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Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression

Significance Juvenile hormone is unique to arthropods and controls numerous essential functions in these organisms. Its receptor, Methoprene-tolerant, turns on genes by directly binding to E-box–like motifs in their regulatory regions. However, how juvenile hormone represses genes has remained unclear. Here, we used the Aedes aegypti female mosquito, in which juvenile hormone is necessary for reproductive maturation, to show that the repressor Hairy is required for the gene-repressive action of juvenile hormone and Methoprene-tolerant. Moreover, we demonstrate that a corepressor, Groucho, supports this Hairy function. Hence, Methoprene-tolerant recruits the evolutionarily conserved Hairy/Groucho molecular system to mediate the juvenile hormone gene-repressive function. The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box–like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.