Identification of a novel transcriptional silencer in the protein-coding region of the human CYP2C9 gene.
A novel regulatory element (27 bp) which confers transcriptional repression was identified within the protein-coding region immediately after the translation start codon in the human cytochrome P450 (CYP) 2C9 gene. Deletion of this element increased transcriptional activity in HepG2 cells by transient transfection assay. Nuclear protein extracts from HepG2 cells and human liver were found in electrophoretic mobility shift assays to bind specifically to the 27 bp element. A putative binding protein was partially purified by DNA-affinity chromatography and was determined by Southwestern blotting to have a molecular weight of approx. 100 kDa. Studies with mutated competitor oligonucleotides established that binding of the nuclear protein to the 27 bp cis-element was dependent upon two 6 bp direct repeats (5'-CTTGTG-3') that were separated by three bases. It is possible that this novel cis-acting element may be involved in the negative regulation of CYP2C9.
Xiang
S
S
Storr Liver Unit, Department of Medicine, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia.
Parsons
H K
HK
Murray
M
M
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D003062:Codon; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004794:Enzyme Repression; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D003062:Codon; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase