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 and encodes cytochrome P450scc, which catalyzes the first step in steroid synthesis. A region of the 5'-flanking DNA of the gene from nucleotides -155 to -131 (-155/-131) is shown to activate transcription in steroidogenic human placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of the gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screening JEG-3 cell and human placental cDNA expression libraries. The open reading frame encodes three zinc fingers of the C(2)H(2) subtype, and separate regions rich in glutamate, proline, and glutamine, which are indicative of a DNA-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 the predicted size. Northern blot analysis demonstrate expression of two TReP-132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal cortex, and testis; and expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates localization of the HA-tagged TReP-132 protein in the nucleus. The expression of exogenous TReP-132 in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 with 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 TReP-132 has an additive effect on promoter activity, and the proteins were demonstrated to interact physically. Thus, these results together indicate the isolation of a novel zinc-finger transcriptional regulating protein of 132 kDa (TReP-132) involved in the regulation of P450scc gene expression.
Gizard
F
F
Oncology and Molecular Endocrinology Research Center, Laval University, Quebec G1K 7P4, Canada.
Lavallée
B
B
DeWitte
F
F
Hum
D W
DW
D000302:Adrenal Cortex / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis* / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism; D014018:Tissue Distribution; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D013256:Steroids; C436322:TRERF1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D002786:Cholesterol Side-Chain Cleavage Enzyme; D012260:Ribonucleases