Journal
NATURE
Volume 573, Issue 7775, Pages 600-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41586-019-1547-y
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Funding
- NIH [R01 AI020047]
- Thrasher Research Fund Early Career Award
- NSF Physics Frontiers Center Award [PHY1427654]
- Welch Foundation [Q-1866]
- USDA Agriculture and Food Research Initiative Grant [2017-05741]
- NIH 4D Nucleome Grant [U01HL130010]
- NIH Encyclopedia of DNA Elements Mapping Center Award [UM1HG009375]
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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 (iE mu)(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 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.
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