspondingly, Wapl depletion in lines activated V-locus contraction and orientation-specific RAG-scanning across the V-locus. Our findings indicate that locus contraction and physiological V-to-DJ joining both are regulated via circumvention of CBE scanning impediments.