LOCALIZED REVISION OF EPIGENETIC LANDSCAPES INDUCED BY DNA DOUBLE-STRAND BREAKS

描述（由申请人提供）：组蛋白的翻译后修饰（PTM）调节许多生物学过程，包括基因转录、DNA复制和染色体分离。组蛋白PTM部分地通过调节染色质结构来使相关基因或多或少地接近介导这些生物过程的核蛋白而起作用。组蛋白PTM还通过与不同蛋白质结构域的相互作用靶向核因子与染色质的特定区域的缔合。DNA代谢的一个重要方面是DNA双链断裂（DSB）的修复，其中组蛋白PTM的功能仍然是一个谜。先前的研究表明，组蛋白变体H2 AX仅在染色质侧翼的DSB中磷酸化，产生称为3-H2 AX的形式。在酵母中，组蛋白PTM的更普遍的变化是在染色质侧翼DSB处诱导的，产生的模式让人想起在可接近的转录基因中发现的模式。然而，在哺乳动物细胞中，几乎没有什么是已知的DSB诱导的组蛋白PTM的变化，由于缺乏有效地引入这些病变在基因组中的定义位置的实验方法。为此，我们创新了基于细胞培养的方法，该方法利用了原代和转化淋巴细胞，其中V（D）J重组酶（RAG）靶向基因组中精确位置的持久性DSB，使我们能够监测细胞对DNA断裂的反应。使用这种新的细胞系统，我们已经表明3-H2 AX在这些RAG-DSB侧翼的染色质中在延伸的距离（>100 kb）上产生。我们现在提出定义组蛋白PTM的星座，响应于相邻染色质中的RAG-DSB，目的是阐明它们在协调DSB修复中的功能。为了实现这一目标，我们将首先使用染色质免疫沉淀筛选RAG-DSB染色质中组蛋白PTM的变化。在初始筛选的指导下，我们将使用包含所有淋巴细胞抗原受体基因座的定制微阵列生成DSB诱导的组蛋白PTM变化的高分辨率地图，这些变化跨越几Mb。这些研究将定义生理性DSB周围表观遗传景观的时间和距离依赖性变化（目标1）。目标1中生成的表观遗传图谱将被纳入策略中，以（i）阐明对特定组蛋白PTM的修订至关重要的DNA损伤反应途径，（ii）确定哪些组蛋白修饰酶负责冲压/擦除DSB诱导的染色质变化，以及（iii）确定DSB是否介导区域染色质的永久性变化。这些开创性的研究将提供一组基础的表观遗传学数据，以揭示组蛋白PTM和DNA修复途径之间的相互作用机制，这些机制共同维持基因组的完整性。这种机制在淋巴细胞中特别重要，其中有效的修复必须针对抗原受体基因座中的程序化DSB，以避免促进癌症的染色体易位。 公共卫生相关性：DNA双链断裂（DSB）是在暴露于遗传毒性物质（如电离辐射）时产生的，是几个重要生理过程（包括淋巴细胞抗原受体基因组装）的中间体。DSB被适当修复而不是进入可能导致致癌染色体易位的替代途径是至关重要的。我们现在建议确定组蛋白修饰，作为中间体协调哺乳动物细胞中DNA-DSB的正常修复。

DESCRIPTION (provided by applicant): The post-translational modification (PTM) of histones regulates many biological processes, including gene transcription, DNA replication, and chromosome segregation. Histone PTMs function, in part, by modulating chromatin structure to render associated genes more or less accessible to nucleoproteins that mediate these biological processes. Histone PTMs also target the association of nuclear factors to specific regions of chromatin via interactions with diverse protein domains. One essential aspect of DNA metabolism in which the function of histone PTMs remains a mystery is the repair of DNA double-strand breaks (DSBs). Prior studies have shown that the histone variant H2AX is phosphorylated exclusively in chromatin flanking DSBs, generating a form called 3-H2AX. In yeast, more general changes in histone PTMs are induced at chromatin flanking DSBs, producing patterns that are reminiscent of those found at accessible, transcribed genes. However, in mammalian cells almost nothing is known about DSB-induced changes in histone PTMs due to the lack of experimental approaches for efficiently introducing these lesions at defined locations in the genome. For this purpose, we have innovated cell culture-based approaches that take advantage of primary and transformed lymphocytes in which the V(D)J recombinase (RAG) targets persistent DSBs at precise locations in the genome, allowing us to monitor the cellular response to DNA breaks. Using this novel cell system, we have shows that 3-H2AX is generated over extended distances (>100 kb) in chromatin flanking these RAG- DSBs. We now propose to define the constellation of histone PTMs that respond to RAG-DSBs in neighboring chromatin with the goal of elucidating their function in coordinating DSB repair. To achieve this goal, we will initially screen RAG-DSB chromatin for changes in a broad panel of histone PTMs using chromatin immunoprecipitation. Guided by the initial screens, we will generate high-resolution maps of DSB-induced changes to histone PTMs spanning several Mb using a custom microarray that contains all lymphocyte antigen receptor loci. These studies will define both time- and distant-dependent changes to the epigenetic landscape surrounding physiologic DSBs (Aim 1). The epigenetic maps generated in Aim 1 will be incorporated into strategies to (i) elucidate DNA damage response pathways that are critical for the revision of specific histone PTMs, (ii) determine which histone modifying enzymes are responsible for stamping/erasing DSB-induced changes in chromatin, and (iii) determine whether DSBs mediate a permanent change in regional chromatin. These pioneering studies will provide a foundational set of epigenetic data to reveal mechanisms of crosstalk between histone PTMs and DNA repair pathways that cooperate to maintain genomic integrity. Such mechanisms are particularly important in lymphocytes, where efficient repair must be directed to programmed DSBs in antigen receptor loci to avoid chromosomal translocations that promote cancer. PUBLIC HEALTH RELEVANCE: DNA double-strand breaks (DSBs) are generated upon exposure to genotoxic agents, such as ionizing radiation, and are intermediates during several important physiologic processes, including lymphocyte antigen receptor gene assembly. It is critical that DSBs are appropriately repaired rather than accessing alternative pathways that potentially result in oncogenic chromosomal translocations. We now propose to identify histone modifications that function as intermediates to coordinate the normal repair of DNA-DSBs in mammalian cells.