Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor superfamily and plays an essential role in the development of endocrine organs. Coactivators and corepressors are important in the transcriptional regulation of gene expression by nuclear receptors. SRC-1 and TIF2 belongs to the SRC family of coactivators and are required for transcriptional regulation by several nuclear receptors including SF-1. Here we demonstrate that another SRC-related coactivator, p/CIP, interacts with SF-1, and that the interaction requires the carboxy-terminal activation function-2 (AF-2) domain of SF-1. By transfection studies performed in an adrenocortical cell line (Y1) and nonsteroidogenic COS-1 cells it is shown that both p/CIP and TIF2 potentates SF-1 mediated transactivation of a reporter construct under the control of the SF-1 binding site (CRS2) from the bovine CYP17 gene. Cotransfection with the catalytic subunit of cAMP-dependent protein kinase (PKA) demonstrated that increased PKA activity further stimulated the positive effect of p/CIP. In contrast, PKA markedly inhibited the positive effect of TIF2 on the transcriptional activity of SF-1. The AF-2 domain of SF-1 is required for the coactivating effect of both p/CIP and TIF2. However, the interaction between TIF2 and SF-1 differed from that of p/CIP and SF-1 with respect to the amino acid requirements within the AF-2 domain. Based on these results, we suggest that TIF2 acts by a mechanism distinct from that by which the other members of the SRC family to regulate the transcriptional activity of SF-1.
Steroidogenic Factor-1 is a member of the orphan nuclear receptor family that acts as a transcription factor to regulate transcription of several genes involved in adrenal and gonadal development and steroidogenesis. A yeast two-hybrid screen with SF-1 as a bait was initiated to search for proteins that interact with SF-1 and further regulate transcription of the SF-1 target genes. An unknown sequence was isolated from a human testis library in this yeast two-hybrid screen. The full-length cDNA was then cloned from a lambda library, and a BLAST search revealed that this gene is localized to the human chromosome 19 and contains 9 exons. The predicted protein contains 615 amino acids and five C2H2 zinc fingers. The interaction with SF-1 is dependent on the activation function-2 domain (AF-2) of SF-1. Northern hybridization and In situ hybridization demonstrated coexpression with SF-1 in the testis and the adrenals. Transfection experiments in adrenal cortical Y1 cells indicate that the possible function of this novel SF-1 interacting protein is repression of the SF-1 target genes Cytochrome P450 steroid hydroxylase-17a (CYP17) and the Cholesterol side chain cleavage cytochrome P450 (CYP11A). ________________________________________