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

Author(s): Yu Zhang [sup.1] [sup.2] [sup.3] [sup.4] [sup.13] , Xuefei Zhang [sup.1] [sup.2] [sup.3] , Zhaoqing Ba [sup.1] [sup.2] [sup.3] , Zhuoyi Liang [sup.1] [sup.2] [sup.3] , Edward W. Dring [...]
The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to J.sub.H segments, before joining upstream V.sub.H segments to DJ.sub.H intermediates.sup.1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain.sup.2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1).sup.3, the D.sub.H segments, and a RAG-bound recombination centre (RC).sup.4. The RC comprises the J.sub.H-proximal D segment (DQ52), four J.sub.H segments, and the intronic enhancer (iE[mu]).sup.5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS).sup.6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented J.sub.H-23RSSs and V.sub.H-23RSSs, respectively.sup.6. Despite 12/23 compatibility, inversional D-to-J.sub.H joining via upstream D-12RSSs is rare.sup.7,8. Plasmid-based assays have attributed the lack of inversional D-to-J.sub.H joining to sequence-based preference for downstream D-12RSSs.sup.9, as opposed to putative linear scanning mechanisms.sup.10,11. As RAG linearly scans convergent CBE-anchored chromatin loops.sup.4,12-14, potentially formed by cohesin-mediated loop extrusion.sup.15-18, we revisited its scanning role. Here we show that the chromosomal orientation of J.sub.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.sub.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. V(D)J recombination in B cells involves cohesin-mediated extrusion of chromatin loops to present DNA targets for cleavage and joining.