ACTIVATION OF CELLULAR SIGNALING PATHWAYS BY DNA DOUBLE STRAND BREAKS

描述(由申请人提供)： 这项建议的主要目的是阐明组蛋白H2AX蛋白通过信号传递细胞对DNA双链断裂(DSB)的反应的机制。DSB是由遗传毒性物质产生的，是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(类开关重组)等生理过程的中间产物。我们已经证明，H_2AX缺陷小鼠表现出基因组不稳定和癌症易感性增加，包括淋巴瘤克隆性易位，其中一些涉及抗原受体基因座。这些数据表明，H_2AX参与了对遗传毒性和生理性DSB的修复和/或细胞反应。在DSB周围的染色质中，H_2AX被磷酸化(生成？-H_2AX)、乙酰化和泛素化；然而，H_2AX和这些其他修饰形式的H_2AX的功能(S)尚不清楚。H_2AX基因缺陷小鼠淋巴细胞减少，在V(D)J重组的抗原受体基因上形成3-H_2AX，提示H_2AX在淋巴细胞抗原受体基因组装过程中产生的DSB的修复和/或细胞反应中起作用。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所必需的，也是激活一个广泛功能的遗传程序以响应这些生理性DSB所必需的。由于ATM是磷酸化H_2AX的主要激酶，我们的数据提出了ATM通过？-H_2AX的磷酸化来调节这些过程的可能性。在这一应用中，我们证明了H2AX不是修复在抗原受体基因组装过程中产生的双链断裂所必需的，而是需要激活基因表达来响应这些生理性双链断裂。因此，我们假设H2AX是激活转录途径响应DSB的重要中间体，至少部分原因是要求在这些DNA损伤周围的染色质中产生3-H2AX。在这里，我们建议使用一种新的基于细胞系的方法，通过这种方法可以在基因组中的精确位置诱导DSB，并阐明在这些断裂处调节β-H_2AX形成的顺式和反式作用因子。此外，我们建议阐明被？-H_2AX激活的转录途径，确定它们是如何被激活的，并确定受这些途径调控的靶基因。

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.