Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate.
We have previously identified a novel xenobiotic responsive element, which has been termed the antioxidant responsive element (ARE), in the 5'-flanking region of the rat quinone reductase gene (Favreau, L. V., and Pickett, C. B. (1991) J. Biol. Chem. 266, 4556-4561). This element is responsible for basal level expression of the gene as well as transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds. In this communication, we demonstrate that hydrogen peroxide can act as an inducer through the ARE sequence, a phenomenon recently demonstrated for the glutathione S-transferase Ya subunit gene (Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639). To further characterize the quinone reductase ARE, we demonstrate by DNase I footprinting that in crude Hep G2 nuclear extracts a trans-acting factor exists which interacts with a region of DNA found within the 31-nucleotide ARE sequence. Furthermore, electrophoretic mobility shift assays demonstrate the presence of a specific DNA-protein complex which can be competed only by double-stranded oligonucleotides containing the ARE sequences from the quinone reductase and glutathione S-transferase Ya subunit genes. Methylation interference and protection assays indicate that several guanine residues found in the sequence GTGACTTGGC are involved in the binding of the nuclear factor(s) to the DNA. Although electrophoretic mobility shift assays indicate that the rat quinone reductase ARE does not contain a high affinity recognition site for in vitro translated c-Jun and c-Fos, 12-O-tetradecanoylphorbol 13-acetate can act as an inducer through the ARE sequence in Hep G2 cells.
Department of Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
L V
LV
Favreau
C B
CB
Pickett
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016762:Genes, fos; D016758:Genes, jun; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis* / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D006861:Hydrogen Peroxide; D016660:NAD(P)H Dehydrogenase (Quinone); D003850:Deoxyribonuclease I; D013755:Tetradecanoylphorbol Acetate