The overall goal of this project is to understand the molecular mechanisms by which phenobarbital (PB) induces cytochrome P450 gene (CYP) expression. P450s catalyze the activation or inactivation of a wide variety of endogenous and exogenous compounds. Clinically, induction of P450s underlies many drug interactions, and the balance between inactivation and activation, which is influenced by induction, determines the ultimate therapeutic or toxic effect of ingested compounds. PB treatment induces the nuclear translocation of the constitutive androstane receptor (CAR). CAR binds to three nuclear receptor (NR) binding sites in a PB responsive enhancer (PBRU) as a heterodimer with RXR and recruits p160 coactivators and other unknown proteins resulting in changes in chromatin structure and gene activation.
The specific aims are to identify the regulatory complex recruited by CAR to the PBRU, to determine changes in chromatin structure at the PBRU correlated with activation of the CYP genes, and to determine the import and export signals of CAR necessary for its nucleocytoplasmic shuttling. To facilitate the isolation of CAR and its protein complexes from mouse liver, a transgenic mouse expressing flag-tagged CAR will be constructed. Nuclear CAR protein complexes in PB-treated animals will be enriched by flag immunoaffinity isolation and proteins in the complex will be identified by mass spectrometry. The relative importance of the three p160 coactivators and the three NR binding sites will be determined. Proteins recruited to the PBRU after PB treatment will be identified by chromatin immunoprecipitation assays and chromatin structure will be probed by sensitivity to cleavage by nucleases and hydroxyl radicals. The functional significance of proteins detected at the PBRU or in CAR complexes will be determined by transient transfections in cultured cells and in hepatocytes in vivo. Nuclear import and export signals and receptors for CAR will be identified by examining the distribution of chimera of CAR fragments and fluorescent proteins in hepatocytes transfected in vivo. These studies, emphasizing in vivo approaches, should provide a description of the regulatory complex at the PBRU, which will provide insight into the mechanism of PB induction of CYP genes, and determine transport signals for nucleocytoplasmic shuttling, which is a key process regulated by PB treatment.
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