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

描述（由申请人提供）：通过RAG 1/RAG 2（RAG）核酸酶从可变（V）、多样性（D）和连接（J）基因片段组装淋巴细胞抗原受体基因对于获得性免疫至关重要。然而，该过程也赋予风险，如通过自身反应性受体的组装和通过涉及免疫球蛋白（IG）或T细胞受体（TCR）基因座的致癌易位的淋巴癌所证明的。虽然IG/TCR组装的研究集中在如何启动RAG切割，但也必须抑制RAG活性以限制多个DNA双链断裂（DSB）和由此产生的基因组不稳定性。30年来人们已经知道，大多数IG/TCR基因的完全装配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重排的异步启动，使得每个等位基因一次重组一个，以及IG/TCR介导的进一步V重排的反馈抑制以维持等位基因排斥是这种控制的两个重要方面。1980年提出，V重组还必须激活瞬时阻止V重排的更直接的信号，为组装的基因表达和信号IG/TCR介导的反馈抑制提供时间。然而，这个额外的长期预测方面的V（D）J重组控制的证据一直缺乏。申请人的实验室最近表明，在IG？重组信号通过共济失调毛细血管扩张突变（ATM）激酶瞬时抑制额外的IG？重新安排他们证明，ATM抑制Rag 1和Rag 2的mRNA和蛋白质水平在响应RAG DSB和强制执行单等位基因IG？表情基于他们的发现，申请人假设RAG DSB通过多种独立机制瞬时抑制额外的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。假设这些互补机制产生故障安全的调控策略，包括抑制：1）通过ATM依赖性但限于发育中的B细胞前体的DSB反应途径的核RAG表达，2）在其他IG等位基因处的反式重组，和3）在切割的IG等位基因上顺式顺序重排。申请人提出剖析这些ATM依赖性限制的潜在分子机制，并确定它们对单等位基因IG表达的实施、IG易位的抑制和IG库的形成的独立贡献。从这个项目中获得的知识将定义保护我们免受淋巴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性疾病的新型免疫学和治疗学的发展。

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.