STEROIDOGENIC FACTOR-I CONTAINS A CARBOXY-TERMINAL TRANSCRIPTIONAL ACTIVATION DOMAIN THAT INTERACTS WITH STEROID-RECEPTOR COACTIVATOR-1

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an i mportant role in the development of the adrenal gland and in sexual di fferentiation. SF-1 regulates the transcription of variety of genes, i ncluding several steroidogenic enzymes, Mullerian inhibiting substance , and gonadotropin genes. In this report, we sought to identify domain s in SF-1 that are required for transactivation and to determine wheth er SF-1 interacts with a subset of known coactivators. Natural variant s of the FTZ-F1 locus include embryonal long terminal repeat-binding p rotein (ELP)-1, ELP-2, and SF-1, which share the DNA-binding domain. A nalyses of the transcriptional activity of these variants revealed tha t the activity of ELP-2 and SF-1 was much greater than ELP-1, which co ntains a distinct carboxy terminus. Further studies were performed usi ng GAL4-SF-1 fusion proteins that were constructed by replacement of t he zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding doma in of GAL4. Elimination of the putative AF-2 domain at the carboxy ter minus of GAL4-SF-1 proteins resulted in a complete loss of transactiva tion. Several lines of evidence demonstrated that SF-1 interacts with steroid receptor coactivator-1 (SRC-1), full-length SRC-1 enhanced GAL 4-SF-1-mediated transactivation, whereas a dominant negative form of S RC-1, consisting of its interaction domain alone, inhibited the activi ty of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 ac tivation domain to the interaction domain of SRC-1 confirmed the inter action between SRC-1 and GAL4-SF-1 and demonstrated that the AF-2 doma in is required for interaction with SRC-1. Furthermore, SRC-1, togethe r with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhanced transcriptional activit y of GAL4-SF-1. We conclude that the carboxy-terminal AF-2 region of S F-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1.