Synergistic action of the transcription factors Kruppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti

Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or repression. Met-mediated gene repression is indirect, requiring involvement of intermediate repressors. Hairy, which functions downstream of Met in the JH gene-repression hierarchy, is one such factor. Kruppel-homolog 1, a zinc-finger transcriptional factor, is directly regulated by Met and has been implicated in both activation and repression of JH-regulated genes. However, the interaction between Hairy and Kr-h1 in the JH-repression hierarchy is not well understood. Our RNAseq-based transcriptomic analysis of the Kr-h1-depleted mosquito fat body revealed that 92% of Kr-h1 repressed genes are also repressed by Met, supporting the existence of a hierarchy between Met and Kr-h1 as previously demonstrated in various insects. Notably, 130 genes are co-repressed by both Kr-h1 and Hairy, indicating regulatory complexity of the JH-mediated PE gene repression. A mosquito Kr-h1 binding site in genes co-regulated by this factor and Hairy was identified computationally. Moreover, this was validated using electrophoretic mobility shift assays. A complete phenocopy of the effect of Met RNAi depletion on target genes could only be observed after Kr-h1 and Hairy double RNAi knockdown, suggesting a synergistic action between these two factors in target gene repression. This was confirmed using a cell-culture-based luciferase reporter assay. Taken together, our results indicate that Hairy and Kr-h1 not only function as intermediate downstream factors, but also act together in a synergistic fashion in the JH/Met gene repression hierarchy. Author summary Juvenile hormone (JH) plays an essential role in preparing Aedes aegypti female mosquitoes for blood feeding, egg development, and pathogen transmission. JH acting through its receptor Methoprene-tolerant (Met) regulates the expression of large gene cohorts. JH mediated gene repression, unlike activation that is directly mediated by Met, is indirect and requires intermediate transcriptional repressors Hairy and Kruppel-homolog 1 (Kr-h1). Here, we demonstrate that Hairy and Kr-h1 can act synergistically in the JH-Met gene repression pathway in Aedes female mosquitoes. These interact directly with regulatory regions of the genes that have both Hairy and Kr-h1 binding sites. Thus, this study has significantly advanced our understanding of the complexity of the JH-mediated gene expression pathway. This research yields valuable information about the JH control of reproductive development of the mosquito A. aegypti, one of the most important vectors of human diseases.