Elucidating Mechanisms of RAG Endonuclease Mediated Feedback Inhibition of V(D)J Recombination

阐明 RAG 核酸内切酶介导的 V(D)J 重组反馈抑制机制

• 批准号: 10538891
• 负责人: CRAIG H BASSING
• 金额: $ 51.72万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-12 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538891
• 关键词: ATM Signaling Pathway; ATM deficient; Alleles; Antigen Receptors; Area; Autoimmunity; B-Lymphocytes; Binding Sites; Biology; Cells; Chromosomes; DNA; DNA Double Strand Break; Data; Double Strand Break Repair; Elements; Ensure; Exclusion; Feedback; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Genetic Recombination; Genetic Transcription; Genomics; IgK; Immune response; Knowledge; Lymphocyte; Malignant lymphoid neoplasm; Mediating; Methods; Modeling; Mus; Pattern; Process; Proteins; Rag1 Mouse; Receptor Gene; Repression; Research; Role; Signal Pathway; Signal Repression; Signal Transduction; Signaling Protein; Specificity; T-Cell Receptor; T-Lymphocyte; Testing; Time; Transcriptional Regulation; Untranslated RNA; V(D)J Recombination; Work; adaptive immunity; ataxia telangiectasia mutated protein; base; cell type; endonuclease; experimental study; insight; mouse model; novel; nuclease; promoter; protein phosphatase inhibitor-2; receptor; repaired; response; thymocyte

PROJECT SUMMARY/ABSTRACT A hallmark of adaptive immunity is mono-allelic expression (allelic exclusion) of B and T cell antigen receptor proteins, which is thought to allow highly-specific immune responses and inhibit autoimmunity. Allelic exclusion is achieved via mono-allelic initiation of RAG1/RAG2 (RAG) nuclease-mediated V gene segment recombination, followed by protein from a V(D)J rearrangement signaling permanent feedback inhibition of V recombination on the other allele. A shortcoming of this control is that it needs time for completion of recombination, expression and signaling of protein, and changes on the second allele. The applicant demonstrated that RAG DNA double strand breaks (DSBs) induced during Vk-to-Jk recombination in pre-B cells signal via the ATM kinase to inhibit Rag1/Rag2 expression, Jk accessibility, and RAG cleavage of the other allele until after the first allele is repaired. He showed that ATM deficiency in mice increases bi-allelic Igk IgH, or TCRb expression. While these data imply that ATM enforces allelic exclusion by signaling DSB feedback inhibition of V recombination, they neither prove this model nor rule out a role for ATM-stimulated DSB repair. The applicant shows new data that ATM enforces Igk allelic exclusion by signaling via the NFkB essential modulator (NEMO) protein, with inhibition of Rag1/Rag2 transcription likely key for inter-allelic control of Vk recombination. The applicant shows that RAG DSBs during IgH D-to-J recombination in pro-B cells or TCRb D-to-J, TCRg, and/or TCRd recombination in DN thymocytes do not require ATM to repress Rag1/Rag2 expression. However, these RAG TCR DSBs signal via ATM to repress expression of a Vb region anti-sense long non-coding RNA, which the applicant shows is expressed only in DN cells where Vb and Db-Jb segments interact and rearrange over vast genomic distances. Based on these data, the applicant hypothesizes that RAG DSBs feedback inhibit V(D)J recombination through complementary mechanisms, including cell type-specific signaling pathways that repress Rag1/Rag2 transcription and antigen receptor locus-specific alterations that suppress V rearrangements. He proposes two independent aims to test fundamental aspects of his model. Aim 1 proposes to elucidate how RAG DSBs induced during different types of rearrangements in different cell types signal repression of Rag1/Rag2 transcription to coordinate initiation of V-to-(D)J recombination between alleles and thereby enforce allelic exclusion. Aim 2 proposes to determine the role of V region anti-sense long non-coding RNAs in promoting long-range V-to-(D)J rearrangements and serving as a DSB-responsive switch to transiently inhibit these rearrangements and thereby orchestrate allelic exclusion. The proposed work will employ powerful mouse models to rigorously elucidate mechanisms by which RAG DSBs trigger transient feedback inhibition of V recombination to help enforce allelic exclusion. The project will provide novel mechanistic insights into one understudied and one completely novel line of research in the field, the latter relevant to the biology all cells. Beyond advancing understanding of a hallmark of adaptive immunity, the findings could identify mechanisms important for suppressing autoimmunity and/or lymphoid malignancies.

项目概要/摘要 适应性免疫的一个标志是 B 和 T 细胞抗原受体的单等位基因表达（等位基因排除） 蛋白质，被认为可以产生高度特异性的免疫反应并抑制自身免疫。等位基因排除 通过 RAG1/RAG2 (RAG) 核酸酶介导的 V 基因片段重组的单等位基因起始来实现， 随后是来自 V(D)J 重排的蛋白质，信号传导 V 重组的永久反馈抑制 另一个等位基因。该对照的缺点是需要时间来完成重组、表达 和蛋白质信号传导，以及第二个等位基因的变化。申请人证明 RAG DNA 双 前 B 细胞中 Vk-to-Jk 重组期间诱导的链断裂 (DSB) 通过 ATM 激酶信号抑制 Rag1/Rag2 表达、Jk 可及性和另一个等位基因的 RAG 切割，直到第一个等位基因修复后。 他发现小鼠体内 ATM 缺陷会增加双等位基因 Igk IgH 或 TCRb 的表达。虽然这些数据意味着 ATM 通过信号 DSB 反馈抑制 V 重组来强制等位基因排除，但它们都没有证明 该模型也不排除 ATM 刺激的 DSB 修复的作用。申请人展示ATM强制执行的新数据 通过 NFkB 必需调节剂 (NEMO) 蛋白发出信号排除 Igk 等位基因，并抑制 Rag1/Rag2 转录可能是 Vk 重组等位基因间控制的关键。申请人表明，RAG DSB 在 pro-B 细胞中的 IgH D-to-J 重组或 DN 胸腺细胞中的 TCRb D-to-J、TCRg 和/或 TCRd 重组 不需要 ATM 来抑制 Rag1/Rag2 表达。然而，这些 RAG TCR DSB 通过 ATM 发出信号来抑制 Vb区反义长非编码RNA的表达，申请人证明其仅在DN中表达 Vb 和 Db-Jb 片段在巨大的基因组距离内相互作用并重新排列的细胞。根据这些数据， 申请人假设 RAG DSB 反馈通过互补抑制 V(D)J 重组 机制，包括抑制 Rag1/Rag2 转录和抗原的细胞类型特异性信号通路 抑制 V 重排的受体位点特异性改变。他提出了两个独立的目标来测试 他的模型的基本方面。目标 1 提出阐明 RAG DSB 在不同类型期间是如何诱导的 不同细胞类型中的重排信号抑制 Rag1/Rag2 转录以协调启动 等位基因之间的 V 到 (D)J 重组，从而强制等位基因排除。目标 2 建议确定 V区反义长非编码RNA在促进长程V-to-(D)J重排和服务中的作用 作为 DSB 响应开关，可暂时抑制这些重排，从而协调等位基因排除。 拟议的工作将采用强大的小鼠模型来严格阐明 RAG DSB 的机制 触发 V 重组的瞬时反馈抑制，以帮助强制等位基因排除。该项目将提供 对该领域一项正在研究的研究和一项完全新颖的研究路线的新颖的机械见解，后者 与生物学相关的所有细胞。除了增进对适应性免疫标志的理解之外，研​​究结果 可以确定抑制自身免疫和/或淋巴恶性肿瘤的重要机制。