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

项目摘要 作为适应性免疫的关键部分，高度多样性抗体库的产生始于有效的免疫应答。 在早期B细胞中通过V（D）J重组组装免疫球蛋白重链位点（IgH） 发展在IgH基因座上，数百个VH广泛分布在2.4Mb上游区域内， DH和JH位于下游0.3Mb结构域内。淋巴细胞特异性RAG 1/RAG 2（RAG） 核酸内切酶通过切割两侧的成对重组信号序列（RSS）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的RSS对，这是广泛的 在染色体距离上分开，空间上接近以进行RAG切割。虽然3D基因组拓扑结构 IgH基因座上的染色质长程相互作用在RAG的调控中起重要作用 由于缺乏高分辨率， 染色质相互作用图和有效的系统，以全面表征假定的调控 元素本提案的总体目标是确定驱动功能性 染色质相互作用介导IgH RAG靶向。我们最近揭示了一种新的机制驱动D 到JH重组，由此粘着蛋白介导的染色质环挤出推动D-JH内的RAG扫描 结构域，以促进生理缺失的D-to-JH连接。有了新的令人信服的证据，我们提出， 这种粘着蛋白介导的动态染色质环也在V到DJH重组期间起作用， 产生多样化的VH库。具有超灵敏的染色质相互作用和V（D）J重组 结合全面的遗传学研究，在Aim 1中，我们将讨论以下功能的重要性： V到DJH重组中的染色质环挤出。在Aim 2中，我们将确定 潜在的粘附素介导的IgH长距离成环。在Aim 3中，我们将鉴定和表征新型IgH顺式- 在指导RAG远程靶向的调控元件。一本小说将大大促进这些研究 我们产生的v-Abl pro-B细胞系显示出有效的IgH长范围染色质环和多样的VH 跨IgH基因座的利用。这将提供一个灵活的基于细胞的系统， IgH顺式调节元件和反式作用因子之间复杂的相互作用，这在体内很难实现 动物模型该项目的完成将为动态3D技术如何提供新的机械见解。 基因组拓扑学利用主要的免疫过程来产生不同的抗体库。

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.