Dynamic regulation ofDrosophilanuclear receptor activity in vivo

Abstract

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue-and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.

Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster

“…In addition, E75A overexpression provokes a phenotype similar to the one induced by DHR3 knockdown. Therefore, as shown in previous studies (Palanker et al, 2006;Reinking et al, 2005;White et al, 1997), E75A might directly counteract DHR3 activity to regulate steroidogenesis. Furthermore, E75 has been proposed to sustain and amplify ecdysone production through a feed-forward effect (Bialecki et al, 2002) suggesting that the developmental arrest observed in E75A mutants is due to the defect in ecdysone auto-amplification.…”

“…4F,G). Considering that E75 has been described to counteract DHR3 activity (Palanker et al, 2006;Reinking et al, 2005) and that DHR3 overexpression provokes developmental arrest at late L2 stage, the developmental arrest due to E75-RNAi or ftz-f1-RNAi could be a consequence of higher DHR3 activity.…”

“…At the late L3 stage, DHR3 downregulates expression of the steroidogenic enzymes Phm, Dib and Sad. Several studies describe DHR3 as a transcriptional activator whose activity is inhibited by E75 (Caceres et al, 2011;Palanker et al, 2006;Reinking et al, 2005;White et al, 1997), suggesting that DHR3 may act indirectly to repress steroidogenesis. We previously reported that βFtz-f1 was necessary for Phm and Dib expression (Parvy et al, 2005).…”

“…These genes include E75 (Eip75B -FlyBase), which encodes three distinct nuclear receptor (NR) isoforms: E75A, B and C (Segraves and Hogness, 1990). DHR3 (Hr46 -FlyBase) is another NR induced by ecdysone (Hiruma and Riddiford, 2004;Horner et al, 1995), which together with E75B regulates transcription of the downstream NR βFtz-f1, the β-isoform encoded by the ftz-f1 gene (Palanker et al, 2006;Reinking et al, 2005;White et al, 1997). The idea of steroidogenesis autoregulation in Drosophila arises from phenotypic analyses showing that E75A mutants fail to produce ecdysteroids during the second larval stage (Bialecki et al, 2002).…”

The unfulfilled/DHR51 gene of Drosophila melanogaster modulates wing expansion and fertility

“…While Northern analysis failed to detect DHR51 transcripts during the last two larval instars or early pupal stages (Sullivan and Thummel, 2003), the hypothesis that DHR51 is expressed in a subset of neurons was supported by a microarray analysis which showed that DHR51 transcripts were enriched in the adult central nervous system (CNS; Chintapalli et al, 2007). Functionally, the DHR51 LBD failed to activate gene transcription supporting the hypothesis that DHR51 may function as a transcriptional repressor (Palanker et al, 2006). This initial molecular genetic characterization of the DHR51 gene documents that DHR51 is expressed in a limited number of heterogeneous cells of the brain and ventral nerve cord throughout development, including interneurons of the mushroom body.…”