Analysis of a phenobarbital-responsive enhancer sequence located in the 5' flanking region of the chicken CYP2H1 gene: identification and characterization of functional protein-binding sites.
We previously identified in the chicken CYP2H1 gene an upstream enhancer domain (-5900/-1100) that responds to phenobarbital. Deletion and restriction enzyme analyses of this domain have now identified two separate enhancer regions that respond to phenobarbital (from -5900 to -4550 and from -1956 to -1400). We have focused here on the latter and in particular a resident 240-base pair (bp) restriction enzyme fragment that retains drug responsiveness. Using deletion analysis and in vitro DNase I footprinting, transcription factor binding sites have been located in the 240-bp fragment. The sites identified are an E-box-like element, a consensus hepatocyte nuclear factor 1 site, a CCAAT box motif, and a novel site. Mutagenesis demonstrated that each site contributed to enhancer activity, although there was a weaker contribution from the CCAAT box, and that no individual site was critical for responsiveness. In keeping with the tissue-restricted expression of the CYP2H1 gene, gel shift experiments established that the proteins binding to these enhancer sites are enriched in chicken liver, kidney, and small intestine. In vitro footprint experiments showed a stronger protection with liver nuclear extracts from drug-treated chickens compared with control extracts on the E-box-like element, the CCAAT box motif, and the novel binding site; however, the basis for this apparent increase in binding remains to be determined. The proteins binding to the 240-bp fragment are different from those recently reported to be required for the activity of the phenobarbital responsive enhancer domains of rodent CYP2 genes.
Dogra
S C
SC
Department of Biochemistry, University of Adelaide, Adelaide, South Australia, Australia.
Davidson
B P
BP
May
B K
BK
D000818:Animals; D001665:Binding Sites; D002642:Chick Embryo; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D016297:Mutagenesis, Site-Directed; D010634:Phenobarbital / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*
D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System; D003850:Deoxyribonuclease I; D010634:Phenobarbital