Mechanisms that Regulate Antibody Class Switch Recombination and Somatic Hypermutation

7.项目概要/摘要。 我们提出了3个具体的目标来阐明调节IgH类转换重组（CSR）和IG的机制 可变区外显子体细胞高突变（SHM）。指导目标1和2的主要前提是染色质 环挤压在促进和调节CSR和SHM中起着关键作用。指导目标3的大前提是 肠道派尔集合淋巴结（PP）GC反应涉及罕见的BCR克隆型的扩增， 水平本征SHMs。这些前提得到了大量已发表的有力数据和初步数据的支持 这在很大程度上来自我们在当前资助期间开发的强大的新技术。 这些试验将用于正在进行的研究，包括高通量CSR试验（CSR-HTGTS） 由于灵敏度和分辨率远远超过先前的测定，因此异常高分辨率的染色质- 相互作用试验（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 在生发中心（GC）B细胞中SHM的序列和顺式作用调控元件， 在一个实施方案中，使用免疫组化组库/SHM测序方法来研究完整GC IgH和IgL组库中的SHM特征。我们 第一个目标是检验以下假设，即粘附素介导的环挤出产生锚定的大IgH环 下游是3 'IgH调节区（3' IgHRR），上游是供体Sµ， 细胞因子/激活剂诱导的I区启动子通过S区的转录对正在进行的 挤压导致Sµ和受体S区域的定向突触，用于定向特异性CSR。 有助于CSR定向特异性的突触下游机制将通过以下方式阐明： 测试设计者核酸酶靶向S区DSB、趋异S区序列和反向S区序列的能力 区域转录单位来介导该活性。目的2提出检测IgH和IgL基因， 假设染色质环挤压并置下游增强子与V（D）J外显子，使它们成为 特殊的SHM位置一个关键的实验方法将是使用我们的乘客系统来分析germinate 具有匹配的IgH或IgL生产和乘客等位基因的中心GC B细胞，源自生理V（D）J 重排，测试突变对乘客等位基因的影响，对SHM和环挤出的影响。 目的3将测试在PP GC B细胞中复发性抗体克隆型表达的生理学影响， 通过内源性库/SHM测序研究发现， 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 假设这些复发性抗体可能参与针对肠道微生物群的免疫应答， 通过表征它们的抗原识别特性和通过它们的异位或消融， 在小鼠模型中表达。这项工作将进一步加深我们对关键抗体的理解， 成熟机制，也可能导致对PP GC衍生抗体的潜在作用的新见解 与正常生理或疾病有关。

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.