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Impact of novel enhancers on Igh repertoire diversity

新型增强子对 Igh 库多样性的影响

基本信息

批准号：
10716628
负责人：
Amy L Kenter
金额：
$ 61.23万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-08 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10716628
关键词：
3-Dimensional Address Antibodies Antibody Repertoire Antigen Receptors Architecture B-Cell Development B-Lymphocytes Binding Bone Marrow Breeding Cell Line Cell Nucleus Cell physiology Cells Chromatin Chromatin Loop Chromatin Structure Chromosome Territory Confocal Microscopy DNA Dependence Development Distal Elements Enhancers Environment Exons Foundations Frequencies Gene Rearrangement Genes Genetic Genetic Recombination Genetic Transcription Genome Genomics Histones IGH@ gene cluster Immune response Immunoglobulins Individual Ions Knockout Mice Link Lymphoid Cell Maps Mediating Methodology Methods Molecular Molecular Conformation Mus Nuclear Nucleic Acid Regulatory Sequences Physical condensation Process Property Proteins Rag1 Mouse Receptor Gene Regulatory Element Series Site Stimulus Structure V(D)J Recombination chromosome conformation capture novel promoter recombinase response spatial relationship superresolution microscopy timeline vaccine development

项目摘要

ABSTRACT Antigen receptor genes are assembled from several gene segments via V(D)J rearrangement during early lymphoid cell development to generate a diverse repertoire of antibodies. During early B cell development in the bone marrow (BM), V(D)J and VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase. VH genes are dispersed through 2.5 Mb of the Igh locus. Locus compaction serves to facilitate spatial proximity between the rearranged DHJH join and distal VH genes. Furthermore, V genes rearrange with very different intrinsic frequencies. However, little is known about the precise looping structure of the Igh locus that leads to locus contraction. We undertook an analysis of the entire Igh locus using chromosome conformation capture (3C) based methodology to systematically characterize three-dimensional (3D) chromatin organization on several genomic scales. We found that the Igh locus is compartmentalized into a topologically associated domain (TAD) that is partitioned into three sub-domains. A set of pro-B cell- specific very-long range looping interactions bridge the sub-domains and are Pax5-dependent. These looping interactions are anchored at Sites I, II, II.5 and III and which are critical facilitators of Igh locus contraction. We have now defined the DNA motifs in these loop anchors and discovered a series of pro-B cell specific novel enhancers (NEs) that participate in a NE-NE-VH gene promoter interactome. We have systematically characterized locus compaction using specific KO mice in combination with chromatin-loop mapping methods and newly constructed NE1 KO mice and cell lines. To begin to understand NE interactome function we asked whether those NEs engaged in E-E and E-Pr looping are in an active state as defined by the H3K27Ac histone marks in single cells. We discovered that the NEs are marked by remarkably large H3K27Ac foci. Here we will 1) systematically characterize NEs individually and in the NE interactome as it relates to VH gene usage during repertoire formation, and 2) examine the relationship between the NE interactome and H3K27Ac foci. The presence of H3K27Ac foci on NEs may indicate the participation of the Igh locus in transcriptional condensates which may define the molecular environment for V(D)J recombination.

摘要抗原受体基因是由几个基因片段通过V（D）J重排组装而成的，早期淋巴细胞发育以产生多种抗体库。早期B细胞在骨髓（BM）中发育，V（D）J和VJ连接发生在IgH和L链基因上，并由RAG重组酶介导。VH基因分散在2.5 Mb的 Igh基因座。基因座压缩用于促进重排的DHJH连接之间的空间接近和远端VH基因。此外，V基因以非常不同的固有频率重排。然而，很少有人知道的精确回路结构的免疫球蛋白基因座，导致基因座收缩。我们采用染色体构象捕获技术对整个Igh基因座进行了分析， (3C)系统地表征三维（3D）染色质的方法在几个基因组尺度上的组织。我们发现免疫球蛋白基因座被划分为一个区域，拓扑关联域（topologically associated domain，简写为Domain）被划分为三个子域。一组前B细胞- 特定的极长距离环相互作用桥接子结构域并且是Pax 5依赖性的。这些循环交互作用锚定在位点I、II、II.5和III，它们是IGH的关键促进者轨迹收缩我们现在已经确定了这些环锚中的DNA基序，并发现了一系列参与NE-NE-VH基因启动子的前B细胞特异性新型增强子（内斯）相互作用体我们使用特定的KO小鼠系统地表征了基因座压实，结合染色质环作图方法和新构建的NE 1 KO小鼠和细胞线为了开始了解NE相互作用组功能，我们询问这些内斯是否参与E-E 和E-Pr环处于活跃状态，如单细胞中H3 K27 Ac组蛋白标记所定义。我们发现内斯以非常大的H3 K27 Ac灶为标志。在这里我们将1）系统地表征NE个体和NE相互作用组中的NE，因为它与VH基因使用有关在剧目形成，和2）检查NE相互作用组之间的关系， H3 K27 Ac病灶。内斯粒细胞上H3 K27 Ac灶的存在可能表明Igh基因座的参与在转录缩合物中，这可能决定了V（D）J重组的分子环境。