The Role of Yeast Mediator in Transcriptional Regulation

描述(由申请人提供)：Mediator是一种全球转录共调节蛋白复合体，在RNA聚合酶II转录基因的激活和抑制中发挥核心作用。介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。目前对介体功能的研究范式是，它作为DNA结合的激活子和一般转录机制之间的直接中介。然而，酵母遗传学研究揭示了该复合体的两个重要功能特性，与这个简单的模型不兼容。第一个是Mediator通过与染色质结构的可能联系来抑制大量基因转录的能力。第二个是激活转录中介体功能所需的额外的共激活复合体和特定的核心启动子序列。尽管在过去的10到15年里，酵母基因技术对其进行了精干的研究，但人们对Mediator促进这些功能的直接机制知之甚少。我们提出的研究满足了一个关键的需求，通过生物化学实验来揭示介体这些新的功能特性的机制基础。我们研究计划的长期目标是对真核细胞中激活或抑制信号与核心转录机制之间的‘沟通’有一个完整的分子理解。我们对酵母介体的研究主要集中在利用纯化的蛋白质和核酸来阐明其作用机制。他们还收集了酵母中大量的遗传、基因组和蛋白质组资源，以在生理环境中了解这些机制。我们的中心假设是，Mediator通过不同于激活剂和RNA聚合酶II之间的直接中介的机制，促进抑制和与其他协同激活剂的合作。本研究项目的具体目标将回答两个关键问题。首先，中介分子促进的转录抑制和基于染色质的转录抑制是如何联系起来的？我们建议进行生化和遗传学实验来阐明抑制的机制，该机制涉及到介体和染色质之间的直接相互作用。第二，SAGA、TFIID和核心启动子序列如何影响介体激活的转录？我们设计了生化实验来确定Mediator如何以及是否直接与不同的共激活剂一起以合作的方式刺激高水平的激活转录。这些研究对于理解用于调节细胞和发育过程的各种转录机制是必不可少的。与公共卫生相关的治疗策略已被提出用于治疗范围广泛的疾病，如癌症、血红蛋白疾病(如镰状细胞性贫血)、阿尔茨海默病和单细胞真核生物感染，如致病真菌。由于缺乏对真核细胞转录调控的复杂机制的认识，这些策略的实施一直受到限制和挫折，无法特定地针对某些基因。通过确定转录机制的中心组成部分如何促进正向和负向调控，我们拟议的研究将为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.