Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic
transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct
binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response
elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic
manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed
to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has
revealed the stage and tissue specificity of NR activation for each of the fly NRs. 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. Dynamic
changes in activation that are indicative of sweeping changes in ligand and/or co-factor
production are also a prominent feature that has been revealed using this approach.
In addition, I went on to characterize the ligand regulated function of a single Drosophila
nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has
demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is
regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75,
reversing its ability to block the activity of its heterodimer partner Drosophila Hormone
Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/26310 |
| Date | 22 February 2011 |
| Creators | Necakov, Aleksandar Sasha |
| Contributors | Krause, Henry M. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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