ACTIVATION OF CELLULAR SIGNALING PATHWAYS BY DNA DOUBLE STRAND BREAKS

描述（由申请人提供）： 该提案的主要目的是阐明组蛋白 H2AX 蛋白发出细胞对 DNA 双链断裂 (DSB) 反应信号的机制。 DSB 由基因毒性剂产生，是 DNA 复制、减数分裂以及淋巴细胞抗原受体基因的生成（VDJ 重组）和多样化（类别转换重组）等生理过程的中间体。我们已经证明，H2AX 缺陷小鼠表现出基因组不稳定和癌症易感性增加，包括淋巴瘤克隆易位，其中一些涉及抗原受体基因座。这些数据表明 H2AX 参与基因毒性和生理 DSB 的修复和/或细胞反应。 H2AX 在 DSB 周围的染色质中被磷酸化（生成 α-H2AX）、乙酰化和泛素化；然而，α-H2AX 和 H2AX 的这些其他修饰形式的功能尚不清楚。 H2AX 缺陷小鼠淋巴细胞减少，并且在经历 V(D)J 重组的抗原受体位点处形成 3-H2AX，这表明 H2AX 在淋巴细胞抗原受体基因组装过程中产生的 DSB 的修复和/或细胞反应中发挥作用。我们已经证明，ATM 的激酶活性是修复抗原受体基因组装过程中产生的 DSB 以及激活响应这些生理 DSB 的广泛功能遗传程序所必需的。由于 ATM 是磷酸化 H2AX 的主要激酶，因此我们的数据提出了 ATM 通过 α-H2AX 磷酸化来介导这些过程的可能性。在此应用中，我们证明 H2AX 不是修复抗原受体基因组装过程中产生的 DSB 所必需的，但需要激活基因表达以响应这些生理 DSB。因此，我们假设 H2AX 是响应 DSB 激活转录途径的重要中间体，至少部分是因为需要在这些 DNA 损伤周围的染色质中生成 3-H2AX。在这里，我们建议使用一种基于细胞系的新型方法，可以在基因组的精确位置诱导 DSB，并阐明在这些断裂处调节 ?-H2AX 形成的顺式作用和反式作用因子。此外，我们建议阐明 ?-H2AX 激活的转录途径，确定它们是如何激活的，并鉴定受这些途径调节的靶基因。公共健康相关性：组蛋白 H2AX 蛋白在细胞对受损 DNA 的反应中至关重要，这对于修复受损 DNA 和消除持续损伤的细胞至关重要，因为它们面临基因组不稳定的风险。我们已经证明，H2AX 在响应 DNA 双链断裂而激活基因表达变化中充当关键的信号传导中间体。在这里，我们建议阐明 H2AX 启动的遗传程序，识别被激活的转录途径，并确定它们是如何被 H2AX 激活的。

DESCRIPTION (provided by applicant): The main objective of this proposal is to elucidate the mechanisms by which the histone H2AX protein signals cellular responses to DNA double strand breaks (DSBs). DSBs are generated by genotoxic agents and are intermediates during physiologic processes such as DNA replication, meiosis and the generation (VDJ recombination) and diversification (class switch recombination) of lymphocyte antigen receptor genes. We have shown that H2AX deficient mice exhibit genomic instability and an increased predisposition cancer, including lymphomas clonal translocations, some of which involve antigen receptor loci. These data indicate that H2AX is involved in the repair of and/or cellular response to genotoxic and physiologic DSBs. H2AX is phosphorylated (generating ?-H2AX), acetylated, and ubiquitylated in chromatin around DSBs; however, the function(s) of ?-H2AX and these other modified forms of H2AX are not known. H2AX deficient mice are lymphopenic and 3-H2AX forms at antigen receptor loci undergoing V(D)J recombination, suggesting that H2AX functions in the repair of and/or cellular response to DSBs generated during lymphocyte antigen receptor gene assembly. We have shown that the kinase activity of ATM is required for the repair of DSBs generated during antigen receptor gene assembly and for the activation of a broadly functional genetic program in response to these physiologic DSBs. Since ATM is the major kinase that phosphorylates H2AX, our data raise the possibility that the ATM functions through the phosphorylation of ?-H2AX to mediate these processes. Within this application, we show that H2AX is not required for the repair of DSBs generated during antigen receptor gene assembly, but is required to activate gene expression in response to these physiologic DSBs. Thus, we hypothesize that H2AX is an important intermediate in activating transcriptional pathways in response to DSBs due, at least in part, to a requirement to generate 3-H2AX in chromatin around these DNA lesions. Here, we propose to use a novel cell line based approach whereby DSBs can be induced at precise locations in the genome and elucidate the cis-acting and trans-acting factors that regulate the formation of ?-H2AX at these breaks. In addition, we propose to elucidate the transcriptional pathways that are activated by ?-H2AX, determine how they are activated, and identify the target genes that are regulated by these pathways. PUBLIC HEALTH RELEVANCE: The histone H2AX protein is centrally important in the cellular responses to damaged DNA, which are critical for the repair of damaged DNA and for the elimination of cells with persistent damage since they are at risk for genomic instability. We have shown that H2AX functions as a critical signaling intermediate in the activation of gene expression changes in response to DNA double strand breaks. Here, we propose to elucidate the genetic program initiated by H2AX, identify the transcriptional pathways that are activated, and determine how they are activated by H2AX.