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获得性免疫至关重要。然而，这一过程也带来了风险，如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)基因座的致癌易位的淋巴癌。虽然对Ig/TCR组装的研究主要集中在RAG裂解是如何启动的，但RAG的活性也必须受到抑制，以限制DNA双链断裂(DSB)和由此导致的基因组不稳定性。34年来人们已经认识到，大多数Ig/TCR基因的完整组装一次发生在一个等位基因上，这一过程被称为等位基因排斥，在所有具有获得性免疫的生物中都是保守的。这一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，以永久阻止在形成有效连接后进一步的V重排。此外，1980年有人提出，V重组必须激活更多的即时信号来瞬时阻断V重组，为编码连接修复、表达和信号反馈抑制提供必要的时间。然而，长期以来预测的监管暂时性方面一直缺乏证据。申请人的实验室最近发现，在Vκ-to-Jκ重组信号过程中，RAG DSB通过共济失调毛细血管扩张突变蛋白激酶(ATM)诱导，瞬时抑制额外的Vκ重排。他们证明，atm下调RAG1mRNA和蛋白水平，以响应RAgκ，并帮助增强单等位基因Ig DSB的表达。根据他们的发现，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，以防止致癌的Ig易位，并确保携带Ig的B淋巴细胞的单特异性。这些机制被认为是为了创建一种失败安全的调控策略，其中包括：1)通过依赖ATM但仅限于发育中的B淋巴细胞的DSB反应通路抑制RAG的表达，2)在第二个未切割的Ig等位基因上反式重组的潜力，以及3)在切割的Ig等位基因上顺式重组。申请人建议剖析这些依赖ATM的V(D)J重组限制的潜在机制，并确定它们对加强单等位Ig表达、抑制Ig易位和形成Ig谱系的独立贡献。从这项研究中获得的知识将确定保护我们免受淋巴癌的分子和途径，以及产生具有多种抗原特异性的B细胞，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的预后、诊断和治疗方法的发展。

 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 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.