This application is for a supplement to GM39360 """"""""Regulation of cytochrome P450 biosynthesis"""""""" for the purpose of constructing a transgenic mouse line expressing constitutive androstane receptor (CAR), tagged with the Flag peptide, in liver. The overall goal of this project is to understand the molecular mechanisms by which phenobarbital (PB) 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. PB induces the translocation from the cytoplasm to the nucleus of the nuclear receptor CAR. CAR, as a heterodimer with the nuclear receptor, RXR, binds to nuclear receptor binding sites in a PB responsive unit (PBRU) in CYP2B genes and constitutively induces gene expression. PB-like inducers may also bind to CAR and enhance its activity.
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, the role of CYP2B gene chromatin structure in PB action, and finally in vivo approaches to establish the validity of the in vitro studies. The major specific aim of the supplement is to construct a transgenic mouse line in which flag-tagged CAR is expressed in the liver. Such a transgenic mouse line will facilitate the isolation from livers of control and PB-treated animals of protein complexes containing CAR to identify CAR interacting proteins; to analyze binding of CAR to the CYP2B genes in vivo by ChIP analysis; and to isolate nuclear and cytoplasmic CAR from control and PB-treated animals to analyze the role of posttranslational modifications in the regulation of CAR. These experiments will greatly increase our understanding of the mechanism of induction of CYPB genes by PB ? ?
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