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 extrusion ^1,2 . 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 site ^3,4 . Functional studies of cohesin complexes in vivo often employ inactivation of particular components ^2,3 . However, inactivation of SA1 or SA2 in mice is limited by mouse embryonic lethality ^5-7 and, for dual SA1/2-inactivation by cycling cell lethality ^8-10 . For these reasons, it remains unknown whether the requirement for SA1/2 in loop extrusion in vitro ^11,12 applies in vivo ^1,2 , and whether these factors have overlapping or distinct functions in regulating this process ³ . Here, we employ G0/G1-arrested v-Abl pro-B lines, which are viable in the absence of cohesin-complex factors ^13,14 , 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 Igκ locus recombination mechanisms ^15,16 . Robust long-range Igh or Igκ 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) inactivation ¹³ . Yet, robust Igκ 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 Igκ counterparts ¹⁵ 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 Igκ to function normally in the absence of internal positioned loop structure across the 3.1-mega-base Vκ locus. We discuss implications of our findings for long-range gene regulation in G0/G1-arrested cells more broadly.