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.

抽象的抗原受体基因由多个基因片段通过 V(D)J 重排组装而成早期淋巴细胞发育产生多种抗体。早期B细胞期间骨髓 (BM) 的发育、V(D)J 和 VJ 连接发生在 IgH 和 L 链基因上，分别由 RAG 重组酶介导。 VH 基因分散在 2.5 Mb 的哎呀轨迹。轨迹压缩有助于促进重新排列的 DHJH 连接之间的空间接近和远端VH 基因。此外，V 基因以非常不同的内在频率重排。然而，对于导致基因座的 Igh 基因座的精确循环结构知之甚少。收缩。我们使用染色体构象捕获对整个 Igh 基因座进行了分析基于 (3C) 的方法来系统地表征三维 (3D) 染色质在多个基因组尺度上进行组织。我们发现 Igh 基因座分为拓扑关联域 (TAD)，分为三个子域。一组pro-B细胞- 特定的超长范围循环相互作用桥接子域并且依赖于 Pax5。这些循环相互作用锚定在位点 I、II、II.5 和 III，它们是 Igh 的关键促进者轨迹收缩。我们现在已经定义了这些环锚中的 DNA 基序并发现了一系列参与 NE-NE-VH 基因启动子的 pro-B 细胞特异性新型增强子 (NE) 相互作用组。我们使用特定的 KO 小鼠系统地表征了位点压缩结合染色质环定位方法和新构建的NE1 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 NE 是否参与 E-E 和 E-Pr 环处于活跃状态，由单细胞中的 H3K27Ac 组蛋白标记定义。我们发现 NE 的标志是非常大的 H3K27Ac 病灶。在这里我们将 1) 系统地单独表征 NE 以及 NE 相互作用组中的特征，因为它与 VH 基因使用相关在曲目形成过程中，2）检查 NE 相互作用组和 H3K27Ac 焦点。 NE 上 H3K27Ac 焦点的存在可能表明 Igh 基因座的参与在转录缩合物中，它可以定义 V(D)J 重组的分子环境。