FUNCTION OF THE TRANSCRIPTIONAL REGULATING PROTEIN OF 132 kDa (TReP-132) ON HUMAN P450scc GENE EXPRESSION

Abstract

Cytochrome P450scc catalyzes the important first step in the steroid synthesis pathway; however, it is clear that additional factors regulating the temporal and spacial specific expression of the CYP11A1 gene remain to be identified. To isolate novel transcription factors that regulate this gene, a cis-acting element of the 5′-flanking region from nucleotides −155 to −131 (−155/−131) was used to screen a human placental λgt11 cDNA expression library, and an interacting clone was isolated. The open reading frame of the cDNA encodes several domains that are characteristic of transcription factors including an acidic region, a region rich in prolines and three zinc-finger motifs. Expression of the cDNA by in vitro transcription/translation and by transient transfection in HeLa cells yielded a protein of 132 kDa, which concurs with the predicted size. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells, stimulate expression of a reporter construct controlled by the P450scc gene 5′-flanking region from nucleotides −1676 to +49. This transcriptional regulating protein of 132 kDa (TReP-132) when expressed in HeLa cells was demonstrated to interact with the −155/−131 region in bandshift analysis, and tandem copies of this region was shown to confer activation of the heterologous HSV thymidine kinase minimal promoter. Coexpression of CBP/p300 with TReP-132 further increased promoter activity, and the proteins were demonstrated to interact physically. RNA analysis demonstrated the highest levels of expression in the adrenal cortex and testis; and transcript expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines, but not in non-steroidogenic HepG2 and HK293 cells. Subsequently it has been shown that TReP-132 interacts with steroidogenic factor-1 (SF-1) through specific domains; and along with the interaction with CBP/p300 these factors are postulated to form a complex to regulate expression of the P450scc gene.

ACKNOWLEDGMENT

We thank John Brozek of IT-OMICS for providing insightful help and advice concerning bioinformatic analysis. This work was supported in part by Operating Grant MT-12901 from the Medical Research Council of Canada and by Scholarship 980149 from the Fonds de la Recherche en Santé du Québec (FRSQ) awarded to D.W.H.