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

NADPH-cytochrome P450 oxidoreductase (CYPOR), a flavoprotein localized in t he nuclear envelope and endoplasmic reticulum of most cell types, is respon sible for transferring electrons from NADPH to the cytochromes P450 as well . as heme oxygenase, squalene epoxidase, and cytochrome b(5). CYPOR is enco ded by a single gene and, similar to many housekeeping genes, has a TATA-le ss, CC-rich promoter with multiple Spl consensus sites. The current work ha s delineated the importance of multiple cis-acting elements contained withi n the proximal promoter for basal expression of the CYPOR gene, Transcripti on factor binding sites within this region included two upstream Spl motifs , a SEC element containing overlapping Sp1/Egr-1/CACCC box motifs, and a no vel site designated the OxidoReductase Upstream element (ORU). Mutational m odification of the ORU element, leading to a loss of protein binding, resul ted in an similar to 90% decrease in transcriptional activity in H4IIE cell s. Similarly, inactivation of the Egr-1/CACCC segment of the SEC element dr amatically reduced promoter activity to less than 10% of wild-type, while m utagenesis of the contiguous Spl site did not affect basal transcription. A lthough 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 S pl 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 bindi ng site closest to the transcription start site was nonfunctional. Furtherm ore, both the SEC and ORU elements were essential for basal expression; how ever, the ORU element exhibited cell-specific differences in regulatory act ivity. Thus, several mechanisms appear to be in place to selectively alter the expression of the CYPOR gene. (C) 2000 Academic Press.