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

V(D)J 重组过程中染色质环化引导 RAG 靶向

基本信息

批准号：
10597767
负责人：
Yu Zhang
金额：
$ 39.13万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10597767
关键词：
3-Dimensional Address Alleles Animal Model Antibodies Antibody Repertoire Antigen Receptors Automobile Driving B-Cell Development B-Lymphocytes Bar Codes Biological Assay Cell Line Cells Chromatin Chromatin Loop Complex Data Distal Elements Ensure Generations Genetic Recombination Genetic study Genome Genomics Heavy-Chain Immunoglobulins IGH@ gene cluster Immune Intercistronic Region Knowledge Lesion Lymphoid Malignant lymphoid neoplasm Maps Mediating Molecular Peptide Signal Sequences Physiological Play Process Rapid screening Receptor Gene Regulatory Element Resolution Role Scanning System T-Lymphocyte T-cell receptor repertoire Testing Time Trans-Activators V(D)J Recombination Variant Work abl Oncogene adaptive immunity base cohesin conditional knockout driving force endonuclease flexibility genomic locus insight mouse model novel pathogen prevent progenitor release factor residence

项目摘要

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.

项目摘要作为适应性免疫的关键部分，高度多样化的抗体库的产生始于有效的抗体库早期 B 细胞期间通过 V(D)J 重组组装免疫球蛋白重链基因座 (IgH) 发展。在 IgH 位点，数百个 VH 广泛分布在 2.4 Mb 上游区域内，并且多个 DH 和 JH 位于下游 0.3 Mb 域内。淋巴特异性 RAG1/RAG2 (RAG) 核酸内切酶通过切割两侧的配对重组信号序列 (RSS) 来启动 V(D)J 重组 V、D 和 J 段。一个主要的知识差距是哪些机制带来了 RSS 对，这些 RSS 对被广泛使用在染色体距离上分开，在空间上接近以进行 RAG 切割。虽然 3D 基因组拓扑 IgH 基因座的长程染色质相互作用在指导 RAG 中发挥重要作用由于缺乏高分辨率，定位和理解潜在机制受到极大阻碍染色质相互作用图谱和有效的系统来全面表征假定的监管元素。该提案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 IgH RAG 靶向。我们最近揭示了一种驱动 D 的新机制到 JH 重组，粘连蛋白介导的染色质环挤出推动 D-JH 内的 RAG 扫描域促进生理缺失的 D-JH 连接。有了新的令人信服的证据，我们建议这种粘连蛋白介导的动态染色质循环也在 V 到 DJH 重组过程中起作用，以确保一代多样化的 VH 曲目。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 Aim1 中，我们将解决以下功能的重要性： V 至 DJH 重组中的染色质环挤压。在Aim2中，我们将确定分子机制潜在的粘连蛋白介导的 IgH 长程循环。在 Aim3 中，我们将鉴定并表征新型 IgH cis- 指导 RAG 远程靶向的监管要素。一部小说将极大地促进这些研究我们生成的 v-Abl pro-B 细胞系显示出高效的 IgH 长程染色质环和多样化的 VH 跨 IgH 位点的利用。这将提供一个灵活的基于细胞的系统来系统地表征 IgH 顺式调节元件和反式作用因子之间复杂的相互作用，这是很难实现的动物模型。该项目的完成将为动态 3D 如何实现提供新的机制见解基因组拓扑利用主要的免疫过程来生成不同的抗体库。