Targeting of somatic hypermutation in the genome

摘要 体细胞高度突变(SHM)产生免疫球蛋白(LG)基因的点突变，对 高亲和力抗体的产生、疫苗效力和对感染的保护。他们的反应是 当激活诱导脱氨酶(AID)使单链DNA中的胞苷脱氨基时， 由RNA聚合酶2(Pol2)转录的上下文。错靶向SHM导致遗传不稳定 以及一系列B细胞恶性肿瘤的发展。了解以下因素和机制 调控和靶向AID/SHM是体液免疫研究的重要目标之一 我们最近的研究产生了一个新的概念 SHM靶向框架，其中基因组循环过程带来SHM靶向(“Divac”)元件 将它们的转录因子(TF)结合到目标基因座，在那里它们阻碍Pol2，从而使AID成为可能 行动。此外，我们的初步工作取得了重大的方法学进展，使我们能够克服 阻碍这一领域进展的障碍。在本提案中，我们利用了这些概念 和技术进步，通过以下两个目标实现我们的核心目标： 目的1：基因发现-在体内发现与SHM相关的新因子和结合Divac元件的新因子。 涉及SHM靶向的因素很少，也没有SHM CRISPR/Cas9筛查的报道。 此外，在体内结合Divac以调节SHM的因子尚不清楚。我们将使用新的快速检测 对SHM(皮疹)细胞系进行CRISPR基因敲除和激活筛选，以确定SHM因子，以及 活体邻近标记和定量蛋白质组学用于识别与主动收缩相关的因素 元素。确定的因素将在多个人类细胞系模型和小鼠身上得到验证。 目标2：假设检验和机制--确定以下因素的贡献和作用机制 SHM中的染色质结构因子、转录因子和转录因子激活域。这是以前没有的 有可能评估细胞基本因子在SHM中的作用。通过将我们新的艾滋病靶向精确度评估(PAAT)分析与Degron技术相结合，我们将严格测试染色质结构的作用 蛋白质、主要的B细胞因子和在目标1中确定的因子在整个基因组的SHM中所起的作用。 作用机制将通过全基因组分析来评估转录参数， 表观遗传标记、因子结合和染色质环路。基因组“艾滋病活动图”将揭示 各因素对LG和非LG基因座的AID/SHM的影响。最后，我们利用一本小说 SHM重建系统检验Divac结合的TFS以SHM为靶点的假设 无序的激活结构域以产生相分离的冷凝物。 总之，我们建议的研究对新技术的发展和 了解抗体基因多样化的机制以及基因组不稳定和癌症的原因。

SUMMARY Somatic hypermutation (SHM) generates point mutations in immunoglobulin (lg) genes and is vital for the generation of high affinity antibodies, vaccine efficacy, and protection against infection. The reaction is initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Mis-targeting of SHM contributes to genetic instability and the development of a range of B cell malignancies. Understanding the factors and mechanisms that regulate and target AID/SHM is one of the most important objectives in the study of humoral immunity and is the central goal of research under this grant Our recent studies have yielded a new conceptual framework for SHM targeting in which genome looping processes bring SHM targeting ("DIVAC") elements with their bound transcription factors (TFs) to the target locus where they stall Pol2, thereby enabling AID action. Further, our preliminary work has yielded major methodological advances that allow us to overcome obstacles that have stymied progress in the field. In this proposal, we take advantage of these conceptual and technical advances to pursue our central goal through the following two aims: Aim 1: Gene Discovery-Identify new factors involved in SHM and that bind DIVAC elements in vivo. Few factors involved in SHM targeting are known and no SHM CRISPR/Cas9 screens have been reported. Furthermore, the TFs that bind DIVAC in vivo to mediate SHM are unknown. We will use novel Rapid Assay for SHM (RASH) cell lines to perform CRISPR knockout and activation screens to identify SHM factors, and in vivo proximity labeling and quantitative proteomics to identify factors that associate with active DIVAC elements. Factors identified will be validated in multiple human cell line models and in mice. Aim 2: Hypothesis Testing and Mechanism-Determine the contribution and mechanism of action of chromatin architecture factors, TFs, and TF activation domains in SHM. It has not previously been possible to assess the role of cell essential factors in SHM. By combining our new Precision Assessment of AID Targeting (PAAT) assay with degron technology, we will rigorously test the role of chromatin architectural proteins, major B cell TFs, and factors identified in Aim 1 for their role in SHM across the entire genome. Mechanism of action will be assessed through genome wide assays that assess parameters of transcription, epigenetic marks, factor binding, and chromatin looping. Genome "AID activity maps" will reveal the contribution of each factor to AID/SHM targeting at lg and non-lg loci. Finally, we take advantage of a novel SHM reconstitution system to test the hypothesis that DIVAC-bound TFs target SHM using intrinsically disordered activation domains to create a phase-separated condensate. Together, our proposed studies are significant for the development of new technologies and for understanding mechanisms of antibody gene diversification and causes of genome instability and cancer.