Mechanisms that Regulate Antibody Class Switch Recombination and Somatic Hypermutation

7.项目摘要/摘要。 我们提出了3个特定的目标来阐明调节IgH类开关重组(CSR)和免疫球蛋白的机制 体细胞高度突变(SHM)。指导目标1和2的主要前提是染色质 环空挤出在促进和调节CSR和SHM方面起着关键作用。指导目标3的大前提是 肠道Peyer‘s Patch(PP)GC反应涉及罕见的BCR克隆型的扩张，这些克隆型获得高 电平本征SHM。这些假设得到了大量强有力的已公布和初步数据的支持 这在很大程度上源于我们在当前筹资期间开发的强大的新技术。 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过以往的分析，一种异常高分辨率的染色质- 相互作用分析(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SHM的序列和顺式作用调控元件，以及a 曲谱库/SHM测序方法研究完整GC、IgH和IGL谱系的SHM特征。我们的 第一个目标将检验这样的假设，即粘附素介导的环路挤出会产生大的锚定的Ig H环 下游为3‘IgH调节区(3’IgHRR)，上游为供体S，其内 细胞因子/激活剂诱导的I区启动子通过S区域转录对正在进行的 挤出导致Sµ和受体S区域的定向突触，以实现定向特定的企业社会责任。 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 设计核酸酶靶向的S区DSB、发散的S区序列和反向S序列的检测能力 区域转录单位来调节这一活动。目标2建议同时测试免疫球蛋白和免疫球蛋白基因 假设染色质环挤压将下游增强子与V(D)J外显子并列，使其成为 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生发 中心GC B细胞，具有匹配的IgH或IGL产生等位基因和乘客等位基因，来源于生理性V(D)J 重排，用于测试突变(S)对乘客等位基因的影响，用于SHM和环路挤出的影响。 目的3将测试反复抗体克隆型表达对PP GC B细胞的生理影响 通过内源性曲目/SHM测序研究发现，也显示了一些反复出现的 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区系的免疫反应 其他肠道抗原，通过表征其抗原识别特性以及通过其异位或被消融 在小鼠模型中的表达。建议的工作应该会促进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的。

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.