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Zhang, Yu

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Zhang

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Yu

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Zhang, Yu

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20192

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Author's Publications: search.filters.isAuthorOfPublication.3fd6a2a9-ba9f-4265-8d31-43e69f8c5687

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    Fundamental Roles of Chromatin Loop Extrusion in Antibody Class Switching
    (Springer Science and Business Media LLC, 2019-10-30) Zhang, Xuefei; Zhang, Yu; Ba, Zhaoqing; Kyritsis, Nia; Alt, Frederick; Casellas, Rafael
    Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμ constant region exons (CHs) with one of six CHs lying 100–200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region. Cytokines and activators induce activation-induced cytidine deaminase (AID) and I-promoter transcription, with 3′ IgH regulatory region (3′ IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3′ IgHRR interactions. Transcription through donor Sμ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sμ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3′ IgHRR enhancers with the 200-kb upstream Sμ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sμ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.
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    Publication
    The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination
    (Springer Science and Business Media LLC, 2019-09) Zhang, Yu; Zhang, Xuefei; Ba, Zhaoqing; Liang, Zhuoyi; Hu, Hongli; Lou, Jiangman; Kyritsis, Nia; Zurita, Jeffrey; Shamim, Muhammad S.; Presser Aiden, Aviva; Lieberman Aiden, Erez; Alt, Frederick W.; Dring, Edward
    RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates1. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1)3, the DHs, and a RAG-bound recombination center (RC)4. The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iE”)5. Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs)6. Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs6. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.