CHARACTERIZATION OF HAMSTER CYP1A1 GENE - INDUCIBLE EXPRESSION AND NEGATIVE REGULATION

A genomic clone encoding the hamster CYP1A1 gene was isolated from a h amster EMBL-3 genomic library and characterized. The CYP1A1 gene conta ined seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307bp of the CY P1A1 gene revealed the occurrence of five consensus xenobiotic respons ive elements (XREs) and one basal transcription element (BTE) in addit ion to the canonical TATA box. For functional analysis, transfection e xperiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5' -flanking region of the CYP1A1 gene and bacterial chloramphenicol acet yltransferase (CAT) gene. External deletion of the upstream region fro m the reporter gene resulted in a stepwise decrease of the CAT activit y, suggesting that XREs were responsible for inducible expression of C YPIAI gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5'-flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3 -fold increase of MC-inducible CAT activity. Using gel retardation ass ays with HepG2 nuclear extract, we demonstrated the presence of a spec ific protein which bound to the NRE fragment. Further competition anal ysis and methylation interference assays revealed that the nuclear pro tein bound to a 22-base fragment (from -688 to -709) of the NRE region , whose sequences were conserved among hamster, human, and rat CYP1A1 genes.