Transcription of eukaryotic protein-encoding genes by RNA polymerase II is modulated by two distinct classes of transcription factors. The first class comprises general transcription factors which are necessary for accurate initiation of transcription. These factors include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIG/J, and TFIIH (11, 82). TFIID is a multiprotein complex consisting of TATA-binding protein (TBP) complexed with a number of TBP-associated factors (73). The binding of TFIID is thought to be the first step in transcriptional initiation (11, 82). The subsequent binding of TFIIB is thought to be involved in bringing RNA polymerase II to the complex through the association of TFIIF (34, 37). This leads to the recruitment of TFIIE and TFIIH and possibly other factors, which ultimately results in the initiation of transcription (11,75,82).The magnitude of transcriptional activity is greatly affected by the second class of transcription factors that generally bind to distal control DNA elements (52). These sequence-specific factors act to either promote or inhibit the formation of an active transcriptional initiation complex. Recent in vitro transcription studies suggest that the entry of TFIIB may be rate limiting for transcriptional initiation and that several transcriptional activators act to recruit or stabilize the interaction of TFIIB with the initiation complex (14, 62). TFIIB contains a potential N-terminal zinc finger structure (amino acids 14 to 36) that may be important for interaction with RNA polymerase II and TFIIF (34), a more C-terminal amphipathic ␣ helix (amino acids 184 to 201), and two imperfect repeats (amino acids 124 to 201 and 218 to 294) (34,37,62) (Fig. 1A). The amphipathic ␣ helix appears to be important for interaction with TBP (34, 37) and several transcriptional activators such as the herpes simplex virus VP16 protein (62).Members of the steroid/thyroid hormone receptor gene family are sequence-specific DNA-binding proteins that play important roles in gene regulation. The steroid hormone receptor subfamily includes the receptors which mediate the effects of glucocorticoids, progestins, mineralocorticoids, androgens, and estrogens (12, 16). The thyroid/retinoid receptor subfamily (16, 21) includes receptors that mediate the effects of thyroid hormone (3,5,3Ј-triiodo-L-thyronine [T3]), all-trans retinoic acid, 9-cis retinoic acid, and 1,25-dihydroxyvitamin D 3 as well as several orphan receptors (e.g., COUP-TF and c-ErbA␣2) whose ligands, if any, are unknown (26,27,44). Steroid/thyroid receptors bind to specific DNA sequences known as hormone response elements (HREs) and mediate ligand-dependent ac-

Many nuclear receptors are capable of recognizing similar DNA elements. The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood. Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity. Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus. The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors. Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction. Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity. Identification of a coactivator with such a unique receptor specificity may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D 3 receptor (VDR), and the receptors that modulate the peroxisomal ␤-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR␤. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXR␤ with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad of T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.