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A duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response.
- Source :
-
The international journal of biochemistry & cell biology [Int J Biochem Cell Biol] 2001 Nov; Vol. 33 (11), pp. 1080-93. - Publication Year :
- 2001
-
Abstract
- We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.
- Subjects :
- Animals
Base Sequence
Cell Nucleus genetics
Cell Nucleus metabolism
Cells, Cultured
Chick Embryo
Cloning, Molecular
DNA Footprinting
Electrophoretic Mobility Shift Assay
Hepatocytes drug effects
Hepatocytes metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Sequence Deletion genetics
Transfection
Cytochrome P-450 Enzyme System genetics
Gene Expression Regulation drug effects
Phenobarbital pharmacology
Promoter Regions, Genetic genetics
Repetitive Sequences, Nucleic Acid genetics
Response Elements genetics
Transcription Factors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1357-2725
- Volume :
- 33
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- The international journal of biochemistry & cell biology
- Publication Type :
- Academic Journal
- Accession number :
- 11551824
- Full Text :
- https://doi.org/10.1016/s1357-2725(01)00076-0