Liver-enriched transcription factors, HNF-1, HNF-3, and C/EBP, are major contributors to the strong activity of the chicken CYP2H1 promoter in chick embryo hepatocytes.
Chicken CYP2H1 promoter constructs express strongly in chick embryo hepatocytes at a level comparable with that of Rous sarcoma viral promoter. We have identified the transcription factors responsible for the active CYP2H1 promoter. Binding sites for transcription factors were located within the first 160 bp of promoter sequence using promoter deletion experiments and DNase I footprint analysis. Sequence analysis revealed characteristic sites for the liver-enriched transcription factors of the HNF-1, HNF-3, and C/EBP families and for the ubiquitous factor, USF. Protein binding to these sites was established by gel mobility shift assays. Mutagenesis and transient transfection experiments demonstrated that these sites, in combination, were responsible for the strong promoter activity with a substantial contribution from HNF-1 and HNF-3. The promoter was also active in mammalian HepG2 and COS-1 cell lines where expression was dependent on the identified transcription factor binding sites but promoter activity in the HeLa cells was low. Transactivation experiments revealed that promoter expression could be activated through the appropriate binding sites by exogenously expressed rat HNF-1alpha or HNF-1beta, rat HNF-3alpha or HNF-3beta and chicken C/EBP alpha. Transcriptional synergism between HNF-1 and C/EBP was observed in these transactivation experiments. A Barbie box-like sequence overlapped the USF element but was not functional. The results demonstrate that liver-enriched transcription factors and USF direct strong expression of the CYP2H1 promoter in transiently transfected cells. By comparison, in vivo expression of this gene in uninduced chick embryo hepatocytes is low but markedly increased by phenobarbital. Drug induction may therefore substantially reflect derepression of this inherently active promoter.
Dogra
S C
SC
Department of Biochemistry, University of Adelaide, South Australia.
May
B K
BK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D006367:HeLa Cells; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008099:Liver / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496499:FOXA1 protein, human; C496502:FOXA2 protein, human; C496501:Foxa1 protein, rat; C496504:Foxa2 protein, rat; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051541:Hepatocyte Nuclear Factor 3-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051539:Hepatocyte Nuclear Factor 1-beta; D003577:Cytochrome P-450 Enzyme System; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I