Hepatocyte nuclear factor 3 is a major determinant of CYP2C6 promoter activity in hepatoma cells

Cytochrome P450 2C6 (CYP2C6) is a developmentally regulated, constitutively expressed form of rat liver microsomal cytochrome P450 that in the liver of adult male rats is induced to a limited extent by phenobarbital. The gene is not expressed at detectable levels in the lung, kidney, or brain. It is expressed and inducible by phenobarbital in differentiated Reuber hepatoma cells that express many hepatocyte-specific genes but not in dedifferentiated derivatives lacking the majority of hepatocyte-specific functions. A 505-base pair proximal segment of the CYP2C6 promoter is highly efficient in driving transcription of a linked chloramphenicol acetyltransferase reporter gene in the differentiated rat hepatoma cell line FGC4, is much less effective in a related dedifferentiated variant H5, and has no measurable activity in nonhepatic C33 human cervical carcinoma cells. The activity of the CYP2C6 promoter in the differentiated hepatoma cells is strongly dependent on hepatocyte nuclear factor (HNF)3, which acts at a complex site just upstream of the TATA motif. Transactivation experiments show that the D-site-binding protein (DBP) may also contribute to CYP2C6 promoter activity, via a site that is adjacent to the proximal HNF3 site. A substantial contribution to promoter activity by the base pair -505 to -316 segment is observed in FGC4 and H5 cells but not in HepG2 cells; deletion of this segment causes a marked diminution in promoter activity only in the former two cell lines. Although footprinting experiments have permitted the definition of three protein binding sites in this region (two HNF3 and one unidentified), mutation of these sites does not diminish promoter activity. The functionally important cis sequences in this region therefore remain to be defined. In HepG2 cells the distal region does not contribute to promoter activity. This most likely accounts for the low promoter activity in HepG2 and implies a deficiency in the relevant trans-acting factor(s).