We used alleles, (as well as 127, 1151C1159]. and a carboxyl-terminal ligand-binding domain name (LBD) (1). Many nuclear receptors function as ligand-regulated transcription factors, including the retinoic acid receptor and the thyroid hormone receptor, whose transcriptional properties require dimerization with a second nuclear receptor, the retinoid X receptor (RXR). Both RXR and its partner recognize and bind target DNA sequences (2C5). In cell culture, RXR heterodimers repress transcription in the absence of ligand by nucleating repressive complexes involving histone deacetylase activity (6C9). In the presence of ligand, corepressors are unable to bind the nuclear receptor complex, which remains bound to target DNA, and a coactivator complex is formed made up of CFTR-Inhibitor-II histone acetyltransferase activity (8, 9). The chromatin structure becomes accessible for general transcriptional machinery and results in gene activation. Ligand-regulated properties of the RXR heterodimers are conserved in invertebrates. The functional ecdysone receptor is composed of a heterodimer between the ecdysone-binding ecdysone receptor (EcR) and the RXR homologue ultraspiracle (USP) (10C13). EcR/USP complexes repress transcription in the absence of ligand and recruit coactivators in the presence of ligand (14C16). The steroid hormone ecdysone, and its more active form 20-hydroxyecdysone, plays a crucial role in larval and metamorphic molts, and the hierarchy of genes responding to ecdysone at these stages has been well characterized and includes itself (17, CFTR-Inhibitor-II 18). Because of the functional conservation between vertebrate and invertebrate RXR heterodimers in cell culture, we can use the easily manipulated and well-studied system to dissect the functional abilities of these complexes and by looking at in development, while confirming the role of as a mediator of the ecdysone response (19, 20). We extend these studies by analyzing the functional abilities of different alleles to activate and repress the ecdysone response genes and the (Stocks and Transgenic Travel Lines. The generation and detection of and mitotic clones in imaginal discs and salivary glands was carried out by using the FRT-Flp system (19, 23). Males of the genotype or is an 8-kb clones were generated by crossing males. Imaginal disk clones were generated as described. As the clones, travel stocks were generated containing males (25). Males from this cross of the genotype were crossed to or EcR isoforms, were used on imaginal discs at a 1:100 dilution each. 15C3 and 10Fil were developed by CFTR-Inhibitor-II Dr. L. Riddiford (26) and obtained from the Developmental Studies Hybridoma Bank. Salivary glands were stained with 15C3 or AD4.4 (gift from D. Hogness; ref. 27), which recognizes EcRB1 at a 1:100 or 1:50 dilution, respectively. To stain for broad complex (BrC), two mouse antibodies were used (gifts from G. Guild, University of Pennsylvania), a Z1 isoform (3C11.OA1) and one that recognizes all BrC isoforms (Mab 2539) (21). Mab 2539 was used diluted 1:100; 3C11.OA1 was used at a dilution of 1 1:100. A monoclonal mouse USP antibody AB11 (gift from F. Kafatos, ref. 28) was used at a 1:100 dilution. Secondary antibodies used were Texas red-conjugated goat anti-rabbit and FITC conjugated goat anti-mouse (The Jackson Laboratory) diluted 1:100. Confocal images were collected on a Nikon/Bio-Rad confocal microscope. Plasmids. Cloning CFTR-Inhibitor-II the travel lines. The reporter plasmids made up of tk-hsp27EcRE, tk-DR1x2, CFTR-Inhibitor-II tk-DR1x3, and pMH100-tk-luc, as well as cytomegalovirus promoter-driven expression plasmids (pCMX) expressing USP, USP3, USP4, VP16:USP, EcRB1, and -galactosidase were described (12, 13, 16, 29, 30). pCMX-based plasmids corresponding to USPL, RXRL, VP16:USP3, VP16:USP4, VP16:USPD, Gal4:EcRB1, or His-USP were constructed by using standard techniques, including various enzyme digestions or PCR amplification. Detailed information is usually available on request. Gel Mobility Shifts. Gel mobility shift experiments were performed by mixing either bacterially expressed His-tagged USP or translated USP proteins with the appropriate DNA in 20 l of binding solution, made up Itga9 of 10 mM Tris-Cl (pH 7.5)/0.05 mM EDTA/40 mM NaCl/20 mM KCl/5% glycerol/0.05% Nonidet P-40/0.2 nmol MgCl2/1 mg BSA/2 mg of poly dI:dC. The mix was incubated at room temperature for 20 min before being loaded onto.