Role of ATM and RAG in maintaining genome stability during Tcra/d rearrangement.

描述(申请人提供)：B和T细胞表达识别几乎无限种抗原的受体。这些受体的多样性是通过一个称为V(D)J重组的过程产生的。这涉及抗原受体基因的重复切割和修复，这些基因分散在几个不同的染色体上。重组受到仔细的调控，以特定的顺序发生在不同的染色体上。这通常是顺利进行的，但有时也会出现错误。已知的是，非同源末端连接(NHEJ)和DNA损伤反应通路的成分缺陷容易导致异常的V(D)J重排事件，从而导致白血病和淋巴瘤。在很大程度上，损伤的范围将取决于每个因素对RAG介导的双链断裂修复的贡献。在这一背景下，个体修复和损伤反应因子的影响已经被广泛研究，但除此之外，对于这些因素是否在调节RAG酶活性或在RAG介导的断裂被引入到一个等位基因后，抗原受体基因和周围基因的可及性以阻止切割的作用，人们知之甚少。这项资助中概述的工作将导致对程序性DNA断裂如何以受控方式引入以保护V(D)J重组期间基因组稳定性的更详细和更机械的理解。我们的目标是观察发育中的T细胞基因组的3D组织，以确定与TCRA/d密切相关的处于风险中的候选细胞，在它们重组的细胞中。此外，我们将确定亲密关系是否通过将活跃的转录基因循环出来并聚集在转录工厂中来培养，这些转录工厂可以丰富RAG蛋白。对核组织和基因活性的联合分析(众所周知，这是RAG靶向的一个重要决定因素)将为我们提供一个起点，从这个起点来揭示单个修复因子的影响。重要的是，这一分析将使我们能够将已知的致癌基因易位事件置于全球背景下，以便我们能够开始了解为什么特定基因是靶标，并与TCRA/d在染色体重排中结合。)

DESCRIPTION (provided by applicant): B and T cells express receptors that recognize an almost infinite variety of antigens. The diversity of these receptors is generated through a process known as V(D)J recombination. This involves repeated cleavage and repair of antigen receptor genes, which are scattered across several different chromosomes. Recombination is carefully regulated and occurs at different chromosomal loci in a specific sequence. This normally proceeds without mishap, but mistakes do sometimes occur. Deficiencies in components of the nonhomologous end joining (NHEJ) and DNA damage response pathways are known to predispose to aberrant V(D)J rearrangement events that can lead to leukemias and lymphomas. In large part, the spectrum of damage will depend on the contribution of each factor to the repair of RAG-mediated double strand breaks. The effects of individual repair and damage response factors in this context have been extensively studied, but beyond this, little is known about whether any of these factors have a role in regulating RAG enzymatic activity or the accessibility of the antigen receptor loci and surrounding genes to halt cleavage after RAG-mediated breaks are introduced on one allele. The work outlined in this grant will lead to a more detailed and mechanistic understanding of how programmed DNA breaks are introduced in a controlled manner to protect genome stability during V(D)J recombination. We aim to look at the 3D organization of the genome in developing T cells to identify 'at risk' candidates that are closely associated with Tcra/d in cells where they are being recombined. Further, we will determine whether intimate relationships are fostered by looping out of actively transcribed genes and congregation in transcription factories that could be enriched for RAG proteins. The combined analyses of nuclear organization and gene activity (which is known to be an important determinant of RAG targeting) will provide us with a starting point from which to unravel the effects of individual repair factors. Importantly, this analysis will allow us to put known oncogenc translocation events into a global context so that we can begin to understand why particular genes are targeted and join with Tcra/d in chromosomal rearrangements. )