Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo-keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4alpha/gamma and HNF-1alpha.
Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5'-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha(1)-antitrypsin gene. A supershift assay using antibodies to human HNF-4alpha, HNF-4gamma and HNF-1alpha showed that HNF-4alpha and HNF-4gamma bound to the HNF-4 motif, and that HNF-1alpha interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4alpha, HNF-4gamma and HNF-1alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.
Ozeki
T
T
Laboratory of Drug Metabolism, Hokkaido University Graduate School of Pharmaceutical Sciences, Sapporo, Hokkaido 060-0812, Japan.
Takahashi
Y
Y
Kume
T
T
Nakayama
K
K
Yokoi
T
T
Nunoya
K
K
Hara
A
A
Kamataki
T
T
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002460:Cell Line; D004247:DNA; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D010088:Oxidoreductases / Q000235:genetics*; D010618:Phenanthrolines / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; C496592:HNF4A protein, human; C496597:HNF4G protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010618:Phenanthrolines; D010750:Phosphoproteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; C025205:1,10-phenanthroline