The overall goal of this project is to understand the molecular mechanisms by which phenobarbital induces cytochrome P450 gene (CYP) expression. Cytochromes P450 form a super family of enzymes responsible for activation or inactivation by oxidative metabolism of a wide variety of endogenous and exogenous compounds. The balance between activation and inactivation, which can be dramatically altered by induction, determines the ultimate therapeutic or toxic activity of an ingested chemical. A model of phenobarbital (PB) action in which PB induces the translocation from the cytoplasm to the nucleus of the nuclear receptor, constitutive androstane receptor (CAR) has been recently developed. CAR, as a heterodimer with the nuclear receptor, RXR, binds to nuclear receptor binding sites in a PB responsive unit (PBRU) at -2.3 Kb to activate hepatic CYP2B gene expression. Other elements in the PBRU contribute to the induction, including NF-1, indicating that a complex of proteins mediates the activation of the gene. The chromatin structure of the CYP2B PBRU and the proximal promoter is altered in hepatic tissue, and PB treatment results in additional protein binding to the PBRU.
The specific aims of this proposal are directed at understanding the mechanism by which CAR/RXR binding to the PBRU activates CYP2B genes beginning with characterization of the proteins binding to the PBRU and their interaction with CAR, then identification and characterization of co-regulator proteins binding to CAR, and finally in vivo approaches to establish the validity of the in vitro studies. The binding of proteins to PBRU sequences will be studied by in vitro footprinting and gel shift assays to assess whether the binding is cooperative, antagonistic, or independent. The influence of chromatin structure on the binding of the proteins will be studied by in vitro assembly of chromatin and footprinting analysis. Potential co-regulators that interact with CAR will be identified by GST-pull downs and yeast two-hybrid analysis. The functional significance of the DNA binding proteins and potential co-regulators will be determined by transient and stable transfections of cultured cells in vitro transcription combined with mutagenesis of the PBRU and co-expression of the factors with CAR/RXR. The role of histone modification in activation of the CYP2B genes will be studied. The in vivo significance of the in vitro experiments will be assessed by determining the binding in vivo of identified factors to the PBRU by the chromatin immunoprecipitation technique and functionally by transient and stable transfections of hepatocytes in vivo by tail vein injection of vector DNA. These studies will establish the nature and function of the complex of proteins responsible for PB activation of CYP2B genes.
Showing the most recent 10 out of 21 publications
