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.

项目总结/摘要 获得性免疫的标志是B和T细胞抗原受体的单等位基因表达（等位基因排斥 蛋白质，这被认为是允许高度特异性免疫反应和抑制自身免疫。等位基因排斥 通过RAG 1/RAG 2（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，其发出V重组的永久反馈抑制信号， 另一个等位基因这种控制的缺点是它需要时间来完成重组、表达 和蛋白质的信号传导，以及第二个等位基因的变化。申请人证明RAG DNA双链 在前B细胞中Vk-至-Jk重组期间诱导的链断裂（DSB）通过ATM激酶发出信号以抑制 Rag 1/Rag 2表达、Jk可接近性和RAG切割另一个等位基因，直到第一个等位基因修复之后。 他表明，小鼠中ATM缺陷增加了双等位基因Igk IgH或TCRb表达。虽然这些数据表明， ATM通过发出V重组的DSB反馈抑制信号来实施等位基因排斥，他们既没有证明 该模型也不排除ATM刺激的DSB修复的作用。申请人显示ATM强制执行的新数据 通过NFkB必需调节剂（NEMO）蛋白的信号传导进行Igk等位基因排斥，并抑制Rag 1/Rag 2 转录可能是Vk重组的等位基因间控制的关键。申请人表明，RAG DSB在 原B细胞中的IgH D-至-J重组或DN胸腺细胞中的TCR b D-至-J、TCR g和/或TCR d重组， 不需要ATM抑制Rag 1/Rag 2表达。然而，这些RAG TCR DSB通过ATM发信号以抑制 Vb区反义长非编码RNA的表达，申请人显示其仅在DN中表达 细胞中Vb和Db-Jb片段在巨大的基因组距离上相互作用和重排。根据这些数据， 本申请人假设RAG DSB反馈通过互补抑制V（D）J重组 机制，包括抑制Rag 1/Rag 2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检验 他的模型的基本方面。目的1阐明不同类型的RAG DSB是如何诱导的， 在不同细胞类型中的重排信号抑制Rag 1/Rag 2转录以协调 等位基因之间的V到（D）J重组，从而强制等位基因排斥。目标2建议确定 V区反义长非编码RNA在促进长距离V-至-（D）J重排和服务 作为DSB响应开关，瞬时抑制这些重排，从而协调等位基因排斥。 拟议的工作将采用强大的小鼠模型，严格阐明RAG DSB的机制， 触发V重组的瞬时反馈抑制以帮助实施等位基因排斥。该项目将提供 新的机械的见解，一个understudied和一个全新的研究领域，后者 与生物学相关的所有细胞。除了推进对适应性免疫标志的理解， 可以确定抑制自身免疫和/或淋巴恶性肿瘤的重要机制。