Characterization of the human cytochrome P4502D6 promoter. A potential role for antagonistic interactions between members of the nuclear receptor family.
The functional mapping of the human cytochrome P4502D6 (CYP2D6) promoter in HepG2 cells revealed the presence of both positive and negative regulatory elements. One of these regulatory elements overlapped a sequence that is highly conserved in most members of the CYP2 family. This element, which consists of a degenerate AGGTCA direct repeat spaced by 1 base pair (DR1) and is known to be a target for members of the steroid receptor superfamily, was found to bind in vitro translated hepatocyte nuclear factor 4 (HNF4) in gel retardation analysis. Using HepG2 nuclear extracts, three protein-DNA complexes were formed on the DR1 element, one of which was confirmed to be dependent on the binding of HNF4. The other DR1 complexes were shown to be due to the interaction of the orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI). Experiments in COS-7 cells showed that HNF4 could activate the CYP2D6 promoter 30-fold. Surprisingly, mutation of the DR1 element produced a relatively minor 23% decrease in activity in HepG2 cells. Additionally, COUP-TFI was shown to inhibit HNF4 stimulation of the CYP2D6 promoter in COS-7 cells, suggesting that COUP-TFI could attenuate the effect of HNF4 in HepG2 cells. However, when HNF4 levels were increased in HepG2 cells by co-transfection, it resulted in the enhancement of CYP2D6 promoter activity, indicating that HNF4 could overcome the repressive effect of COUP-TFI. Therefore, the contribution of the DR1 element in controlling the transcription of the CYP2D6 gene depends on the balance between positively and negatively acting transcription factors.
Cairns
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Molecular Pharmacology Unit, Imperial Cancer Research Fund, Biomedical Research Centre, Ninewells Hospital, Dundee, DD1 9SY, Scotland, United Kingdom.
Smith
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McLaren
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Wolf
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D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019389:Cytochrome P-450 CYP2D6 / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D019389:Cytochrome P-450 CYP2D6; D015500:Chloramphenicol O-Acetyltransferase