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 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 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 (iEmicro)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, respectively⁶. 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-12RSSs⁹, 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.