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

项目摘要/摘要 获得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(等位基因排斥) 蛋白质，这被认为允许高度特异的免疫反应，并抑制自身免疫。等位基因排斥 是通过RAG1/RAG2(RAG)核酸酶介导的V基因片段重组的单等位基因启动实现的， 其次是V(D)J重排的蛋白质，发出永久反馈抑制V重组的信号 另一个等位基因。这种控制的一个缺点是它需要时间来完成重组、表达 和蛋白质的信号传递，以及第二个等位基因的变化。申请者证明了破烂的DNA加倍 前B细胞VK-to-JK重组过程中诱导的链断裂(DSB)信号通过ATM激酶抑制 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之后。 他指出，小鼠缺乏ATM会增加双等位免疫球蛋白IgH或TCRb的表达。虽然这些数据意味着 ATM通过发出DSB反馈抑制V重组来强制等位基因排斥，他们都没有证明 这种模式也不排除ATM刺激的DSB修复的作用。申请人展示自动柜员机强制执行的新数据 通过抑制Rag1/Rag2通过NFkB必需调节器(NEMO)蛋白的信号传递来排除IgK等位基因 转录可能是VK重组等位基因间控制的关键。申请人须证明破损的DSB在 前B细胞中的IgH D-to-J重组或糖尿病肾病胸腺细胞中的TCRb D-to-J、TCRG和/或TCRd重组 不需要ATM来抑制Rag1/Rag2的表达。然而，这些RAG TCRDSB通过ATM发送信号来抑制 申请人所示的VB区反义长非编码RNA的表达仅在dN中表达 VB和DB-JB片段在基因组距离遥远的地方相互作用和重排的细胞。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。目的1旨在阐明RAG DSB在不同类型中是如何诱导的 不同细胞类型的重排信号抑制Rag1/Rag2转录以协调 等位基因之间的V-to-(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在促进长程V-to-(D)J重排和服务中的作用 作为DSB反应的开关，暂时抑制这些重排，从而协调等位基因排斥。 这项拟议的工作将使用强大的小鼠模型来严格阐明RAG DSB 触发V重组的瞬时反馈抑制，以帮助强制执行等位基因排斥。该项目将提供 对该领域一个未被充分研究的研究和一个全新的研究路线的新的机械洞察力，后者 与生物学相关的所有细胞。除了促进对获得性免疫的一个标志的理解之外，这些发现 可以确定抑制自身免疫和/或淋巴系统恶性肿瘤的重要机制。

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.