The fundamental role of chromatin loop extrusion in physiological V(D)J recombination

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to J H segments, before joining upstream V H segments to DJ H intermediates 1 . In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain 2 at the 3′ end of Igh contains an internal subdomain that spans the 5′ CBE anchor (IGCR1) 3 , the D H segments, and a RAG-bound recombination centre (RC) 4 . The RC comprises the J H -proximal D segment (DQ52), four J H segments, and the intronic enhancer (iEμ) 5 . Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS) 6 . D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented J H -23RSSs and V H -23RSSs, respectively 6 . Despite 12/23 compatibility, inversional D-to-J H joining via upstream D-12RSSs is rare 7 , 8 . Plasmid-based assays have attributed the 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 . As RAG linearly scans convergent CBE-anchored chromatin loops 4 , 12 – 14 , potentially formed by cohesin-mediated loop extrusion 15 – 18 , we revisited its scanning role. Here we show that the chromosomal orientation of J H -23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3′ Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of J H segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3′ Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3′ Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.