bHLH-PAS heterodimer of methoprene-tolerant and Cycle mediates circadian expression of juvenile hormone-induced mosquito genes

Abstract

Juvenile hormone (JH) governs a great diversity of processes in insect development and reproduction. It plays a critical role in controlling the gonadotrophic cycles of female mosquitoes by preparing tissues for blood digestion and egg development. Here, we show that in female Aedes aegypti mosquitoes JH III control of gene expression is mediated by a heterodimer of two bHLH-PAS proteins-the JH receptor methoprene-tolerant (MET) and Cycle (CYC, AAEL002049). We identified Aedes CYC as a MET-interacting protein using yeast two-hybrid screening. Binding of CYC and MET required the presence of JH III. In newly eclosed female mosquitoes, the expression of two JH-responsive genes, Kr-h1 and Hairy, was dependent on both the ratio of light to dark periods and JH III. Their expression was compromised by in vivo RNA interference (RNAi) depletions of CYC, MET, and the steroid receptor coactivator SRC/FISC. Moreover, JH III was not effective in induction of Krh1 and Hairy gene expression in vitro in fat bodies of female mosquitoes with RNAi-depleted CYC, MET or SRC/FISC. A sequence containing an E-box-like motif from the Aedes Kr-h1 gene promoter specifically interacted with a protein complex, which included MET and CYC from the female mosquito fat body nuclear extract. These results indicate that a MET/CYC heterodimer mediates JH III activation of Kr-h1 and Hairy genes in the context of light-dependent circadian regulation in female mosquitoes during posteclosion development. This study provides an important insight into the understanding of the molecular basis of JH action.hormone receptor | insect hormone | transcriptional control M osquitoes serve as vectors of harmful human diseases because blood feeding is required for their egg development; disease pathogens use hematophagous female mosquitoes for obligatory stages of their life cycles. An insect-specific sesquiterpenoid juvenile hormone III (JH III) is essential for a newly eclosed female mosquito to reach a competence stage for blood feeding and egg maturation (1, 2). Although several aspects of the JH III-dependent development have been characterized (3-6), our understanding of molecular mechanisms underlying JH regulation of female mosquito posteclosion development is limited.Methoprene-tolerant (MET), which is a basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) protein, was discovered in Drosophila melanogaster (7), and its role in mediating a JH response has been established (8). MET binds to JH with a high affinity, suggesting that it is the JH receptor (9, 10). As a bHLH protein, MET requires either a homo-or heterodimer partner for its activity (11). Studies in Aedes aegypti, the beetle Tribolium castaneum, and the silkworm Bombyx mori have shown a bHLH-PAS domain-containing steroid receptor coactivator (SRC/FISC/ Taiman) to interact with MET (10,[12][13][14]. Whether, an additional bHLH transcription factor with DNA-binding properties is required as a Met partner remains to be established.Using yeast two-hybrid (Y2H) screening, we identified an Aedes ortholo...

Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein

“…aegypti, MET also heterodimerizes in a JH-dependent manner with Cycle (CYC), another bHLH-PAS protein (41). The MET-CYC complex in newly emerged adult female mosquitoes is required for circadian expression of some JH-induced genes, including AaKr-h1 (41).…”

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex

“…Met is a member of the family of basic helixloop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factors that requires homo-or heterodimerization for DNA binding and transcriptional regulation (7). In the Aedes aegypti mosquito, Met forms a heterodimer with other bHLH-PAS factors, the steroid receptor coactivator (SRC/FISC), or Cycle (CYC) in a JHdependent manner (8,9). JH-mediated Met-CYC binding has been quantitatively simulated using the two-hybrid yeast β-galactosidase assay in a yeast cell, Y187 (9).…”

“…In the Aedes aegypti mosquito, Met forms a heterodimer with other bHLH-PAS factors, the steroid receptor coactivator (SRC/FISC), or Cycle (CYC) in a JHdependent manner (8,9). JH-mediated Met-CYC binding has been quantitatively simulated using the two-hybrid yeast β-galactosidase assay in a yeast cell, Y187 (9). Thus, it is possible to test both JHAs and JHANs for their interactions with Met and its partners using this assay.…”

Microarrays reveal discrete phases in juvenile hormone regulation of mosquito reproduction

“…This suppression appears to be indirect, likely requiring synthesis of repressive transcription factors that then act on these genes. One candidate is the known repressive bHLH transcription factor Hairy, which is known to be activated by the JH-Met-Cycle complex in the mosquitoes (7). How Hairy might suppress these genes is not known.…”