The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to J(H)s, before joining upstream V(H)s to DJ(H) intermediates(1). In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain(2) at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1)(3), the D(H)s, and a RAG-bound recombination center (RC)(4). The RC comprises J(H)-proximal D (DQ52), 4 J(H)s, and the intronic enhancer (“iEμ”)(5). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs)(6). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented J(H)-23RSSs and V(H)-23RSSs(6). Despite 12/23 compatibility, inversional D to J(H) joining via upstream D-12RSSs is rare(7,8). Plasmid-based assays attributed lack of inversional D to J(H) joining to sequence-based preference for downstream D-12RSSs(9), as opposed to putative linear scanning mechanisms(10,11). Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops(4,12-14), potentially formed by cohesin-mediated loop extrusion(15-18), we revisited a scanning role. Here, we report that J(H)-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of J(H)s, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.