Juvenile hormone (JH) is one of the principal hormones that regulate insect development and reproduction. Accumulating evidence suggests that Methoprene-tolerant (Met) protein is a nuclear receptor of JH. Work by others has shown that Met is capable of binding JH at physiological concentration. An RNAi knockdown of Met causes down-regulated expression of JH-responsive genes and a phenotype similar to that observed in JH-deficient insects, suggesting that Met is required for mediating physiological and molecular responses to JH.
The work in this report aims to understand the mechanisms underlying gene regulation by JH via Met. Met is a bHLH-PAS (basic-helix-loop-helix Per-ARNT-Sim) family protein. Many proteins in this family function as heterodimers formed with other proteins of this family. In a yeast two-hybrid screening, we discovered that another bHLH-PAS family protein, FISC, interacts with Met in the presence of JH. FISC is also required for JH functions as an RNAi knockdown of FISC down-regulated JH-responsive genes. To elucidate how Met and FISC mediate JH functions in gene regulation, we employed molecular biology techniques and characterized the formation of a JH-Met-FISC complex and its actions in activating gene expression using mosquito Aedes aegypti as a model. My results demonstrated that Met and FISC forms a complex when JH is present via their conserved N-terminal domains. The complex then binds to E box-like sequences presented in the promoter of JH-responsive genes to activate gene expression. This mechanism also applies to the fruit fly Drosophila melanogaster, suggesting that it is a conserved action of JH in insects. Further studies showed that DNA-binding by Met and FISC requires the basic regions of the bHLH domains of both proteins. Lastly we identified a consensus binding-site of Met and FISC. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/53504 |
| Date | 30 December 2013 |
| Creators | Li, Meng |
| Contributors | Biochemistry, Zhu, Jinsong, Gillaspy, Glenda E., Larson, Timothy J., Tu, Zhijian Jake |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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