A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression

Florence Gizard,Bernard Lavallée,Fredérique Dewitte,Dean W Hum

doi:10.1074/jbc.m100113200

Abstract

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.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF297872
Isolation of TReP-132 cDNA Clone—The promoter region of the human P450scc gene contains a sequence between nucleotides Ϫ155 and Ϫ131 (Ϫ155/Ϫ131) that was previously demonstrated to confer increased transcriptional activity when fused to a heterologous HSV thymidine kinase minimal promoter (TK32)
The regulation of P450scc gene expression is a key determinant of steroid synthesis in steroidogenic tissues, considering that the encoded cytochrome P450scc catalyzes the first step in the steroid synthesis pathway

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF297872. Subsequent studies in mouse Leydig MA-10 [14], I-10 [15], human placenta JEG-3 [16], and adrenal NCI-H295 [17] cells identified regions of the 5Ј-flanking DNA that conferred basal promoter activity and response to cAMP. Positive cis-acting elements have been identified, which confer expression of P450scc gene promoter activity in steroidogenic cell lines, but not in nonsteroidogenic cells. The region between Ϫ155 and Ϫ131 was found to significantly increase promoter activity in JEG-3 cells [1, 16] These putative cis-acting elements in the 5Ј-flanking region of the human P450scc gene have been demonstrated to interact with multiple proteins, which include CREB/ATF [19], NF1and Sp1-like proteins, and in particular the steroidogenic factor-1 (SF-1). Huang et al [24] identified two proteins, LBP-1b and LBP-9, which interact with the Ϫ155/Ϫ131 element and regulate reporter activity via this element

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Conclusion