A novel zinc finger protein TReP-132 interacts with CBP/p300 to regulate human CYP11A1 gene expression

The human CYP11A1 gene is expressed specifically in steroidogenic tissues a nd encodes cytochrome P450sec, which catalyzes the first step in steroid sy nthesis. A region of the 5 ' -flanking DNA of the gene from nucleotides -15 5 to -131 (-155/-131) is shown to activate transcription in steroidogenic h uman placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of t he gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screen ing JEG-3 cell and human placental cDNA expression libraries. The open read ing frame encodes three zinc fingers of the C2H2 subtype, and separate regi ons rich in glutamate, proline, and glutamine, which are indicative of a DN A-binding protein involved in gene transcription. Expression of the cDNA in vitro and in HeLa cells yields a protein of 132 kDa, which concurs with th e predicted size. Northern blot analysis demonstrate expression of two TReP -132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal corte x, and testis; and expression is also found in the steroidogenic JEG-3, NCI -H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates loca lization of the HA-tagged TReP-132 protein in the nucleus. The expression o f exogenous TReP-132 in HeLa cells was demonstrated to interact with the -1 55/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter constr uct controlled by the P450sec gene 5 ' -flanking region from nucleotides -1 676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 wi th the Gal4 DNA-binding domain was able to significantly increase promoter activity of a reporter construct via Gal4-binding sites upstream of the E1b minimal promoter. Coexpression of CREB-binding protein (CBP)/p300 with TRe P-132 has an additive effect on promoter activity, and the proteins were de monstrated to interact physically. Thus, these results together indicate th e isolation of a novel zinc-finger transcriptional regulating protein of 13 2 kDa (TReP-132) involved in the regulation of P450sec gene expression.