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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要自适应免疫的标志是B和T细胞抗原受体的单相表达（等位基因排除）蛋白质被认为允许高度特异性的免疫反应并抑制自身免疫性。等位基因排除通过rag1/rag2（rag）核酸酶介导的v基因段重组的单相关启动来实现然后是v（d）J重新排列信号的蛋白质，永久反馈抑制V对另一个等位基因。该控制的缺点是它需要时间完成重组，表达以及蛋白质的信号传导，以及第二等位基因的变化。申请人证明了抹布DNA双重 pre-b细胞中VK-to-JK重新组合在BER-B细胞中诱导的链断裂（DSB）通过ATM激酶信号以抑制 RAG1/RAG2表达，JK可访问性和其他等位基因的RAG裂解，直到修复第一个等位基因后。他表明，小鼠的ATM缺乏会增加BI-calilalic IGK IGH或TCRB表达。虽然这些数据暗示 ATM通过信号DSB的反馈抑制V重组来强制执行等位基因排除，它们既没有证明该模型也不排除ATM刺激的DSB修复的作用。申请人显示了ATM强制执行的新数据通过NFKB必需调节剂（NEMO）蛋白信号传导IGK等位基因排除，并抑制RAG1/RAG2 转录可能是对VK重组的互行控制的关键。申请人表明抹布DSB在 DN胸腺细胞中的Pro-B细胞或TCRB D-TO-J，TCRG和/或TCRD重组的IGH D-TO-J重组不需要ATM抑制RAG1/RAG2表达。但是，这些RAG TCR DSB通过ATM信号以压抑 Vb区域反义长长的非编码RNA的表达，申请人显示仅在DN中表达 VB和DB-JB段的细胞在巨大的基因组距离上相互作用和重新排列。基于这些数据，申请人假设RAG DSBS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受体基因座特异性改变，抑制V重排。他提出了两个独立目标来测试他的模型的基本方面。 AIM 1提议阐明RAG DSB在不同类型中的诱导方式 RAG1/RAG2转录的不同细胞类型中的重排信号抑制以坐标的启动 v-to-（d）等位基因之间的重组，从而强制执行等位基因排除。 AIM 2建议确定 V区域反义长度非编码RNA在促进远程V-TO-（D）J重排和服务中的作用作为DSB响应的转换，可以暂时抑制这些重排，从而编排等位基因排除。拟议的工作将采用强大的鼠标模型来严格阐明RAG DSB的机制触发对V重组的瞬态反馈抑制，以帮助执行等位基因排除。该项目将提供新的机械洞察力对一个研究过的研究和一项完全新颖的研究系列，后者与所有细胞的生物学有关。除了促进对自适应免疫标志的理解之外，可以确定对于抑制自身免疫和/或淋巴恶性肿瘤重要的机制。