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 JH segments, before joining upstream VH segments to DJH intermediates1 . In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1)3 , the DH segments, and a RAG-bound recombination centre (RC)4 . The RC comprises the JH -proximal D segment (DQ52), four JH 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 JH -23RSSs and VH -23RSSs, respectively6 . Despite 12/23 compatibility, inversional D-to-JH joining via upstream D-12RSSs is rare7,8 . Plasmid-based assays have attributed the lack of inversional D-to-JH joining to sequence-based preference for downstream D-12RSSs9 , as opposed to putative linear scanning mechanisms10,11 . As RAG linearly scans convergent CBE-anchored chromatin loops4,12-14 , potentially formed by cohesin-mediated loop extrusion15-18 , we revisited its scanning role. Here we show that the chromosomal orientation of JH -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 JH 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.