Spatiotemporal control of recombination by the RAG proteins and ATM

描述（由申请人提供）：淋巴样肿瘤是人类最常见的恶性肿瘤之一，原因尚不清楚；在过去的二十年中，它们的发病率一直在增加。虽然这些疾病由多种病因引起，但涉及抗原受体位点的染色体易位是一个共同的潜在机制。B淋巴细胞和T淋巴细胞的发育是由V (D)J重组驱动的，这是一个抗原受体位点上的基因片段反复重排以产生大量抗原受体基因库的过程。由于V(D)J重组需要每天数百万次对广泛分散的基因片段进行切割和连接，即使是很小的错误率也会带来相当大的易位风险。这种风险在B细胞中进一步增加，B细胞经历另一种称为类开关重组（CSR）的基因组重排过程，以改变免疫球蛋白（Ig）分子的效应功能。鉴于异常修复双链断裂（DSBs）的有害后果，V (D)J重组和CSR都必须严格调节切割底物的可及性以及DNA损伤反应和修复机制的活性。尽管详细的遗传和生化研究已经确定了这些过程中涉及的因素，但这些“批量技术”无法提供对其时空运作的深入了解；直到最近，显微镜技术才允许我们观察这些过程，因为它们发生在特定的细胞中。利用3D FISH和其他技术，我的实验室最近证明了同源Ig等位基因以一种特定阶段的方式进行物理配对，这种方式与这些基因座重组的顺序阶段相似。令人惊讶的是，这种等位基因间的关联是由V(D)J重组酶和DNA损伤检查点蛋白ATM介导的：在一个Ig等位基因上引入双链断裂，诱导ATM依赖的另一个等位基因重新定位到中心周围异染色质，以防止进一步的切割（Nature Immunology, in press）。值得注意的是，缺乏atm的小鼠和人类容易发生某些复发性的致癌易位，其机制尚不清楚；我们的工作揭示了ATM功能的时空方面。我们提出同源配对的等位基因进行重组，通过确保断裂末端与同源等位基因对齐，而不是与其他位点接触，保护基因组的稳定性。我们希望更深入地研究这一机制，研究ATM和RAG蛋白如何控制位点构象和核位置。

DESCRIPTION (provided by applicant): Lymphoid neoplasms are among the most common malignancies in humans, and, for reasons that remain obscure; their incidence has been increasing over the past two decades. Although these disorders arise from diverse etiologies, chromosomal translocations involving the antigen receptor loci are a common underlying mechanism. B and T lymphocyte development is driven by V (D)J recombination, a process by which gene segments at the antigen receptor loci are repeatedly rearranged to create a vast repertoire of antigen receptor genes. Because V(D)J recombination entails the cleavage and joining of widely dispersed gene segments many millions of times each day, even a miniscule error rate still carries considerable risk of translocation. This risk is further increased in B cells, which undergo yet another genomic rearrangement process known as class switch recombination (CSR) to change the effector function of the immunoglobulin (Ig) molecule. Given the deleterious consequences of aberrantly repaired double strand breaks (DSBs), both V (D)J recombination and CSR must tightly regulate accessibility of substrates for cleavage and the activities of the DNA damage response and repair machineries. Although detailed genetic and biochemical studies have identified the factors involved in these processes, these "bulk techniques" cannot provide much insight into their spatiotemporal workings; only recently have microscopic techniques allowed us to view these processes as they occur in specific cells. Using 3D FISH and other techniques, my lab recently demonstrated that homologous Ig alleles physically pair up in a stage-specific manner that parallels the sequential stages of recombination of these loci. Surprisingly, this interallelic association is mediated by the V(D)J recombinase and the DNA damage checkpoint protein ATM: introduction of a double-strand break at one Ig allele induces ATM-dependent repositioning of the other allele to pericentromeric heterochromatin to prevent further cleavage (Nature Immunology, in press). Notably, ATM-deficient mice and humans are prone to certain recurrent oncogenic translocations, by mechanisms that have remained unclear; our work sheds light on the spatiotemporal aspects of ATM function. We propose that homologous pairing of alleles undergoing recombination protects genomic stability by ensuring that broken ends are aligned with homologous alleles rather than in contact with other loci. We want to delve more deeply into this mechanism, investigating how ATM and the RAG proteins exert control over locus conformation and nuclear location. PUBLIC HEALTH RELEVANCE: Chromosomal translocations underlie a number of tumors of the lymphoid lineage. Recent advances in microscopy and FISH techniques allows us to visualize DNA breaks and interactions between chromosomes in individual cells, tracing their movements in and out of euchromatic regions in the nucleus. We recently discovered that the V (D)J recombinase and ATM mediate pairing of homologous Ig alleles and movement of one allele to pericentromeric heterocrhomatin as control points to preserve genomic stability during recombination. We propose to further these studies to understand precisely how these factors achieve these unexpected effects.