Molecular mechanisms of E75 activation by 20-hydroxyecdysone and juvenile hormone

Travis Joseph Bernardo, Fordham University

Abstract

Many key developmental and physiological processes in Drosophila are directed by the steroid 20-hydroxyecdysone and the sesquiterpenoid juvenile hormone. Despite their importance, much information is missing with regard to the molecular mechanisms underlying the activation of gene expression by these two hormones. The early genes occupy a unique position in the ecdysone regulatory hierarchy. They exhibit a rapid, synchronous, and widespread hormonal activation that is distinctive in comparison to other ecdysone target genes. However, to date no comprehensive analysis has been undertaken to determine whether ecdysone signaling in the early genes is mediated through unique molecular mechanisms. Using chromatin immunoprecipitation, DNase I hypersensitivity, and chromosome conformation capture in conjunction with computational techniques, it was determined that the E75 gene possesses multiple functional ecdysone response elements. These elements interact over long distances in vivo with the E75 promoters, demonstrating that the ecdysone utilizes chromatin looping to regulate transcription and suggests that EcREs can function cooperatively to facilitate the rapid activation of E75 by ecdysone. Based on our laboratory's finding that the nuclear receptor FTZ-F1 is a necessary component of the intracellular JH signaling pathway responsible for the activation of E75A, I sought to better understand the role of this factor in JH signaling. Through the use of chromatin immunoprecipitation, synthetic F1RE and two-hybrid luciferase reporter assays, and computational homology modeling, I determined that FTZ-F1 is physically bound to E75A during the JH response and that FTZ-F1 interacts JH-dependently with the candidate JH receptors MET and GCE. This interaction enables the F1RE to function as a JH-responsive element and suggests that FTZ-F1 may function as a recruitment factor for the JH receptor. Furthermore, I explored the mechanism of interaction between FTZ-F1 and MET/GCE and identified a novel LIxxL motif enabling MET and GCE to participate in an AF2/NR box interaction with FTZ-F1, thus providing a mechanistic explanation for the interaction of the JH receptors with a nuclear receptor protein.^

Subject Area

Biology, Molecular

Recommended Citation

Bernardo, Travis Joseph, "Molecular mechanisms of E75 activation by 20-hydroxyecdysone and juvenile hormone" (2013). ETD Collection for Fordham University. AAI3588203.
https://fordham.bepress.com/dissertations/AAI3588203

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