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研究揭示V(D)J重组中染色质环挤压的作用
2019-09-12 13:48

美国波士顿儿童医院Frederick W. Alt等研究人员揭示了染色质环挤压在V(D)J重组中的基本作用。2019年9月11日,《自然》在线发表了这一成果。

由于RAG线性扫描会聚的CBE锚定染色质环,可能由cohesin介导的环挤出形成,研究人员重新审视了其扫描作用。研究人员发现JH-23RSS的染色体方向程序RC结合RAG线性扫描3'Igh子域中的上游染色质,用于会聚定向的D-12RSS,从而调解除基于RC的DQ52之外的所有D区段的缺失连接,通过扩散相关机制加入。在没有JH区段的情况下形成的基于DQ52的RC中,由下游DQ52-RSS结合的RAG扫描下游恒定区含外显子的3'Igh亚结构域,其中扫描可通过重复的Igh开关序列转录,以及基于3'Igh CBE的环锚,被靶向结合的无酶活的Cas9所阻止。每个扫描障碍在受阻区域内局部增加对潜在底物序列的RAG活性。RC中染色质相互作用的高分辨率图谱显示,这种局部RAG靶向与环路挤压过程的相应障碍相关,该过程驱使染色质经过RC结合的RAG。

据介绍,RAG核酸内切酶通过将D区段连接至JH区段,在将上游VH区段连接至DJH中间体之前,来启动前体B细胞中的Igh V(D)J装配。在小鼠前体B细胞中,Igh 3'末端的CTCF结合元件(CBE)锚定的染色质环结构域含有跨越5'CBE锚(IGCR1)、DH区段和RAG结合的重组中心(RC)。RC包括JH-近端D区段(DQ52)、四个JH区段和内含子增强子(iEμ)。稳定的RAG介导的切割局限于成对的V(D)J区段,其侧翼为互补重组信号序列(12RSS和23RSS)。D区段在下游和上游侧接12RSS,其分别介导与会聚定向的JH-23RSS和VH-23RSS的缺失连接。尽管12/23的兼容性,通过上游D-12RSS进行的反向D-to-JH连接很少见。与推定的线性扫描机制相反,基于质粒的分析归因于缺乏倒置D-to-JH与基于序列的下游D-12RSS9的偏好相联系。

附:英文原文

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

Author: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, Frederick W. Alt

Issue&Volume: 2019-09-11

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 loops4,12,13,14, potentially formed by cohesin-mediated loop extrusion15,16,17,18, 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.

DOI: 10.1038/s41586-019-1547-y

Source:https://www.nature.com/articles/s41586-019-1547-y

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:69.504
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

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