A Genetic Screen for Ecdysone Receptor Coactivators and Corepressors

蜕皮激素受体辅激活剂和辅阻抑剂的遗传筛选

• 批准号: 0077841
• 负责人: Peter Cherbas
• 金额: $ 31.79万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077841&HistoricalAwards=false
• 关键词: Genetic; Screen; Ecdysone; Receptor; Coactivators

ABSTRACT The nuclear receptors are an ancient and widespread family of transcription factors that includes the receptors for the vertebrate steroid hormones, those for many other small lipophilic hormones (for example thyroid hormone and vitamin D), and many orphan receptors that may not be associated with hormones. The family also includes the polypeptides EcR and USP that together comprise the insect ecdysone receptor. A general picture of nuclear receptor function has emerged from extensive biochemical studies: Receptors bind to specific DNA target sequences where they may activate or repress transcription from a nearby promoter by binding to protein coactivators and corepressors that link the DNA-tethered receptor to the transcriptional apparatus. Binding of a ligand (steroid or other small lipophilic signaling molecule) to the receptor converts the receptor from one that binds corepressor to one that binds coactivator. The transcriptional activities regulated by the nuclear receptors are central to normal development. Moreover studies of nuclear receptor coactivators and corepressors are providing valuable clues to the rate-limiting steps in chromosomal gene activation and to processes that lead to tissue-specific regulation by hormones and other regulatory molecules. The ecdysone receptor in insects is a heterodimeric nuclear receptor composed of the protein EcR and USP. It mediates transcriptional responses to ecdysone, the steroid hormone that coordinates molting and metamorphosis in insects (and other arthropods). Many lines of evidence indicate that it is similar (though not identical) in both structure and function to the largest group of nuclear receptors, the type II or heterodimeric receptors and prior work has probed molecular aspects of the EcR/USP transcriptional response. Because of its well-developed genetics and because the Drosophila genome has been sequenced, the opportunity exists to begin to use genetic methods to identify proteins that interact with nuclear receptors. This project will develop a genetic screen using flies engineered to be sensitive to small fluctuations in the concentrations of proteins that interact with EcR/USP. The objective of the proposed experiments is the identification, by genetic means, of proteins that interact genetically (and physically) with EcR/USP. It is anticipated that the genes identified will include coactivators and co-repressors, chaperones, chromatin-remodeling factors, and other proteins that may be required for EcR/USP function. By distinguishing between global and tissue-specific interactions, the screen may provide insights into the roles of multiple, functionally similar corepressors and coactivators known from vertebrate studies. This screen has the potential to increase our understanding of gene expression and nuclear receptor function by: (1) Adding new molecules to the list of proteins that interact with nuclear receptors; (2) Identifying critical intermediates (coactivators and corepressors) in Drosophila where they can be integrated into on-going studies of gene regulation in development; (3) Applying a whole organism genetic approach to the analysis of a process (nuclear receptor interactions) that has heretofore been approachable only in cultured cells and by biochemistry; (4) Shedding light on tissue-and stage-specific aspects of coactivator and corepressor function

核受体是一个古老而广泛的转录因子家族，包括脊椎动物类固醇激素受体，许多其他小的亲脂性激素（如甲状腺激素和维生素D）的受体，以及许多可能与激素无关的孤儿受体。 该家族还包括多肽EcR和USP，它们一起构成昆虫蜕皮激素受体。 广泛的生物化学研究已经揭示了核受体功能的一般情况：受体与特定的DNA靶序列结合，在那里它们可以通过与蛋白质共激活子和共抑制子结合来激活或抑制附近启动子的转录，所述蛋白质共激活子和共抑制子将DNA束缚的受体与转录装置连接起来。 配体（类固醇或其他小的亲脂性信号分子）与受体的结合将受体从结合辅阻遏物的受体转化为结合辅激活物的受体。 由核受体调节的转录活性对正常发育至关重要。 此外，对核受体辅激活子和辅阻遏子的研究为染色体基因激活的限速步骤以及激素和其他调节分子导致组织特异性调节的过程提供了有价值的线索。 昆虫蜕皮激素受体是由蛋白质EcR和USP组成的异二聚体核受体。 它介导蜕皮激素的转录反应，蜕皮激素是一种类固醇激素，在昆虫（和其他节肢动物）中协调蜕皮和变态。 许多证据表明，它在结构和功能上都与最大的核受体II型或异二聚体受体相似（尽管不相同），并且先前的工作已经探测了EcR/USP转录反应的分子方面。 由于果蝇的遗传学已经很发达，而且果蝇的基因组已经测序，因此有机会开始使用遗传学方法来鉴定与核受体相互作用的蛋白质。 该项目将开发一种基因筛选，使用对与EcR/USP相互作用的蛋白质浓度的微小波动敏感的果蝇。 拟议实验的目的是通过遗传手段鉴定与EcR/USP发生遗传（和物理）相互作用的蛋白质。 预计鉴定的基因将包括共激活子和共阻遏子、分子伴侣、染色质重塑因子和EcR/USP功能可能需要的其他蛋白质。 通过区分全球和组织特异性的相互作用，屏幕可以提供洞察的作用，多个，功能相似的辅阻遏物和辅激活物已知的脊椎动物研究。该筛选有可能通过以下方式增加我们对基因表达和核受体功能的理解：（1）在与核受体相互作用的蛋白质列表中添加新分子;（2）识别关键中间体（辅激活子和辅抑制子）的研究，以便将它们整合到正在进行的发育过程中基因调控的研究中;（3）应用整个生物体遗传学方法来分析迄今为止仅在培养细胞和生物化学中可接近的过程（核受体相互作用）;（4）阐明辅激活子和辅阻遏子功能的组织和阶段特异性方面