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.

概括 体细胞超突变（SHM）在免疫球蛋白（LG）基因中产生点突变，对 产生高亲和力抗体，疫苗功效和防止感染的保护。反应是 当激活诱导脱氨酶（AID）脱氨基的单链DNA中的胞苷时开始 RNA聚合酶2（POL2）的转录背景。 SHM的错误靶向有助于遗传不稳定性 以及一系列B细胞恶性肿瘤的发展。了解因素和机制 规范和目标辅助/SHM是体液免疫研究中最重要的目标之一，是 根据这笔赠款的研究的核心目标我们最近的研究产生了一个新的概念 SHM靶向基因组循环过程带来SHM靶向（“ DIVAC”）元素的框架 及其绑定的转录因子（TF）到目标基因座的降低POL2，从而实现了援助 行动。此外，我们的初步工作取得了重大的方法论，使我们能够克服 在现场阻碍进步的障碍。在此提案中，我们利用这些概念 以及通过以下两个目标实现我们的核心目标的技术进步： 目标1：基因发现 - 识别SHM中涉及的新因素，并结合体内DIVAC元素。 涉及SHM靶向的因素很少，并且没有据报道SHM CRISPR/CAS9屏幕。 此外，在体内结合DIVAC与介导SHM的TF是未知的。我们将使用新颖的快速测定 SHM（皮疹）细胞系执行CRISPR敲除和激活屏幕以识别SHM因子，并且 体内接近标记和定量蛋白质组学，以识别与活动DIVAC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中得到验证。 目标2：假设检验和机制确定的作用贡献和机制 SHM中的染色质体系结构因子，TFS和TF激活域。以前还没有 可以评估细胞基本因素在SHM中的作用。通过将我们对援助靶标（PAAT）测定的新精确评估与Degron技术相结合，我们将严格测试染色质体系结构的作用 蛋白质，主要B细胞TFS以及AIM 1中确定的因子在整个基因组中在SHM中的作用。 作用机理将通过评估转录参数的基因组广泛的测定来评估， 表观遗传标记，因子结合和染色质循环。基因组“援助活动地图”将揭示 每个因素对LG和非LG基因座的AID/SHM靶向的贡献。最后，我们利用一本小说 SHM重建系统测试了Divac结合的TFS靶向SHM的假设 无序的激活域以创建相位分离的冷凝物。 我们提出的研究共同对开发新技术和对 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