A novel positive regulatory element that enhances hamster CYP2A8 gene expression mediated by xenobiotic responsive element

CYP2A8 is a major form of cytochrome P-450 inducible by 9-methylcholanthren e in Syrian hamster liver. To identify DNA elements necessary for the trans criptional activation of the CYP2A8 gene, we analyzed the regulatory region of the CYP2A8 gene and conducted transient transfection experiments of CYP 2A8-luciferase fusion plasmids in primary cultures of hamster hepatocytes. We analyzed up to -5 kb of the 5'-flanking region and found the region suff icient for the 3-methylcholanthrene-inducible gene expression. This region contained a consensus sequence for xenobiotic responsive element (XRE) betw een -2366 and -2349, which was shown to be essential for induction of the g ene expression. Furthermore, we found a novel positive regulatory element f or XRE-mediated gene expression (PREX) located upstream of the XRE. This el ement is not identified in any genes inducible by 5-methylcholanthrene so f ar reported. Without PREX, the XRE-mediated promoter activity was enhanced nearly 10-fold, whereas with PREX, the activity was enhanced 20-fold over t he basal level. Gel mobility shift assays revealed specific binding of nucl ear proteins to PREX. Mutations and deletions of PREX caused a loss of the binding and promoter-enhancing activities, respectively. Moreover, transien t expression experiments showed that the enhancing activity of PREX was not observed in Drosophila Schneider's line 2 cells, which were shown to lack the PREX binding proteins.