Spatiotemporal control of recombination by the RAG proteins and ATM

描述（由申请方提供）：类肉瘤是人类最常见的恶性肿瘤之一，由于尚不清楚的原因，其发病率在过去二十年中一直在增加。尽管这些疾病的病因多种多样，但涉及抗原受体基因座的染色体易位是常见的潜在机制。B和T淋巴细胞发育由V（D）J重组驱动，V（D）J重组是抗原受体基因座处的基因片段重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的广泛分散的基因片段的切割和连接，即使是极小的错误率仍然具有相当大的易位风险。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别转换重组（CSR）的基因组重排过程，以改变免疫球蛋白（IG）分子的效应子功能。考虑到异常修复的双链断裂（DSB）的有害后果，V（D）J重组和CSR必须严格调节用于切割的底物的可及性以及DNA损伤应答和修复机制的活性。虽然详细的遗传和生物化学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。利用三维荧光原位杂交和其他技术，我的实验室最近证明，同源IG等位基因以阶段特异性的方式物理配对，与这些位点重组的顺序阶段平行。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等位基因处引入双链断裂诱导另一个等位基因向着丝粒周围异染色质的ATM依赖性重新定位，以防止进一步切割（Nature Immunology，出版中）。值得注意的是，ATM缺陷的小鼠和人类容易发生某些复发性致癌易位，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，经历重组的等位基因的同源配对通过确保断裂末端与同源等位基因对齐而不是与其他基因座接触来保护基因组稳定性。我们希望更深入地研究这一机制，研究ATM和RAG蛋白如何控制基因座构象和核定位。 公共卫生相关性：染色体易位是许多淋巴系肿瘤的基础。显微镜和FISH技术的最新进展使我们能够可视化单个细胞中染色体之间的DNA断裂和相互作用，追踪它们在细胞中的进出运动。 核内的常染色质区。我们最近发现V（D）J重组酶和 ATM介导同源IG等位基因配对和一个等位基因向着丝粒周围的移动 作为控制点，以在重组期间保持基因组稳定性。我们建议进一步研究，以准确了解这些因素如何实现这些意想不到的效果。

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.