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 repre ssion was identified within the protein-coding region immediately afte r the translation start codon in the human cytochrome P450 (CYP) 2C9 g ene. Deletion of this element increased transcriptional activity in He pG2 cells by transient transfection assay. Nuclear protein extracts fr om HepG2 cells and human liver were found in electrophoretic mobility shift assays to bind specifically to the 27 bp element. A putative bin ding 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 est ablished 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 se parated by three bases. It is possible that this novel cis-acting elem ent may be involved in the negative regulation of CYP2C9. (C) 1998 Els evier Science B.V.