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通过V（d）J的重排从几个基因段组装早期淋巴样细胞的发育产生多种抗体曲目。在早期B细胞中骨髓（BM），V（d）J和VJ连接的发育发生在IGH和L链基因上，分别由RAG重组酶介导。 VH基因通过2.5 MB分散 IGH基因座。基因座压实可促进重新排列的DHJH连接之间的空间接近度和远端VH基因。此外，V基因以非常不同的固有频率重新排列。但是，关于导致基因座的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基因座进行分析（3C）系统地表征三维（3D）染色质的方法在几个基因组量表上组织。我们发现IGH基因座被分为拓扑相关的域（TAD）分为三个子域。一组Pro-B细胞 - 特定的非常长的范围循环相互作用桥接了子域，并且依赖于PAX5。这些循环相互作用锚定在I，II，II.5和III的站点，它们是IGH的关键促进者基因座收缩。现在，我们已经定义了这些循环锚中的DNA图案，并发现了一个系列参与NE-NE-VH基因启动子的Pro-B特异性新型增强子（NES） Interactome。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映射方法以及新构建的NE1 KO小鼠和细胞线。为了开始了解NE互动函数，我们询问了那些从事E-E的NES是否参与 E-PR循环处于由单个细胞中H3K27AC组蛋白标记定义的活性状态。我们发现NE的标记为大型H3K27AC焦点。在这里我们将1）系统地对NE进行单独和NE相互作用的表征，因为它与VH基因使用有关在曲目形成期间，以及2）检查NE Interactome与 H3K27AC焦点。 H3K27AC对NES的存在可能表明IGH基因座的参与在转录冷凝物中，可以定义V（d）J重组的分子环境。