20 Hydroxyecdysone Suppression of Juvenile Hormone Response

Ecdysteroids and iuvenile hormone (JH) are major hormones that regulate various developmental events during an insect.s life cycle. The diverse biological effects of ecdysteroids are mediated by ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). The mechanisms by which JH signal is transduced to control the expression of genes is not understood. The biological effects of JH and ecdysteroids suggest a cross talk between these two hormones. JH response elements (JHRE) identified in the promoter region of JH- and 20-hydroxyecdysone (20E)-responsive JH esterase (jhe) gene and ecdysone receptor (EcR) deficient Drosophila melanogaster cell line (L57) were used to develop a robust system. In L57 cells, a reporter gene placed under the control of JHRE is induced by JH, and the JH-induced expression is suppressed by 20E in a dose- and time-dependent manner. The nuclear proteins isolated from L57 cells specifically bound to JHRE and this binding was prevented by pretreatment of nuclear proteins with 20E. This 20E suppression of JH-induced gene expression is mediated through EcR. Preliminary studies showed that EcR does not directly bind to JHRE containing promoter. Ligand-binding but not DNA-binding of EcR is required for 20E suppression of JH action. These results suggest that the EcR suppresses JH-induced gene expression though a .nonclassical. mode of action, i.e., without binding to ecdysone response elements directly. This is the first example of a system where a nonclassical action for EcR is suggested; we propose to utilize this system to study the mechanisms involved in the nonclassical action of EcR and its cross talk with the components of JH signal transduction cascade. The two specific objectives of this proposal are (1) to study the mechanism for 20E suppression of JH response and (2) to identify and characterize proteins that are important in cross talk between 20E and JH. Mutations to select amino acids in the ligand-binding domain of D. melanogaster EcR will be used to verify nonclassical action of EcR. RNA interference (RNAi)-mediated silencing of usp, Drosophila hormone receptors 38 and 78, and Seven-up genes will be employed to study their role in 20E suppression of JH response. A yeast two-hybrid assay will be used to identify proteins that interact with EcR. The EcR-interacting proteins will be characterized through (i) RNAi-mediated silencing of their expression in L57 cells, (ii) pull down and two-hybrid assays, (iii) studies on expression of RNA in D. melanogaster tissues and (iv) analysis of JH and 20E response in D. melanogaster mutants for genes coding for the identified proteins. Despite recent progress in understanding of the molecular basis of ecdysteroid action, it is still not known how a systemic ecdysteroid signal can exert temporal and tissue-specific biological effects. Although, JH plays important roles in insect development and reproduction, the molecular basis of JH action is not known. Very little is known about the nonclassical action of EcR and cross talk between ecdysteroids and JH. Results from the proposed studies will fill some of these gaps and help in developing environmentally friendly pest management methods and efficient gene switches. Training of postdoctoral fellows, graduate and undergraduate students is proposed. Undergraduate students can carry out most of the proposed work very well. The PI has a good source of undergraduate students through his participation in the University of Kentucky agriculture biotechnology undergraduate training program. Results from the proposed research will be presented at local, national and international meetings and published promptly in leading scientific and non-technical journals. Efforts will be made to recruit undergraduate and graduate students and postdoctoral fellows from underrepresented minorities and women groups.