ACTIVATION OF CELLULAR SIGNALING PATHWAYS BY DNA DOUBLE STRAND BREAKS

描述（由申请人提供）： 本提案的主要目的是阐明组蛋白H2 AX蛋白信号细胞响应DNA双链断裂（DSB）的机制。DSB由遗传毒性剂产生，是生理过程中的中间体，如DNA复制、减数分裂和淋巴细胞抗原受体基因的产生（VDJ重组）和多样化（类别转换重组）。我们已经表明，H2 AX缺陷小鼠表现出基因组不稳定性和增加的易感性癌症，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，H2 AX参与修复和/或细胞反应的遗传毒性和生理DSB。H2 AX被磷酸化（产生？H2 AX）、乙酰化和泛素化;然而，H2 AX和H2 AX的这些其他修饰形式是未知的。H2 AX缺陷型小鼠是淋巴细胞减少的，并且在经历V（D）J重组的抗原受体基因座处形成3-H2 AX形式，表明H2 AX在淋巴细胞抗原受体基因组装期间产生的DSB的修复和/或细胞应答中起作用。我们已经表明，ATM的激酶活性是修复抗原受体基因组装过程中产生的DSB和激活响应于这些生理DSB的广泛功能的遗传程序所必需的。由于ATM是磷酸化H2 AX的主要激酶，我们的数据提出了ATM通过磷酸化？H2 AX介导这些过程。在此应用中，我们表明，H2 AX是不需要的抗原受体基因组装过程中产生的DSB的修复，但需要激活基因表达，以响应这些生理DSB。因此，我们假设，H2 AX是一个重要的中间体，在激活转录途径响应DSB，由于，至少部分地，在这些DNA损伤周围的染色质中产生3-H2 AX的要求。在这里，我们建议使用一种新的细胞系为基础的方法，DSB可以诱导在基因组中的精确位置，并阐明顺式作用和反式作用的因素，调节？H2 AX在这些休息。此外，我们建议阐明的转录途径，激活？- H2 AX，确定它们是如何被激活的，并确定由这些途径调节的靶基因。公共卫生相关性：组蛋白H2 AX蛋白在对受损DNA的细胞反应中是重要的，这对于修复受损DNA和消除具有持续性损伤的细胞至关重要，因为它们处于基因组不稳定性的风险中。我们已经表明，H2 AX功能作为一个关键的信号中间体在激活基因表达的变化，响应DNA双链断裂。在这里，我们建议阐明H2 AX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H2 AX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.