A Genetic Screen for Ecdysone Receptor Coactivators and Corepressors

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的受体，许多其他小的亲脂激素(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。该家族还包括共同组成昆虫蜕皮激素受体的多肽ECR和USP。广泛的生化研究揭示了核受体功能的大体图景：受体与特定的DNA靶序列结合，通过与蛋白质共激活因子和辅阻遏物结合来激活或抑制附近启动子的转录，这些蛋白质辅活制子和辅阻遏物将DNA连接的受体连接到转录装置。配体(类固醇或其他小的亲脂信号分子)与受体的结合将使受体从结合辅阻遏子的受体转变为结合辅活化子的受体。核受体调控的转录活动是正常发育的核心。此外，对核受体辅活化子和辅阻遏子的研究正在为染色体基因激活的限速步骤以及由激素和其他调节分子进行组织特异性调控的过程提供有价值的线索。昆虫蜕皮激素受体是一种异源二聚体核受体，由ECR和USP蛋白组成。它调节对蜕皮激素的转录反应，蜕皮激素是一种类固醇激素，协调昆虫(和其他节肢动物)的蜕皮和变态。许多证据表明，它在结构和功能上与最大的核受体--II型或异二聚体受体相似(尽管不完全相同)，先前的工作已经探索了ECR/USP转录反应的分子方面。由于果蝇的遗传学很发达，而且已经对果蝇基因组进行了测序，因此有机会开始使用遗传方法来识别与核受体相互作用的蛋白质。该项目将开发一种使用苍蝇的基因筛查，该苍蝇被设计成对与ECR/USP相互作用的蛋白质浓度的微小波动敏感。拟议实验的目标是通过遗传手段识别与ECR/USP在遗传上(和物理上)相互作用的蛋白质。预计已确定的基因将包括共激活因子和共抑制因子、伴侣、染色质重塑因子以及ECR/USP功能可能需要的其他蛋白质。通过区分全局和组织特异性的相互作用，该筛查可以提供对脊椎动物研究中已知的多个功能相似的辅助抑制因子和辅助激活因子的作用的见解。这一筛选有可能增加我们对基因表达和核受体功能的了解：(1)在与核受体相互作用的蛋白质列表中增加新的分子；(2)识别果蝇中的关键中间体(辅助激活因子和辅助抑制因子)，在那里它们可以整合到正在进行的发育中基因调控的研究中；(3)应用全生物遗传学方法来分析一个过程(核受体相互作用)，这是迄今为止只有在培养细胞中和通过生物化学才能接近的；(4)揭示辅助激活因子和辅助抑制因子功能的组织和阶段特有的方面。

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