Regulation of Cyp2a5 transcription in mouse primary hepatocytes: roles of hepatocyte nuclear factor 4 and nuclear factor I.
The cytochrome P4502a5 (Cyp2a5) gene is expressed principally in liver and olfactory mucosa. In the present study, the transcriptional mechanisms of hepatocyte-specific expression of Cyp2a5 were studied in mouse primary hepatocytes. The Cyp2a5 5'-flanking region -3033 to +10 was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis revealed two major activating promoter regions localized at proximal 271 bp and at a more distal area from -3033 to -2014 bp. The proximal activation region was characterized further by DNase I footprinting, and a single clear footprint was detected in the studied area centred over a sequence similar to the NF-I (nuclear factor I)-binding site. The binding of NF-I was confirmed using an EMSA (electrophoretic mobility-shift assay). A putative HNF-4 (hepatocyte nuclear factor 4)-binding site was localized at the proximal promoter by computer analysis of the sequence, and HNF-4alpha was shown to interact with the site using an EMSA. The functional significance of HNF-4 and NF-I binding to the Cyp2a5 promoter was evaluated by site-directed mutagenesis of the binding motifs in reporter constructs. Both mutations strongly decreased transcriptional activation by the Cyp2a5 promoter in primary hepatocytes, and double mutation almost completely abolished transcriptional activity. Also, the functionality of the distal activation region was found to be dependent on the intact HNF-4 and NF-I sites at the proximal promoter. In conclusion, these results indicate that HNF-4 and NF-I play major roles in the constitutive regulation of hepatic expression of Cyp2a5.
Ulvila
Johanna
J
Department of Pharmacology and Toxicology, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland.
Arpiainen
Satu
S
Pelkonen
Olavi
O
Aida
Kaoru
K
Sueyoshi
Tatsuya
T
Negishi
Masahiko
M
Hakkola
Jukka
J
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites; D019556:COS Cells / Q000201:enzymology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D018983:DNA Footprinting / Q000379:methods; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496592:HNF4A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C496488:Hnf1a protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D003850:Deoxyribonuclease I