The Role of Yeast Mediator in Transcriptional Regulation

描述（由申请人提供）：中介体是一种全球性的转录共调控蛋白复合物，在RNA聚合酶II转录基因的激活和抑制中起核心作用。中间体的高度保守的组成和功能，以及其复杂的机制，使酿酒酵母成为深入分子研究的理想模型。目前关于中介功能的范式是，它作为dna结合激活子和一般转录机制之间的直接中介。然而，酵母遗传学研究已经揭示了复合体的两个重要功能特性，这些特性与这个简单的模型不兼容。首先是中介体通过与染色质结构的可能联系抑制大量基因转录的能力。第二是额外的共激活子复合物和特定的核心启动子序列需要在激活转录中介者的功能。尽管在过去的10到15年里，酵母遗传技术已经进行了很好的研究，但对Mediator促进这些功能的直接机制知之甚少。我们提出的研究通过生化实验来揭示这些新功能特性的机制基础，满足了迫切需要。我们研究计划的长期目标是获得对真核细胞中激活或抑制信号与核心转录机制之间“通信”的完整分子理解。我们建议对酵母介质的研究集中在使用纯化蛋白和核酸来阐明机制。他们还整理了酵母中大量的遗传、基因组和蛋白质组学资源，以便在生理背景下理解这些机制。我们的中心假设是，中介促进抑制并通过不同于作为激活剂和RNA聚合酶II之间的直接中介的机制与其他共激活剂合作。当前研究项目的具体目标将回答两个关键问题。首先，介质促进和基于染色质的转录抑制是如何联系在一起的？我们提出生化和遗传实验来阐明一种抑制机制，涉及中介和染色质之间的直接相互作用。其次，SAGA、TFIID和核心启动子序列如何影响中介激活的转录？我们设计了生化实验，以确定如何以及如果中介直接与不同的共激活剂工作，以合作的方式刺激高水平的活化转录。这些研究对于了解用于调节细胞和发育过程的转录机制的全部范围至关重要。已经提出了操纵某些基因表达的治疗策略，用于治疗范围广泛的疾病，如癌症、血红蛋白病（如镰状细胞性贫血）、阿尔茨海默病和单细胞真核生物（如致病真菌）感染。由于缺乏对真核细胞中转录调控的复杂机制的认识，这些策略实施中的一个限制和挫折是无法特异性靶向某些基因。通过确定转录机制的核心组件如何促进正调控和负调控，我们提出的研究将为设计操纵基因表达的努力增加一个重要的新考虑。

DESCRIPTION (provided by applicant): Mediator is a global transcriptional co-regulatory protein complex that plays a central role in the activation and repression of RNA polymerase II transcribed genes. The highly conserved composition and function of Mediator, as well as the intricacies of its mechanism, make Saccharomyces cerevisiae an ideal model for incisive molecular studies. The current paradigm for Mediator function is that it serves as a direct intermediary between DNA-bound activators and the general transcription machinery. However, yeast genetic studies have revealed two important functional properties of the complex that are not compatible with this simple model. The first is Mediator's ability to repress transcription of a large number of genes via a possible link to chromatin structure. The second is the additional co-activator complexes and specific core promoter sequences required for Mediator function in activated transcription. Although ably studied by yeast genetic techniques over the past 10 to 15 years, very little is known about the direct mechanisms used by Mediator to facilitate these functions. Our proposed research meets a critical need by using biochemical experiments to reveal the mechanistic basis of these novel functional properties of Mediator. The long-term goal of our research program is to obtain a complete molecular understanding of the 'communication' between activation or repression signals and the core transcription machinery in eukaryotic cells. Our proposed studies of yeast Mediator center on the use of purified proteins and nucleic acids to elucidate mechanisms. They also marshal the substantial genetic, genomic and proteomic resources in yeast to understand these mechanisms in a physiological context. Our central hypothesis is that Mediator facilitates repression and co-operates with other co-activators through mechanisms that are distinct from serving as a direct intermediary between activators and RNA Polymerase II. The specific aims of the current research project will answer two key questions. First, how are Mediator-facilitated and chromatin-based transcription repression linked? We propose biochemical and genetic experiments to elucidate a mechanism of repression that involves direct interactions between Mediator and chromatin. Second, how do SAGA, TFIID, and core promoter sequence affect Mediator activated transcription? We have designed biochemical experiments to determine how and if Mediator directly works with different co-activators to stimulate high levels of activated transcription in a cooperative manner. These studies are essential to understand the full range of transcriptional mechanisms used to regulate cellular and developmental processes. PUBLIC HEALTH RELEVANCE Therapeutic strategies that manipulate the expression of certain genes have been proposed for diseases as wide ranging as cancer, hemoglobinpathies (such as sickle-cell anemia), Alzheimer's disease, and infections by single celled eukaryotes such as pathogenic fungi. A limitation and frustration in the implementation of these strategies has been the inability to specifically target certain genes due to a lack of appreciation for the complex mechanisms of transcriptional regulation in eukaryotic cells. By determining how a central component of the transcription machinery facilitates both positive and negative regulation, our proposed research will add an important new consideration to efforts designed to manipulate gene expression.