The overall goal of this proposal is to understand the molecular mechanisms by which the classical inducer, phenobarbital (PB), increases gene expression of cytochromes P450. The specific objectives of this proposal are to identify and characterize the cis-and trans-acting factors that mediate phenobarbital induction in the CYP2B and CYP2C subfamilies. Progress in this area has been hindered by the lack of continuous cell culture model systems for phenobarbital induction. However, the laboratory has demonstrated that transfection of DNA into rat liver in situ has been shown to be a simple and reproducible method for expression of CYP genes and was used to confirm that a CYP2B2 DNA fragment could mediate phenobarbital responsiveness. Deletion and site-specific mutation will be used to identify the phenobarbital-responsive elements (PBRE) in the CYP2B2 DNA fragment. Classical recombinant DNA techniques will be used to identify regions important for regulation. The effect of phenobarbital on the chromatin structure of the PBRE will also be analyzed, and binding of the regulatory factors in native chromatin will be determined. Homologous recombination in mice will be used to assess the in vivo function of the identified elements. Rabbit and mouse CYP2C genes will be screened for PBRE's by sequence similarity and by the in situ transfection assay, and ultimately by targeted deletions in mice. Characteristics of phenobarbital induction of CYP2C genes in the mouse will be compared with CYP2B genes to determine if different induction mechanisms are involved for the two genes. These studies should provide a detailed understanding of a functional PBRE at the molecular level.
Showing the most recent 10 out of 21 publications
