Robust V(D)J Recombination in the Absence of SA1/2-mediated Chromatin Loops

Abstract

The cohesin ring complex organizes and mediates functional activity of the genome through chromatin loop extrusion1,2. Cohesin comprises a core SMC1-SMC3-RAD21 ring associated with multiple regulatory proteins, including SA1 and SA2 paralogs that bind mutually-exclusively to a RAD21 site3,4. Functional studies of cohesin complexes in vivo often employ inactivation of particular components2,3. However, inactivation of SA1 or SA2 in mice is limited by mouse embryonic lethality5-7 and, for dual SA1/2-inactivation by cycling cell lethality8-10. For these reasons, it remains unknown whether the requirement for SA1/2 in loop extrusion in vitro11,12 applies in vivo1,2, and whether these factors have overlapping or distinct functions in regulating this process3. Here, we employ G0/G1-arrested v-Abl pro-B lines, which are viable in the absence of cohesin-complex factors13,14, to dissect SA1 and SA2 functions in V(D)J recombination, a strictly G0/G1-specific process that harnesses loop extrusion to effect distinct Igh and Ig{kappa} locus recombination mechanisms15,16. Robust long-range Igh or Ig{kappa} V(D)J recombination continued unabated in the absence of SA1 or SA2. However, inactivation of both abrogated most positioned chromatin loops genome-wide and also abolished long-range Igh V(D)J recombination with a phenotype similar to RAD21 (cohesin) inactivation13. Yet, robust Ig{kappa} V(D)J recombination continued upon dual SA1/2-inactivation, which along with Hi-C findings, demonstrates ongoing loop-extrusion in the absence of SA1/2. Correspondingly, replacement of weak Igh V(D)J recombination signal sequences (RSSs) that target V(D)J recombination activity, with strong Ig{kappa} counterparts15 restored long-range Igh V(D)J recombination in SA1/2 inactivated cells. We conclude that SA1/2 together maintain loop extrusion impediments and genome-wide architecture in G0/G1-arrested cells; and, in their absence, loop extrusion proceeds unimpeded. Remarkably, strong RSSs allow Ig{kappa} to function normally in the absence of internal positioned loop structure across the 3.1-mega-base Ig{kappa} locus. We discuss implications of our findings for long-range gene regulation in G0/G1-arrested cells more broadly.