Synergistic action of E74B and ecdysteroid receptor in activating a 20-hydroxyecdysone effector gene

A number of insect effector genes activated by the steroid hormone 20-hydroxyecdysone (20E) are dually controlled by the ecdysteroid receptor (EcR/USP) and products of ecdysteroid early responsive genes (E74, E75, and Broad). However, the molecular mechanism of this dual action is poorly understood. Here we examined transcriptional activation of the vitellogenin (Vg) gene in the yellow fever mosquito, Aedes aegypti, by EcR/USP and E74 in response to an elevation of 20E titers. There are two isoforms of the Aedes E74 gene, AaE74A and AaE74B, which have a common C-terminal Ets DNA-binding domain and isoform-specific N termini in the female mosquito. Inhibiting expression of AaE74B but not AaE74A by RNA interference led to substantial reduction in the Vg gene expression. AaE74B and the ecdysteroid receptor synergistically enhanced 20E-induced transcription of the Vg promoter. This action required the E74-binding sites and the ecdysone response elements in the Vg 5' regulatory region. Two-hybrid assays and coimmunoprecipitation analyses demonstrated direct interaction between AaE74B and AaEcR/AaUSP. Moreover, disruption of this interaction by a dominant negative E74 mutant abolished the enhanced activation of Vg. Therefore, the cooperative interaction between AaE74B and the ecdysteroid receptor is required for high-level expression of the Vg gene in vivo. The synergistic activation is accomplished through their 20E-dependent protein-protein interaction on the gene promoter. This study reveals how the 20E direct-indirect regulation of an effector gene is achieved at the molecular level.