DNA Double Strand Break Mediated Feedback Inhibition of V(D)J Recombination

描述（由申请人提供）：通过RAG1/RAG2 （RAG）核酸酶组装来自可变(V)、多样性(D)和连接(J)基因片段的淋巴细胞抗原受体基因对适应性免疫至关重要。然而，自身反应性受体的组装和涉及免疫球蛋白（Ig）或T细胞受体（TCR）位点的致癌易位的淋巴样癌也证明了这一过程也会带来风险。虽然Ig/TCR组装的研究主要集中在RAG切割是如何开始的，但RAG的活性也必须受到限制，以限制多重DNA双链断裂（dsb）和由此产生的基因组不稳定性。30年来，我们已经知道大多数Ig/TCR基因的完整组装一次发生在一个等位基因上，这表明控制等位基因之间V(D)J重组的机制具有根本的重要性。V型重排的异步启动，即每个等位基因一次重组一个，以及Ig/ tcr介导的对进一步V型重排的反馈抑制，以维持等位基因的排除，是这种控制的两个重要方面。1980年有人提出，V重组还必须激活更多的即时信号，这些信号暂时阻止V重排，为组装的基因表达和Ig/ tcr介导的反馈抑制信号提供时间。然而，关于V(D)J重组控制的这一长期预测的额外方面的证据一直缺乏。申请人实验室最近证实，在Ig？重组信号通过共济失调毛细血管扩张突变（ATM）激酶短暂抑制额外的Ig？重组。他们证明，ATM既抑制Rag1和Rag2 mRNA和蛋白水平，又抑制RAG DSBs的单等位基因Ig？表达式。基于他们的发现，申请人假设RAG DSBs通过多种独立的机制暂时抑制额外的V重组，以防止致癌Ig易位并确保携带Ig的B细胞的单特异性。这些互补机制被假设为产生一种失效保险的调节策略，包括抑制：1)依赖于atm但仅限于B细胞前体发育的DSB反应途径的核RAG表达，2)其他Ig等位基因的反式重组，以及3)裂解的Ig等位基因的顺式顺序重排。申请人建议剖析这些依赖于atm的限制的潜在分子机制，并确定它们对单等位基因Ig表达、抑制Ig易位和形成Ig库的独立贡献。从该项目获得的知识将确定保护我们免受淋巴细胞癌和产生具有多种抗原特异性的B淋巴细胞的分子和途径，从而导致自身免疫。从长远来看，这些知识将促进针对特定人类免疫疾病的新型预后和治疗方法的发展。

DESCRIPTION (provided by applicant): Assembly of lymphocyte antigen receptor genes from variable (V), diversity (D), and joining (J) gene segments by the RAG1/RAG2 (RAG) nuclease is vital for adaptive immunity. However, this process also confers risk as evidenced by assembly of auto-reactive receptors and by lymphoid cancers with oncogenic translocations involving immunoglobulin (Ig) or T cell receptor (TCR) loci. While studies of Ig/TCR assembly have focused on how RAG cleavage is initiated, RAG activity also must be restrained to limit multiple DNA double strand breaks (DSBs) and resultant genomic instability. It has been known for 30 years that complete assembly of most Ig/TCR genes occurs on one allele at a time, indicating fundamental importance of mechanisms that control V(D)J recombination between alleles. Asynchronous initiation of V rearrangements, such that each allele recombines one at a time, and Ig/TCR-mediated feedback inhibition of further V rearrangements to maintain allelic exclusion are two important facets of this control. It was proposed in 1980 that V recombination also must activate more immediate signals that transiently prevent V rearrangements, providing time for assembled genes to be expressed and signal Ig/TCR-mediated feedback inhibition. However, evidence for this additional long-predicted facet of V(D)J recombination control has been lacking. The applicant's lab recently showed that RAG DSBs induced during Ig? recombination signal through the Ataxia Telangiectasia mutated (ATM) kinase to transiently inhibit additional Ig? rearrangements. They demonstrated that ATM both inhibits Rag1 and Rag2 mRNA and protein levels in response to RAG DSBs and enforces mono-allelic Ig? expression. Based on their discoveries, the applicant hypothesizes that RAG DSBs transiently inhibit additional V recombination through multiple independent mechanisms to safeguard against oncogenic Ig translocations and ensure monospecificity of Ig-bearing B cells. These complementary mechanisms are hypothesized to generate a failsafe regulatory strategy that includes suppression of: 1) nuclear RAG expression by DSB response pathways that are ATM-dependent but restricted to developing B cell precursors, 2) recombination in trans at other Ig alleles, and 3) sequential rearrangements in cis on the cleaved Ig allele. The applicant proposes to dissect the underlying molecular mechanisms for these ATM-dependent restrictions and determine their independent contributions to enforcement of mono-allelic Ig expression, suppression of Ig translocations, and shaping Ig repertoire. Knowledge acquired from this project will define molecules and pathways that protect us from lymphoid cancers and production of B lymphocytes with multiple antigen specificities, resulting in autoimmunity. In the long-term, such knowledge will foster the development of novel prognostics and therapeutics for specific human immunological disorders.