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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039360-13
Application #
6179558
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Long, Rochelle M
Project Start
1988-02-01
Project End
2001-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
13
Fiscal Year
2000
Total Cost
$210,567
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
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Xia, Jun; Kemper, Byron (2007) Subcellular trafficking signals of constitutive androstane receptor: evidence for a nuclear export signal in the DNA-binding domain. Drug Metab Dispos 35:1489-94
Zhang, Quanyuan; Bae, Yangjin; Kemper, Jongsook Kim et al. (2006) Analysis of multiple nuclear receptor binding sites for CAR/RXR in the phenobarbital responsive unit of CYP2B2. Arch Biochem Biophys 451:119-27
Xia, Jun; Kemper, Byron (2005) Structural determinants of constitutive androstane receptor required for its glucocorticoid receptor interacting protein-1-mediated nuclear accumulation. J Biol Chem 280:7285-93
Bae, Yangjin; Kemper, Jongsook Kim; Kemper, Byron (2004) Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP). DNA Cell Biol 23:81-91
Rivera-Rivera, Ilia; Kim, Jongsook; Kemper, Byron (2003) Transcriptional analysis in vivo of the hepatic genes, Cyp2b9 and Cyp2b10, by intravenous administration of plasmid DNA in mice. Biochim Biophys Acta 1619:254-62
Min, Gyesik; Kemper, J Kim; Kemper, Byron (2002) Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo. J Biol Chem 277:26356-63
Kim, J; Min, G; Kemper, B (2001) Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PB J Biol Chem 276:7559-67
Liu, S; Rivera-Rivera, I; Bredemeyer, A J et al. (2001) Functional analysis of the phenobarbital-responsive unit in rat CYP2B2. Biochem Pharmacol 62:21-8
Kim, J; Rivera-Rivera, I; Kemper, B (2000) Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital. Nucleic Acids Res 28:1126-32

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