Novel transcriptional regulation of the human CYP3A7 gene by Sp1 and Sp3 through nuclear factor kappa B-like element.
Human CYP3A7 and CYP3A4 are expressed in fetal and adult livers, respectively, although the 5'-flanking regions of the two genes show 90% homology. The purpose of this study was to clarify the mechanism(s) responsible for the transcriptional regulation of the CYP3A7 gene in human hepatoma HepG2 cells that showed fetal phenotypes. Transfection studies using a series of the CYP3A7 or CYP3A4 promoter-luciferase chimeric genes identified a nuclear factor kappaB (NF-kappaB)-like element between nucleotides -2326 and -2297 that conferred the transcriptional activation of the CYP3A7 gene. A 1-base pair mismatch within the corresponding region of the CYP3A4 gene was sufficient for a differential enhancer activity. A gel shift assay using nuclear extracts from HepG2 cells showed that Sp1 and Sp3 bound to the NF-kappaB-like element of the CYP3A7 but not CYP3A4 gene. Specific activation of the CYP3A7 promoter by Sp1 and Sp3 was confirmed by a co-transfection of the p3A7NF-kappaB or p3A4NF-kappaB reporter gene with Sp1 or Sp3 expression plasmid into Drosophila cells, which lacked endogenous Sp family. Additionally, introduction of mutations into binding sites for hepatocyte nuclear factor 3beta, upstream stimulatory factor 1, and a basic transcription element in the proximal promoter attenuated luciferase activity to 20% of the level seen with the intact CYP3A7 promoter. Thus, we conclude that the expression of the CYP3A7 gene in HepG2 cells is cooperatively regulated by Sp1, Sp3, hepatocyte nuclear factor 3beta, and upstream stimulatory factor 1.
Saito
T
T
Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, N12W6, Kita-ku, Sapporo, Hokkaido 060-0812, Japan.
Takahashi
Y
Y
Hashimoto
H
H
Kamataki
T
T
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; D016328:NF-kappa B; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C080333:transcription factor nuclear factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051705:Sp3 Transcription Factor; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A