Mechanisms that Regulate Antibody Class Switch Recombination and Somatic Hypermutation

调节抗体类别转换重组和体细胞超突变的机制

• 批准号: 10392890
• 负责人: Frederick W. Alt
• 金额: $ 53.1万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392890
• 关键词: Address; Alleles; Antibodies; Antigens; Appearance; B-Cell Activation; B-Cell Antigen Receptor; B-Lymphocytes; Biological Assay; C57BL/6 Mouse; ChIP-seq; Chromatin; Chromatin Loop; Chromosome Pairing; Chronic; Data; Development; Disease; Downstream Enhancer; Elements; Engineering; Enhancers; Exons; Formulation; Funding; Genes; Genetic Transcription; Human; Immune response; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Inbred Strains Mice; Intestines; Knock-in; Lead; Location; Mediating; Methods; Modeling; Mus; Mutation; Nature; Nucleic Acid Regulatory Sequences; Pattern; Peyer' s Patches; Physiological; Physiology; Play; Promoter Regions; Property; Publishing; Recurrence; Regulatory Element; Resolution; Role; Run-On Assays; Site; Specificity; Structure of germinal center of lymph node; System; Testing; V(D)J Recombination; Work; activation-induced cytidine deaminase; base; cohesin; complementarity-determining region 3; cytokine; embryonic stem cell; experimental study; global run on sequencing; gut microbiota; insight; microbiome; mouse model; new technology; nuclease; response; targeted nucleases

7. Project Summary/Abstract. We propose 3 specific aims to elucidate mechanisms regulating IgH class switch recombination (CSR) and Ig variable region exon somatic hypermutation (SHM). The major premise guiding Aims 1 and 2 is that chromatin loop extrusion plays a key role in promoting and regulating CSR and SHM. The major premise guiding Aim 3 is that intestinal Peyer's Patch (PP) GC responses involve expansion of rare BCR clonotypes that acquire high level intrinsic SHMs. These premises are supported by a wealth of strong published and preliminary data derived in large part from powerful new technologies that we developed during the current funding period. These assays, which will be used for ongoing studies, include a high throughput CSR assay (CSR-HTGTS) with sensitivity and resolution far beyond that of prior assays, an exceptionally high-resolution chromatin- interaction assay (3C-HTGTS), a rapid ES cell-based V(D)J knock-in passenger allele system to test impacts of sequences and cis-acting regulatory elements on SHM in germinal center (GC) B cells, and a repertoire/SHM sequencing method to study SHM features across complete GC IgH and IgL repertoires. Our first aim will test the hypothesis that cohesin-mediated loop extrusion generates large IgH loops anchored downstream by the 3'IgH regulatory region (3'IgHRR) and upstream by donor Sµ within which cytokine/activator-induced I region promoter transcription through S regions generates impediments to ongoing extrusions resulting in directional synapsis of Sµ and acceptor S regions for orientation-specific CSR. Mechanisms downstream of synapsis that contribute to orientation-specificity of CSR will be elucidated by testing ability of designer nuclease-targeted S region DSBs, divergent S region sequences, and inverted S region transcription units to mediate this activity. Aim 2 proposes to test, for both IgH and IgL genes, the hypothesis that chromatin loop extrusion juxtaposes downstream enhancers with V(D)J exons to make them a privileged SHM location. A key experimental approach will be to use our passenger system to assay germinal center GC B cells with matched IgH or IgL productive and passenger alleles, derived from physiological V(D)J rearrangements, for effects of test mutation(s) on the passenger allele for effects on SHM and loop extrusion. Aim 3 will test the physiological impact of recurrent antibody clonotypes expression in PP GC B cells that we discovered through endogenous repertoire/SHM sequencing studies that also revealed some recurrent clonotypes to be microbiome-dependent. To elucidate physiological implications, we will further test the hypothesis that these recurrent antibodies may be involved in an immune response against gut microbiota or other gut antigens by characterizing their antigen recognition properties and by their ectopic or ablated expression in mouse models. The proposed work should advance our understanding of key antibody maturation mechanisms and may also lead to new insights into potential roles of PP GC-derived antibodies with respect to involvement in normal physiology or disease.

7。项目摘要/摘要。 我们提出了3个具体目的，以阐明调节IGH类转换重组（CSR）和IG的机制 可变区域外显子体细胞超突变（SHM）。主要的前提指导目标1和2是染色质 循环延伸在促进和控制CSR和SHM中起关键作用。主要的前提指导目标3是 肠Peyer的贴片（PP）GC响应涉及扩大稀有BCR clonotypes 等级固有的SHM。这些前提得到了大量发表和初步数据的支持 在很大程度上是我们在当前资助期间开发的强大新技术。 这些分析将用于正在进行的研究，包括高吞吐量CSR分析（CSR-HTGTS） 具有敏感性和分辨率远远超出了先前的测定，一种异常高分辨率的染色质 - 相互作用测定（3C-HTGTS），一种基于ES的快速电池V（D）J敲击乘客等位基因系统，以测试影响 在生发中心（GC）B细胞中SHM上的序列和顺式作用调节元件，A 曲目/SHM测序方法研究了完整的GC IGH和IGL曲目的SHM特征。我们的 第一个目标将检验以下假设，即粘蛋白介导的环延伸产生锚定的大型IGH回路 3'igh监管区域（3'ighrr）下游，上游由供体Sµ 细胞因子/激活剂诱导的I区域启动子通过S区域引起的障碍 导致sμ和受体s区域的定向突触的挤出因素特异性CSR。 突触下游的机制有助于CSR的方向特异性。 设计器核酸酶靶向的S区域DSB，不同的S区域序列和倒置S的测试能力 区域转录单元介导此活动。 AIM 2提案，以测试IGH和IGL基因，即 假设染色质环延伸与V（d）J外显子并列下游增强剂以使其成为一个 特权SHM位置。一种关键的实验方法是使用我们的乘客系统测定生发 中心GC B细胞具有匹配的IGH或IGL生产性和乘客等位基因，源自物理V（d）J 重排，以实现测试突变对乘客等位基因的影响，以影响SHM和LOOP延伸。 AIM 3将测试我们的PP GC B细胞中复发性抗体clonotypes表达的物理影响。 通过内源性曲目/SHM测序研究发现，该研究也揭示了一些经常性 clonotypes是微生物组依赖性的。为了阐明生理意义，我们将进一步测试 假设这些复发性抗体可能参与针对肠道菌群的免疫反应或 其他肠道抗原通过表征其抗原识别特性以及异位或消融 在鼠标模型中的表达。拟议的工作应提高我们对关键抗体的理解 成熟机制，也可能导致对PP GC衍生抗体潜在作用的新见解 关于正常生理或疾病的参与。