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

Yu Zhang, Xuefei Zhang, Zhaoqing Ba, Zhuoyi Liang, Edward W. Dring, Hongli Hu, Jiangman Lou, Nia Kyritsis, Jeffrey Zurita, Muhammad S. Shamim, Aviva Presser Aiden, Erez Lieberman Aiden and Frederick W. Alt ()
Additional contact information
Yu Zhang: Boston Children’s Hospital
Xuefei Zhang: Boston Children’s Hospital
Zhaoqing Ba: Boston Children’s Hospital
Zhuoyi Liang: Boston Children’s Hospital
Edward W. Dring: Boston Children’s Hospital
Hongli Hu: Boston Children’s Hospital
Jiangman Lou: Boston Children’s Hospital
Nia Kyritsis: Boston Children’s Hospital
Jeffrey Zurita: Boston Children’s Hospital
Muhammad S. Shamim: Baylor College of Medicine
Aviva Presser Aiden: Baylor College of Medicine
Erez Lieberman Aiden: Baylor College of Medicine
Frederick W. Alt: Boston Children’s Hospital

Nature, 2019, vol. 573, issue 7775, 600-604

Abstract: Abstract 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.

Date: 2019
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DOI: 10.1038/s41586-019-1547-y

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