Molecular Mechanisms Of Juvenile Hormone Action Via USF To Promote Vitellogenin Expression In Locusta Migratoria

Vitellogenesis,prerequisite to successful insect reproduction has been considered as the potential target for insect pest control and for taking advantage of beneficial insects.Juvenile hormone(JH)stimulates vitellogenesis in a variety of insects,but the underlying molecular mechanisms remain poorly understood.In the preliminary study of this dissertation research,we found that JH-induced vitellogenin(Vg)expression during vitellogenic phase was dependent on insulin in the migratory locust,Locusta migratoria.We hypothesize that the nutrient signaling is essential to JH-dependent vitellogenesis and that insulin and JH signaling pathways coordinate Vg expression in vitellogenic stage.This dissertation research therefore set out to elucidate the molecular basis of Vg regulation coordinated by insulin and JH.Analysis of the upstream promoter region of Vg gene revealed a conserved E-box motif(CACGCG)that is recognized by Upstream Stimulatory Factor(USF),a basic-Helix-Loop-Helix(bHLH-zip)transcription factor.Based on the published literatures,it was speculated that this transcription factor is likely to play a crucial role in Vg expression regulated by insulin and JH.By using the approaches of biochemistry,molecular biology,cell biology and genetics,we aimed to unveil the role of USF in the regulation of Vg expression coordinated by insulin and JH.RNAi-mediated knockdown of USF led to significantly reduced levels of Vg,blocked ovarian development and arrested oocyte maturation.A polyclonal USF antibody was raised by immunizing New Zealand white rabbits with prokaryotic recombinant USF protein.Western blot showed that USF phosphorylation positively correlated with Vg expression levels.Interestingly,insulin induced USF expression,whereas JH stimulated USF phosphorylation.Further bioinformatic analysis and site-directed mutagenesis demonstrated that JH activated USF phosphorylation at amino acid residue threonine 182(T182)via protein kinase A(PKA).Immunocytochemistry and microscopic imaging revealed that JH treatment caused translocation of overexpressed USF-RFP-His from cytoplasm to nucleus,which depended upon JH and PKA-mediated phosphorylation of USF at T182.Co-IP,ChIP and EMSA demonstrated that phosphorylated USF interacted with JH nuclear receptor,Met and bound to the DNA element containing E-box motif in the promoter of Vg gene,consequently promoting Vg transcription.The results of this dissertation research indicate that USF expression and function are regulated by both insulin and JH pathways.In vitellogenic stage of locusts,insulin upregulates the expression of USF,while JH triggers phosphorylation of USF at T182.Phosphorylated USF enters into nucleus,dimerizes with Met and binds to the E-box motif in Vg promoter,consequently activates the transcription of Vg.These findings provide new insights into the understanding of non-genomic action of JH.The results also point to a previously unidentified mechanism by which JH and insulin coordinately act on USF to activate Vg expression.