Redox-mediated p300 regulation of hepatocyte NF-kB

描述(申请人提供)：细胞氧化还原状态调节选定的转录因子的活性，如核因子-kB。氧化还原依赖的核因子-kB激活的顺式和反式调节的传统一级范式已经被广泛探索。然而，现在清楚的是，可诱导的基因转录也需要跨调节区的染色质结构的重组。研究表明，转录因子，如核因子-kB，结合到它们的靶部位需要招募具有组蛋白乙酰转移酶(HAT)活性的转录共激活因子(S)。这些共激活蛋白反过来又受其磷酸化状态的调节。在氧化应激的特定情况下，关于核因子-kB依赖转录中氧化还原敏感的染色质重塑和组蛋白修饰的高阶表观遗传机制知之甚少。在白介素1β(IL-1β)刺激CCL9.1小鼠肝细胞的模型中，我们已经证明，过氧化应激通过诱导组蛋白超乙酰化和染色质重塑来增强NF-kB结合位点(NT-114)，从而增强诱导型一氧化氮合酶(INOS)的转录。这种表观遗传现象不会仅在IL-1β或过氧化氢的作用下发生。以前的研究还没有研究肝细胞iNOS启动子上的核因子-kB活化过程中依赖氧化还原的染色质重塑。在R21的应用中，使用肝细胞iNOS作为氧化还原敏感基因表达的模型，我们建议表征氧化还原依赖的转录辅助激活因子，它通过染色质重塑和组蛋白超乙酰化来增强核因子-kB的激活。在进一步研究之前，必须确定这种共激活蛋白及其与氧化还原介导的染色质重塑的功能相关性。初步研究将集中在p300转录辅助激活蛋白/HAT蛋白上。鉴于这一试验性项目的新颖性和高风险性质，我们认为R21融资机制特别相关。

DESCRIPTION (provided by applicant): Cellular redox state regulates activity of selected transcription factors, such as NF-kB. The traditional first-order paradigms of cis- and trans- regulation of redox-dependent NF-kB activation have been extensively explored. However, it is now clear that inducible gene transcription also requires reorganization of chromatin structure across regulatory regions. Studies demonstrate that binding of transcription factors, such as NF-kB, to their target sites requires recruitment of transcriptional co-activator(s) with histone acetyltransferase (HAT) activity. These co-activator proteins are, in turn, regulated by their phosphorylation state. In the specific instance of oxidative stress, exceedingly little is known of the higher-order epigenetic mechanisms involving redox-sensitive chromatin remodeling and histone modification in NF-kB dependent transcription. In a model of interleukin-1beta (IL-1beta) stimulated CCL9.1 murine hepatocytes, we have demonstrated that peroxide-mediated oxidative stress enhances inducible nitric oxide synthase (iNOS) transcription by inducing histone hyperacetylation and chromatin remodeling at an otherwise silent NF-kB binding site (nt -114) to enhance NF-kB binding. This epigenetic phenomenon does not occur with IL-1beta or peroxide alone. Previous studies have not investigated redox-dependent chromatin remodeling in NF-kB activation at the hepatocyte iNOS promoter. In this R21 application, using hepatocyte iNOS as a model of redox-sensitive gene expression, we propose to characterize the redox-dependent transcriptional co-activator which enhances NF-kB activation through chromatin remodeling and histone hyperacetylation. This co-activator protein and its functional relevance to redox-mediated chromatin remodeling must be characterized before additional studies can proceed. Initial studies will focus upon the p300 transcriptional co-activator/HAT protein. Given the novel and high risk nature of this experimental venture, we believe the R21 funding mechanism to be particularly relevant.