Peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator 1␣ (PGC-1␣) is a transcriptional coactivator that is a key component in the regulation of energy production and utilization in metabolic tissues. Recent work has identified PGC-1␣ as a strong coactivator of the orphan nuclear receptor estrogen-related receptor ␣ (ERR␣), implicating ERR␣ as a potential mediator of PGC-1␣ action. To understand the role of ERR␣ in PGC-1␣ signaling, a parallel approach of high-throughput screening and gene-expression analysis was used to identify ERR␣ small-molecule regulators and target genes. We report here the identification of a potent and selective ERR␣ inverse agonist that interferes effectively with PGC-1␣͞ERR␣-dependent signaling. This inverse agonist inhibits the constitutive activity of ERR␣ in both biochemical and cell-based assays. Also, we demonstrate that monoamine oxidase B is an ERR␣ target gene whose expression is regulated by PGC-1␣ and ERR␣ and inhibited by the ERR␣ inverse agonist. The discovery of potent and selective ERR␣ modulators and their effect on PGC-1␣ signaling provides mechanistic insight into gene regulation by PGC-1␣. These findings validate ERR␣ as a promising therapeutic target in the treatment of metabolic disorders, including diabetes and obesity.

The actions of the male sex hormones testosterone and dihydrotestosterone are mediated by the intracellular androgen receptor (AR) 1 (reviewed in Refs. 1 and 2). In the absence of hormone, the receptor is sequestered in the cytosol with molecular chaperone proteins, which dissociate upon hormone binding. The hormone-bound receptor translocates to the nucleus and is targeted to specific genes through the recognition and binding to the DNA response element, 5Ј-AGA/TACA/TnnnT/ AGTTCT/C-3Ј, which in turn leads to activation of gene transcription (3-10). The activated receptor also represses gene expression through protein-DNA interactions at negative response elements (11,12) or through interactions with other transcription factors (13)(14)(15)(16)(17).In addition to the well characterized DNA-binding domain (DBD) and ligand-binding domain (LBD), regions of the proteins important for transactivation have been mapped to the amino-terminal domain (NTD; 18 -21). These studies have revealed a modular nature for the AR-transactivation domain, with the region between amino acids 142 and 485, containing the TAU-1/AF-1 and TAU-5/AF-5 determinants, being critical for receptor-dependent activation (20, 21). Sequences within the AR-NTD have been shown to mediate protein-protein interactions with the carboxyl-terminal LBD (22-28), the general transcription factors TFIIF (29) and TFIIH (30), members of the p160 family of nuclear receptor coactivator proteins (31-34), and the general coactivator CREB-binding protein (35,36).TFIIF is a tetramer of two subunits, RAP30 and RAP74. TFIIF recruits TFIIE and TFIIH to the preinitiation complex (PIC) and interacts directly with the RNA polymerase II enzyme and prevents pausing of the enzyme during subsequent transcription elongation (37-39). Previously, we have demonstrated that the isolated transactivation function of the human AR, amino acids 142 to 485, interacts with the large subunit of TFIIF, termed RAP74, and that this interaction was capable of reversing AR-dependent squelching of basal transcription under cell-free conditions (29). More recently, we have shown that binding of RAP74 results in the AR-transactivation domain adopting a protease-resistant conformation (40).In the present study we have extended these observations to map the region(s) of RAP74 involved in this interaction with the AR. Using a series of deletion constructs of RAP74 we show that sequences within both the amino-and carboxyl-terminal domains of the protein are sufficient to bind the AR-transactivation function and to reverse receptor-dependent squelching of transcription. In the context of the holo-TFIIF, the carboxylterminal binding site may be the main binding site. Introduction of point mutations into the AR-transactivation domain revealed that sequences near the amino terminus are important for RAP74 binding. These mutations fail to disrupt the interaction of the AR with the p160 coactivator protein SRC-1a. Thus, TFIIF and SRC-1a interact with distinct regions of the AR-transactivation domain. The impl...