Reconstruction of ligand-dependent transactivation of Choristoneura fumiferana ecdysone receptor in yeast

Ecdysteroids play an important role in regulating development and reproduct ion in insects. Interaction of 20-hydroxyecdysone (20E) with ecdysone recep tor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the ac tivation of 20E-responsive genes. In this paper we describe a ligand-mediat ed transactivation system in yeast using the spruce budworm Choristoneura f umiferana ecdysone receptor (CfEcR), Coexpression of C. fumiferana USP (CfU SP) with CfEcR in yeast led to constitutive transcription of the reporter g ene. However, deletion of the A/B domain of CfUSP abolished constitutive ac tivity observed for the CfUSP:CfEcR complex. Replacement of USP with its ma mmalian homolog retinoid X receptors (RXRs) abolished the constitutive acti vity of the heterodimer but it did not restore EcR ligand-mediated transact ivation. These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast. A ligand-mediated transactivat ion was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:DeltaA/BUSP complexes. Deletion of the A/B domain of EcR in the con text of DeltaA/BEcR:RXR:GRIP1 or DeltaA/BEcR:DeltaA/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation. This is the first example o f highly efficient ligand-dependent transactivation of insect EcR in yeast. Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in ins ect tissue culture cells and whole insect systems. The results open the way to develop assays that can be used to screen novel species-specific ecdyso ne agonist/antagonist insecticides.