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DNA双链断裂（DSB）和由此产生的基因组不稳定性。34年来，人们已经认识到，大多数IG/TCR基因的完全组装一次发生在一个等位基因上，这是通过一种称为等位基因排斥的过程来实现的，该过程在具有适应性免疫的所有生物体中是保守的。这种基本控制机制的两个方面是等位基因之间V重排的异步启动和IG/TCR介导的反馈，以在形成生产性连接后永久地阻断进一步的V重组。此外，1980年有人提出，V重组必须激活更多的即时信号才能暂时阻断V重组，为编码连接修复、表达、然后信号反馈抑制提供必要的时间。然而，这一长期预测的监管瞬态方面的证据一直缺乏。申请人的实验室最近显示，在Vκ至J κ重组信号期间，通过共济失调毛细血管扩张突变（ATM）蛋白激酶诱导RAG DSB，以瞬时抑制额外的Vκ重排。他们证明ATM下调RAG 1和RAG 2 mRNA和蛋白水平以响应RAG DSB，并有助于加强单等位基因IGκ表达。基于他们的发现，申请人假设RAG和其他类型的DSB通过多种独立但互补的机制瞬时抑制V重排，以防止致癌IG易位并确保携带Ig的B淋巴细胞的单特异性。假设这些机制产生故障安全调节策略，其包括抑制：1）通过ATM依赖性但限于发育中的B淋巴细胞的DSB应答途径的RAG表达，2）在第二个未切割的IG等位基因处的反式重组潜力，和3）在切割的IG等位基因上顺式顺序重组。申请人提出剖析V（D）J重组的这些ATM依赖性限制的潜在机制，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。从这项研究中获得的知识将定义保护我们免受淋巴癌的分子和途径，以及具有多种抗原特异性的B细胞的产生，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免疫学、诊断学和治疗学的发展。

 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 DNA double strand breaks (DSBs) and resultant genomic instability. It has been recognized for 34 years that complete assembly of most Ig/TCR genes occurs on one allele at a time, enforced by a process called allelic exclusion that is conserved among all organisms with adaptive immunity. Two facets of this fundamental control mechanism are asynchronous initiation of V rearrangements between alleles and Ig/TCR-mediated feedback to permanently block further V recombination after a productive join is formed. In addition, it was proposed in 1980 that V recombination must activate more immediate signals to transiently block V recombination, providing the time necessary for coding joins to be repaired, expressed, and then signal feedback inhibition. However, evidence for this long-predicted transient aspect of regulation has been lacking. The applicant's lab recently showed that RAG DSBs induced during Vκ-to-Jκ recombination signal via the Ataxia Telangiectasia mutated (ATM) protein kinase to transiently inhibit additional Vκ rearrangements. They demonstrated that ATM down-regulates RAG1 and RAG2 mRNA and protein levels in response to RAG DSBs, and helps enforce mono-allelic Igκ expression. Based on their findings, the applicant hypothesizes that RAG and other types of DSBs transiently suppress V rearrangements via multiple independent but complementary mechanisms to safeguard from oncogenic Ig translocations and ensure mono-specificity of Ig-bearing B lymphocytes. These mechanisms are hypothesized to create a failsafe regulatory strategy that includes the suppression of: 1) RAG expression by DSB response pathways that are ATM-dependent but restricted to developing B lymphocytes, 2) recombination potential in trans at the second non-cleaved Ig allele, and 3) sequential recombination in cis on the cleaved Ig allele. The applicant proposes to dissect underlying mechanisms for these ATM-dependent restrictions of V(D)J recombination and determine their independent contributions to enforcement of mono-allelic Ig expression, suppression of Ig translocations, and shaping Ig repertoire. The knowledge acquired from this research will define molecules and pathways that protect us from lymphoid cancers, as well as production of B cells with multiple antigen specificities, resulting in autoimmunity. In the long-term, such knowledge will foster the development of novel prognostics, diagnostics, and therapeutics for specific human immunological disorders.