Targeting of somatic hypermutation in the genome

靶向基因组中的体细胞超突变

• 批准号: 10642885
• 负责人: David G. Schatz
• 金额: $ 50.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642885
• 关键词: 3-Dimensional; Genome; Immunoglobulin Somatic Hypermutation; Kininogenase; NFIB gene

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.

总结 体细胞超突变（SHM）在免疫球蛋白（Ig）基因中产生点突变，并且对于免疫球蛋白（Ig）的表达至关重要。 高亲和力抗体的产生、疫苗效力和抗感染保护。反应 当活化诱导的脱氨酶（AID）将细胞中单链DNA中的胞苷脱氨时， RNA聚合酶2（Pol 2）的转录背景。SHM的错误靶向导致遗传不稳定性 以及一系列B细胞恶性肿瘤的发展。了解影响因素和机制 调节和靶向AID/SHM是体液免疫研究中最重要的目标之一， 我们最近的研究产生了一个新的概念， 用于SHM靶向的框架，其中基因组成环过程带来SHM靶向（“DIVAC”）元件 它们将转录因子（TF）结合到靶基因座上，在靶基因座上它们使Pol 2停滞，从而使AID成为可能。 行动上此外，我们的初步工作在方法上取得了重大进展，使我们能够克服 阻碍该领域进展的障碍。在本提案中，我们利用这些概念性的 和技术进步，通过以下两个目标实现我们的中心目标： 目的1：基因发现-确定参与SHM并在体内结合DIVAC元件的新因子。 涉及SHM靶向的因素很少，也没有报道过SHM CRISPR/Cas9筛选。 此外，在体内结合DIVAC以介导SHM的TF是未知的。我们将使用新的快速检测 对于SHM（RASH）细胞系进行CRISPR敲除和活化筛选以鉴定SHM因子，以及 体内邻近标记和定量蛋白质组学鉴定与活性DIVAC相关的因子 元素将在多种人细胞系模型和小鼠中验证所确定的因子。 目的2：假设检验和机制-确定的贡献和作用机制 SHM中的染色质结构因子、TF和TF激活结构域。它以前没被 可能评估细胞必需因子在SHM中的作用。通过将我们新的AID靶向精确评估（PAAT）检测与降解决定子技术相结合，我们将严格测试染色质结构在细胞内的作用。 蛋白质、主要B细胞TF和Aim 1中鉴定的在整个基因组中SHM中的作用的因子。 作用机制将通过评估转录参数的全基因组测定来评估， 表观遗传标记、因子结合和染色质环。基因组“艾滋病活动地图”将揭示 每个因子对AID/SHM靶向Ig和非Ig基因座的贡献。最后，我们利用一本小说 SHM重建系统，以测试DIVAC结合的TF靶向SHM的假设， 无序的激活域以产生相分离的冷凝物。 总之，我们提出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性和癌症的原因。

项目成果

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

• DOI: 10.4049/jimmunol.2100923
• 发表时间: 2022-01-01
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Tarsalainen A;Maman Y;Meng FL;Kyläniemi MK;Soikkeli A;Budzyńska P;McDonald JJ;Šenigl F;Alt FW;Schatz DG;Alinikula J
• 通讯作者: Alinikula J

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation.

拓扑相关结构域描绘了对体细胞超突变的易感性。

• DOI: 10.1016/j.celrep.2019.11.039
• 发表时间: 2019
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Senigl,Filip;Maman,Yaakov;Dinesh,RaviK;Alinikula,Jukka;Seth,RashuB;Pecnova,Lubomira;Omer,ArinaD;Rao,SuhasSP;Weisz,David;Buerstedde,Jean-Marie;Aiden,ErezLieberman;Casellas,Rafael;Hejnar,Jiri;Schatz,DavidG
• 通讯作者: Schatz,DavidG

Dancing with DNA: AID embraces flexible partners.

与 DNA 共舞：AID 拥抱灵活的合作伙伴。

• DOI: 10.1038/s41422-023-00823-1
• 发表时间: 2023
• 期刊: Cell research
• 影响因子: 44.1
• 作者: Wang,Jianshu;Schatz,DavidG
• 通讯作者: Schatz,DavidG