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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