Functional Analysis of the Estrogen-related Receptor in Drosophila

描述（由申请人提供）：用小的亲脂性化合物改变核受体（NR）转录活性的能力使其成为药物干预的理想靶点。由于这种治疗潜力，NR已经成为二十多年来深入研究的焦点。尽管这些努力，但是，还有很多关于他们的分子作用机制的了解。我建议使用果蝇作为体内模型来检查雌激素相关受体（ERR）的调节和功能，NR首先确定由于其与雌激素受体（ER）的相似性。脊椎动物ERRs调节脂肪代谢和线粒体生物合成，ERRa突变小鼠对饮食诱导的肥胖有抵抗力。ERRa和ERRy也可作为乳腺肿瘤的生物标志物，用于预测临床结果和对激素治疗的反应性。然而，这些研究由于存在三种不同的脊椎动物ERR基因而变得复杂。相比之下，果蝇基因组只有一个ERR拷贝（dERR），这为研究ERR的调控和功能提供了一个简化的系统。我们的实验室已经产生了dERR无效突变体，使用一系列代谢测定和微阵列的初步研究表明，dERR作为一种重要的代谢调节剂，在碳水化合物代谢和线粒体功能中起主要作用。在拟议的研究中，这些dERR突变缺陷将使用代谢测定和代谢组学分析进行广泛表征。此外，我将使用电泳迁移率变动分析和染色质免疫沉淀，以确定是否dERR直接调节基因参与碳水化合物代谢。尽管脊椎动物的错误反应被认为会影响控制糖分解的基因，但这是一个人们知之甚少且未被充分认识的功能。脊椎动物ERRs也被研究了它们在雌激素信号传导中的潜在作用，ERRa的表达响应雌激素而上调。类似地，幼虫dERR表达和激活的开始似乎与类固醇激素20-羟基蜕皮激素（20 E）的脉冲相协调。使用功能丧失遗传学研究、北方印迹和器官培养，我将确定dERR表达是否受20 E调节，20 E是否可以激活dERR配体结合结构域，以及dERR是否在20 E触发的转录级联中起作用。这些研究将为ERR功能的分子机制提供新的见解。考虑到脊椎动物ERRs参与了包括肥胖和II型糖尿病以及乳腺癌等人类疾病主要风险因素的过程，拟议的研究将为NRs的这一亚类如何影响人类健康提供有价值的见解。

DESCRIPTION (provided by applicant): The ability to alter the transcriptional activity of nuclear receptors (NRs) with small lipophilic compounds makes them ideal targets for pharmaceutical intervention. As a result of this therapeutic potential, NRs have been the focus of intensive research for over twenty years. In spite of these efforts, however, a great deal remains to be learned regarding their molecular mechanisms of action. I propose to use Drosophila as an in vivo model to examine the regulation and function of the Estrogen-Related Receptor (ERR), a NR first identified due to its similarity with the estrogen receptor (ER). Vertebrate ERRs regulate fat metabolism and mitochondrial biogenesis, and ERRa mutant mice are resistant to diet-induced obesity. ERRa and ERRy also serve as biomarkers in breast tumors, and are used to predict clinical outcomes and responsiveness to hormone treatments. These studies, however, are complicated by the presence of three distinct vertebrate ERR genes. In contrast, the Drosophila genome possesses only one copy of ERR (dERR), providing a simplified system in which to study ERR regulation and function. Our lab has generated dERR null mutants, and preliminary studies using a range of metabolic assays and microarrays indicate that dERR acts as an essential metabolic regulator, with primary roles in carbohydrate metabolism and mitochondrial function. In the proposed research, these dERR mutant defects will be extensively characterized using metabolic assays and metabolomic profiling. Furthermore, I will use electrophoretic mobility shift assays and chromatin immunoprecipitation to determine if dERR directly regulates genes involved in carbohydrate metabolism. Although vertebrate ERRs are thought influence genes that control the breakdown of sugars, this is a poorly understood and underappreciated function. Vertebrate ERRs have also been studied for their potential role in estrogen signaling, and expression of ERRa is upregulated in response to estrogen. Similarly, the onset of larval dERR expression and activation appears to be coordinated with pulses of the steroid hormone 20-hydroxyecdysone (20E). Using loss-of-function genetic studies, northern blots, and organ culture, I will determine if dERR expression is regulated by 20E, if 20E can activate the dERR ligand- binding domain, and if dERR acts in the transcriptional cascades triggered by 20E. The studies proposed here will provide new insights into the molecular mechanisms of ERR function. Considering that vertebrate ERRs are involved in processes that comprise major risk factors for human disease, such as obesity and type II diabetes, as well as breast cancer, the proposed research will provide valuable insights into how this subclass of NRs can impact human health.