IDENTIFICATION OF POSITIVE AND NEGATIVE REGULATORY ELEMENTS OF THE HUMAN CYTOCHROME P4501A2 (CYP1A2) GENE

We previously demonstrated an enhancer-like positive regulatory elemen t within a 259-bp sequence (-2352 to -2094 bp) of the human CYPIA2 gen e in HepG2 cells. Three protein binding sites were identified by DNase I footprinting analyses within the 259-bp sequence: protected region A PRA; -2283 to -2243 bp), PRE (-2218 to -2187 bp), and PRC (-2124 to -2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995 ). In the present study, the functional significance of those protecte d regions was examined. Transfection experiments with deletion and sub stitution mutants defined the PRE and PRC as containing positive and n egative regulatory elements, respectively. Human breast carcinoma MCF- 7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter- or thymidine kinase promoter-lu ciferaae reporter gene constructs. HNF-1, which contributes to the liv er specificity of genes, enhanced reporter gene activity in a PRC sequ ence-dependent manner. These results suggested that PRC could exist bo und to a repressor which was displaceable by other transcription facto rs such;as HNF-1. Results obtained by transfection of HepG2 hepatoma c ells with various PRE substitution mutant-luciferase gene fusion const ructs indicated that the entire sequence of PRE was necessary for prom oter activity. Consequently, the regulation of CYP1A2 expression is ve ry complex, requiring a number of both positive and negative regulator y factors. (C) 1997 Academic Press.