Functional Analysis of the Estrogen-related Receptor in Drosophila

描述（由申请人提供）：用小亲脂化合物改变核受体（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，近二十年来，核磁共振一直是深入研究的焦点。然而，尽管有了这些努力，关于它们的分子作用机制仍有许多有待了解。我建议使用果蝇作为体内模型来研究雌激素相关受体（ERR）的调节和功能，ERR是由于与雌激素受体（ER）相似而首次发现的NR。脊椎动物ERRs调节脂肪代谢和线粒体生物发生，而ERRa突变小鼠对饮食诱导的肥胖具有抗性。ERRa和ERRy也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调控和功能提供了一个简化的系统。我们的实验室已经生成了dERR无效突变体，并且使用一系列代谢测定和微阵列进行的初步研究表明，dERR是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分析和代谢组学分析广泛表征。此外，我将使用电泳迁移转移试验和染色质免疫沉淀来确定dERR是否直接调节涉及碳水化合物代谢的基因。虽然脊椎动物的err被认为影响控制糖分解的基因，但这是一个知之甚少和未被重视的功能。脊椎动物的ERRs也被研究在雌激素信号传导中的潜在作用，ERRa的表达在雌激素的反应中上调。同样，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（20E）的脉冲相协调。通过功能缺失基因研究、northern blots和器官培养，我将确定20E是否调节dERR表达，20E是否可以激活dERR配体结合域，以及dERR是否在20E触发的转录级联反应中起作用。本文提出的研究将为ERR功能的分子机制提供新的见解。考虑到脊椎动物err参与了人类疾病（如肥胖、II型糖尿病以及乳腺癌）的主要危险因素的过程，拟议的研究将为这类nr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.