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. 项目总结/摘要。 我们提出了 3 个具体目标来阐明调节 IgH 类别转换重组 (CSR) 和 Ig 的机制 可变区外显子体细胞超突变（SHM）。指导目标 1 和 2 的主要前提是染色质 循环挤出在促进和调节 CSR 和 SHM 方面发挥着关键作用。指导目标 3 的大前提是 肠道派尔集结 (PP) GC 反应涉及罕见 BCR 克隆型的扩展，这些克隆型获得高 级内在 SHM。这些前提得到大量已发表的强有力的初步数据的支持 很大程度上源自我们在当前资助期间开发的强大新技术。 这些检测将用于正在进行的研究，包括高通量 CSR 检测 (CSR-HTGTS) 其灵敏度和分辨率远远超出以前的检测方法，具有异常高分辨率的染色质 相互作用测定 (3C-HTGTS)，一种基于 ES 细胞的快速 V(D)J 敲入乘客等位基因系统，用于测试影响 生发中心 (GC) B 细胞中 SHM 的序列和顺式作用调节元件，以及 谱库/SHM 测序方法可研究完整 GC IgH 和 IgL 谱库的 SHM 特征。我们的 第一个目标将检验粘连蛋白介导的环挤出产生锚定的大 IgH 环的假设 下游为 3'IgH 调节区 (3'IgHRR)，上游为供体 Sμ，其中 细胞因子/激活剂诱导的 I 区启动子通过 S 区转录会阻碍正在进行的转录 挤压导致 Sμ 和受体 S 区域定向突触，以实现定向特异性 CSR。 有助于 CSR 方向特异性的突触下游机制将通过以下方式阐明 设计核酸酶靶向的 S 区 DSB、分歧 S 区序列和倒置 S 的测试能力 区域转录单位介导此活动。目标 2 建议测试 IgH 和 IgL 基因 假设染色质环挤出将下游增强子与 V(D)J 外显子并置，使它们成为 优越的 SHM 位置。一个关键的实验方法是使用我们的乘客系统来分析生发 中心 GC B 细胞，具有匹配的 IgH 或 IgL 生产和过客等位基因，源自生理 V(D)J 重排，测试突变对过客等位基因的影响，以及对 SHM 和环挤出的影响。 目标 3 将测试我们研究的 PP GC B 细胞中反复出现的抗体克隆型表达的生理影响。 通过内源性库/SHM 测序研究发现，该研究还揭示了一些复发性 克隆型依赖于微生物组。为了阐明生理意义，我们将进一步测试 假设这些反复出现的抗体可能参与针对肠道微生物群的免疫反应或 其他肠道抗原，通过表征其抗原识别特性以及其异位或消融 在小鼠模型中的表达。拟议的工作应该增进我们对关键抗体的理解 成熟机制，也可能带来对 PP GC 衍生抗体潜在作用的新见解 关于参与正常生理或疾病。