Molecular basis for cell-specific regulation of the NADPH-cytochrome P450 oxidoreductase gene

NADPH-cytochrome P450 oxidoreductase (CYPOR), a flavoprotein localized in the nuclear envelope and endoplasmic reticulum of most cell types, is responsible for transferring electrons from NADPH to the cytochromes P450 as well. as heme oxygenase, squalene epoxidase, and cytochrome b(5). CYPOR is encoded by a single gene and, similar to many housekeeping genes, has a TATA-less, CC-rich promoter with multiple Spl consensus sites. The current work has delineated the importance of multiple cis-acting elements contained within the proximal promoter for basal expression of the CYPOR gene, Transcription factor binding sites within this region included two upstream Spl motifs, a SEC element containing overlapping Sp1/Egr-1/CACCC box motifs, and a novel site designated the OxidoReductase Upstream element (ORU). Mutational modification of the ORU element, leading to a loss of protein binding, resulted in an similar to 90% decrease in transcriptional activity in H4IIE cells. Similarly, inactivation of the Egr-1/CACCC segment of the SEC element dramatically reduced promoter activity to less than 10% of wild-type, while mutagenesis of the contiguous Spl site did not affect basal transcription. Although both Spl sites contained within the minimal promoter were required for optimal expression in H4IIE cells, loss of these sites was compensated for by those Sp1 motifs located upstream of position 206, suggesting that Spl was acting as a position-independent enhancer. Hence, the CYPOR promoter was distinguished from the majority of TATA-less promoters in that Spl was not a primary transcriptional regulator and by the fact that the Spl binding site closest to the transcription start site was nonfunctional. Furthermore, both the SEC and ORU elements were essential for basal expression; however, the ORU element exhibited cell-specific differences in regulatory activity. Thus, several mechanisms appear to be in place to selectively alter the expression of the CYPOR gene. (C) 2000 Academic Press.