Recruitment and Dynamics of the Mediator Complex

该项目将为分子相互作用提供新的见解，这些分子相互作用决定了基因在活细胞中如何打开和关闭。 该项目的重点是一种名为Mediator的大型蛋白质复合物，它选择性地打开酵母和哺乳动物细胞中的基因子集。 将讨论两个核心问题。 首先，Mediator是如何选择和开启其基因靶点的？ 第二，与在哺乳动物细胞中的作用相比，在酵母中的介体作用如何？ 在这个项目中使用的比较方法将产生广泛的影响，在“简单”酵母与“复杂”哺乳动物细胞的基因活性的基本理解。 其中一些结果将以“大数据”的形式出现-描述整个基因组中分子相互作用的大数据集-这些数据集将存放在公共档案馆，科学家和公众可以自由查阅。 此外，该项目还将为本科生、一名博士后研究员和两名女研究生提供培训，其中一名是代表性不足的少数民族成员。介体蛋白复合物由20多种相互作用的蛋白质组成，存在于所有有核细胞（真核生物）中。 已知介体通过与负责RNA合成（转录）的机制相关联而发挥关键的基因激活作用。 然而，Mediator如何定位其基因靶点，以及当它找到它们时它会做什么，还没有完全了解。 该项目将在三个目标中解决这些问题。 第一个将测试属于一般转录机制的蛋白质帮助Mediator找到其目标的想法。 该方法将使用一种称为ChIP-seq的方法，在正常酵母细胞和一般转录机制的组分受损的突变体中比较Mediator与其靶点的关联，该方法可以识别蛋白质与整个基因组中DNA的关联。 第二个目标将跟进以前的结果，这些结果表明，中介者与不同目标的关联与微分动力学有关--中介者找到目标的速度有多快，以及它再次离开的频率和速度有多快。这一想法将使用染色质免疫沉淀（ChIP，ChIP-seq的基础）的变化进行测试，这些变化提供了结合动力学的信息。 前两个目标将使用面包酵母（酿酒酵母）的模型系统来完成，该模型系统易于操作，但在分子组成上与哺乳动物细胞非常相似。 在第三个目标中，将通过“敲低”这些亚基的表达并通过高通量测序测量对全基因组转录的影响来测试哺乳动物细胞中介体的特定亚基的功能受损对基因转录的影响。 在酵母中，这是一种有效的策略，用于了解介体结构如何与其功能相关。 比较在哺乳动物细胞中获得的结果将揭示与酵母的相似性和差异，从而揭示使用酵母系统获得的知识的价值和局限性。

This project will provide new insights into molecular interactions that govern how genes are turned on and off in living cells. The project focuses on a large protein complex called Mediator, which selectively turns on subsets of genes in both yeast and mammalian cells. Two central questions will be addressed. First, how does Mediator select and turn on its gene targets? Second, how does Mediator action in yeast compare to its action in mammalian cells? The comparative approach used in this project will have broad impact on fundamental understanding of gene activity in "simple" yeast versus "complex" mammalian cells. Some of the results will be in the form of "big data"--large data sets that describe molecular interactions across an entire genome--and these will be deposited in public archives where they can be freely accessed by scientists and the public. In addition, the project will provide training for undergraduates, a postdoctoral fellow, and two female graduate students, one of whom is a member of an underrepresented minority.The Mediator protein complex consists of over twenty interacting proteins and is found in all cells with nuclei (eukaryotes). Mediator is known to play a critical gene activating role via association with the machinery responsible for RNA synthesis (transcription). However, how Mediator locates its gene targets and exactly what it does when it finds them, are not completely understood. This project will address these issues in three aims. The first will test the idea that proteins belonging to the general transcription machinery help Mediator to find its targets. The approach will be to compare Mediator association with its targets in normal yeast cells and in mutants in which components of the general transcription machinery are impaired, using a method called ChIP-seq that identifies where proteins associate with DNA across an entire genome. The second aim will follow up on previous results that suggested that Mediator association with different targets is related to differential dynamics--how fast Mediator finds its targets and how often and rapidly it leaves again. This idea will be tested using variations of chromatin immunoprecipitation (ChIP, the basis of ChIP-seq) that provide information on binding dynamics. These first two aims will be done using the model system of baker's yeast (Saccharomyces cerevisiae), which is easy to work with but is quite similar to mammalian cells in its molecular makeup. In the third aim, the effect of impairing function of specific subunits of Mediator in mammalian cells on gene transcription will be tested by "knocking down" expression of those subunits and measuring the effect on genome-wide transcription by high throughput sequencing. In yeast, this has been a productive strategy for understanding how Mediator structure relates to its function. Comparing the results obtained in mammalian cells will reveal similarities and differences with yeast, and thereby reveal the value and limitations of knowledge gained using the yeast system.