Role of the liver-enriched transcription factor HNF-1 alpha in expression of the CYP2E1 gene.
The role of the trans-acting factor HNF-1 alpha in activating CYP2E1 gene expression was confirmed by transient co-transfection of an HNF-1 alpha expression plasmid and the CYP2E1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Only HNF-1 alpha, and not HNF-1 beta, HNF-4, C/EBP alpha, C/EBP beta, or DBP, was able to activate the CYP2E1 promoter. The extent of activation was proportional to the number of copies of the HNF-1 binding sequence upstream of the promoter. Removal or mutation of the HNF-1 binding sequence led to inactivation of the promoter in response to HNF-1 alpha. Gel-shift Western blot analysis using a synthetic HNF-1 binding sequence derived from CYP2E1 and rat liver nuclear extract revealed that the protein-DNA complex obtained with adult rat liver nuclear extract consisted of both HNF-1 alpha and HNF-1 beta proteins. The shifted bands produced by nuclear extracts from adult, where the endogenous CYP2E1 gene is active, and fetal rat liver, where the gene is inactive, were found to migrate differently, suggesting that the population of factors, possibly including different ratios of HNF-1 alpha and HNF-1 beta proteins, may change during development. However, the co-transfection study did not show cooperativity between the two factors. Elements upstream of the HNF-1 binding site were found to affect the activity of the promoter negatively in the transfection assay. DNase I hypersensitive site mapping revealed a hypersensitive site in this inhibiting element in the adult rat liver sample but not in liver from newborn animals.
Liu
S Y
SY
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Gonzalez
F J
FJ
D000375:Aging / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D005333:Fetus / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D003850:Deoxyribonuclease I