Juvenile hormone regulates Aedes aegypti Krüppel homolog 1 through a conserved E box motif

• AaKr-h1 is a JH-response gene in Aag-2 cells.
• JH induction of AaKr-h1 requires the presence of AaMet.
• A conserved 13 nucleotide JHRE identified in the promoter of Aakr-h1 is required for JH induction of this gene.
• bHLH domain of AaMet is required for JH induction of Aakr-h1.
• The expression of both AaKr-h1α and AaKr-h1β is induced by JH III.

Juvenile hormone (JH) plays important roles in regulation of many physiological processes including development, reproduction and metabolism in insects. However, the molecular mechanisms of JH signaling pathway are not completely understood. To elucidate the molecular mechanisms of JH regulation of Krüppel homolog 1 gene (Kr-h1) in Aedes aegypti, we employed JH-sensitive Aag-2 cells developed from the embryos of this insect. In Aag-2 cells, AaKr-h1 gene is induced by nanomolar concentration of JH III, its expression peaked at 1.5 h after treatment with JH III. RNAi studies showed that JH induction of this gene requires the presence of Ae. aegypti methoprene-tolerant (AaMet). A conserved 13 nucleotide JH response element (JHRE, TGCCTCCACGTGC) containing canonical E box motif (underlined) identified in the promoter of AaKr-h1 is required for JH induction of this gene. Critical nucleotides in the JHRE required for JH action were identified by employing mutagenesis and reporter assays. Reporter assays also showed that basic helix loop helix (bHLH) domain of AaMet is required for JH induction of AaKr-h1. 5' rapid amplification of cDNA ends method identified two isoforms of AaKr-h1, AaKr-h1α and AaKr-h1β, the expression of both isoforms is induced by JH III, but AaKr-h1α is the predominant isoform in both Aag-2 cells and Ae. aegypti larvae.

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