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）调节许多生物过程，包括基因转录，DNA复制和染色体分离。组蛋白PTMs的部分功能是通过调节染色质结构，使相关基因或多或少地接近介导这些生物过程的核蛋白。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特定区域的关联。DNA代谢的一个重要方面是DNA双链断裂（DSBs）的修复，其中组蛋白PTMs的功能仍然是一个谜。先前的研究表明，组蛋白变体H2AX仅在dsb的染色质侧被磷酸化，产生一种称为3-H2AX的形式。在酵母中，组蛋白PTMs更普遍的变化是在dsb侧的染色质上引起的，产生的模式让人想起那些在可接近的转录基因上发现的模式。然而，在哺乳动物细胞中，由于缺乏有效地将这些病变引入基因组中特定位置的实验方法，对dsb诱导的组蛋白PTMs变化几乎一无所知。为此，我们创新了基于细胞培养的方法，利用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。利用这种新颖的细胞系统，我们已经证明3-H2AX在这些RAG- dsb侧翼的染色质上产生的距离很长（约100 kb）。我们现在提议定义对邻近染色质中的rag -DSB有反应的组蛋白ptm群，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法筛选RAG-DSB染色质在广泛组蛋白ptm中的变化。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位点的定制微阵列，生成dsb诱导的组蛋白PTMs跨越几个Mb的高分辨率地图。这些研究将定义围绕生理dsb的表观遗传景观的时间和远程依赖变化（目的1）。Aim 1中生成的表观遗传图谱将被纳入以下策略中：(i)阐明DNA损伤反应途径，这对修改特定组蛋白PTMs至关重要；（ii）确定哪些组蛋白修饰酶负责抑制/消除dsb诱导的染色质变化；（iii）确定dsb是否介导区域染色质的永久变化。这些开创性的研究将提供一套基础的表观遗传学数据，以揭示组蛋白ptm和DNA修复途径之间的串扰机制，这些机制合作维持基因组的完整性。这种机制在淋巴细胞中尤为重要，在淋巴细胞中，必须针对抗原受体位点上的程序化dsb进行有效修复，以避免促进癌症的染色体易位。

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.