Chromatin looping directed RAG targeting during V(D)J recombination

项目摘要 作为获得性免疫的关键部分，高度多样化的抗体库的产生首先是高效的 B细胞早期通过V(D)J重组组装免疫球蛋白重链基因(IGH) 发展。在IGH基因座，数百个VHS广泛分布在2.4Mb的上游区域和多个 DHS和JHS位于下行0.3 Mb域内。淋系特异性RAG1/RAG2(RAG) 核酸内切酶通过切割成对的重组信号序列(RSS)侧翼启动V(D)J重组 V、D和J段。一个主要的知识差距是什么机制带来了RSS对，这是广泛存在的 在染色体距离上分开，进入空间接近的地方进行碎屑切割。尽管3D基因组拓扑学 此外，免疫球蛋白基因座上的长距离染色质相互作用在引导rag中起着重要的作用。 由于缺乏高分辨率，因此很大程度上阻碍了对潜在机制的了解 染色质相互作用图和有效系统来全面表征假定的调控 元素。这项建议的总体目标是确定驱动功能的分子机制 染色质相互作用介导免疫球蛋白RAG靶向。我们最近揭示了一种驱动D的新机制 到JH重组，由此粘附素介导的染色质环挤出推动D-JH内的RAG扫描 结构域促进生理缺失的D-to-JH连接。有了新的令人信服的证据，我们建议 这种粘附素介导的动态染色质环也在V到DJH的重组过程中运行，以确保 产生了不同的VH曲目。具有超敏感的染色质相互作用和V(D)J重组 检测结合全面的遗传学研究，在Aim1中，我们将解决功能重要性的问题 染色质在V向DJH重组中环挤压。在AIM2中，我们将确定分子机制 潜在的粘附素介导的免疫球蛋白长距离环行。在Aim3中，我们将识别和表征新的IgH顺式- 指导RAG远程瞄准的监管要素。一本小说将极大地促进这些研究 我们建立的V-Abl Pro-B细胞株表现出高效的IgH、长距离染色质环路和多样化的VH IGH区域的利用率。这将提供一种灵活的基于细胞的系统来系统地表征 IgH顺式调控元件和反式作用因子之间复杂的相互作用，这在 动物模型。该项目的完成将提供关于如何动态3D的新的机械见解 基因组拓扑利用一个主要的免疫过程来产生不同的抗体谱系。

Project Abstract As a critical part of adaptive immunity, generation of a highly diverse antibody repertoire begins with efficient assembly of the immunoglobulin heavy chain locus (IgH) through V(D)J recombination during early B cell development. At IgH locus, hundreds of VHs are widely-spread within a 2.4 Mb upstream region and multiple DHs and JHs are located within a downstream 0.3 Mb domain. The lymphoid-specific RAG1/RAG2 (RAG) endonuclease initiates V(D)J recombination by cutting paired recombination signal sequences (RSSs) flanking V, D, and J segments. A major knowledge gap is what mechanisms bring the RSS pairs, which are widely separated in chromosomal distance, into spatial proximity for RAG cleavage. Although 3D genome topology and long-range chromatin interaction at IgH locus has been implicated to play important roles in directing RAG targeting, understanding the underlying mechanisms is greatly hindered due to lack of high-resolution chromatin interaction maps and efficient systems to comprehensively characterize the putative regulatory elements. The overall objective of this proposal is to determine the molecular mechanisms driving functional chromatin interactions mediating IgH RAG targeting. We have recently revealed a novel mechanism driving D to JH recombination, whereby cohesin-mediated chromatin loop extrusion propels RAG scanning within D-JH domain to promote physiologically deletional D-to-JH joining. With new compelling evidence, we propose that this cohesin-mediated dynamic chromatin looping also operates during V to DJH recombination to ensure generation of a diverse VH repertoire. With ultra-sensitive chromatin interaction and V(D)J recombination assays combined with comprehensive genetic studies, in Aim1, we will address the functional importance of chromatin loop extrusion in V to DJH recombination. In Aim2, we will determine the molecular mechanisms underlying cohesin-mediated IgH long-range looping. In Aim3, we will identify and characterize novel IgH cis- regulatory elements in directing RAG long-range targeting. These studies will be greatly facilitated by a novel v-Abl pro-B cell line we generated that shows efficient IgH long-range chromatin looping and diverse VH utilization across IgH locus. This will provide a flexible cell-based system to systematically characterize complex interplay between IgH cis-regulatory elements and trans-acting factors, which is difficult to achieve in animal models. Completion of this project will provide new mechanistic insights on how the dynamic 3D genome topology harnesses a major immune process for generation of diverse antibody repertoires.