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. ? ? ?
