Regulation of DNA Damage Induced Genes by Yeast TAFIIs

描述(由申请人提供)：基因表达的调节是细胞中的一个基本过程，这个过程中的变化与人类的许多疾病状态有关。它在多个水平上受到调控，确定这些事件是如何协调的是理解基因表达的核心。酵母CCR4-NOT复合体最初被描述为一般转录因子TFIID的调节因子。值得注意的是，它现在已经涉及到基因调控的多个方面，包括mRNA降解、翻译沉默、组蛋白甲基化和蛋白质泛素化。它的众多功能令人着迷，表明它从基因产物的出生到死亡都调节着基因的表达。我们对TFIID和DNA损伤诱导基因的研究使我们对这个复合体进行了表征，因为它似乎在调节DNA损伤应激反应中发挥作用。在过去的资助期间，我们确定它是一种真正的延长因子，直接激活受阻RNA聚合酶II(RNAPII)。有趣的是，CCR4-NOT似乎通过一种不同于其他迄今表征的延长因子的机制发挥作用；因此，分析其功能有可能揭示因素如何调节RNAPII的新见解。我们已经开发了许多强大的分析方法来充分利用酵母的生物化学、强大的遗传学和基因组学来表征其作用机制以及它在体内基因表达中所起的作用。这项建议中描述的研究的目标是调查CCR4结合的停滞伸长复合体的结构--而不是更好地了解它是如何促进伸长的；探索它与其他伸长因子的合作以调节RNAPII的活性；通过绘制CCR4-非突变体基因组中RNAPII停滞的变化图，分析该复合体在体内的生理功能。该项目的长期目标是了解这种多功能复合体如何调节基因调控的多个步骤，以维持真核细胞的正常发育和基因组的完整性。

DESCRIPTION (provided by applicant): The regulation of gene expression is a fundamental process in cells, and alterations in this process have been linked to numerous disease states in humans. It is regulated at multiple levels, and determining how these events are coordinated is central to understanding gene expression. The yeast Ccr4-Not complex was originally described as a regulator of the general transcription factor TFIID. Remarkably, it has now been implicated in multiple aspects of gene regulation including mRNA degradation, translational silencing, histone methylation and protein ubiquitylation. Its myriad of functions is quite fascinating and suggests it regulates gene expression from the birth to death of a gene product. Our study of TFIID and DNA damage inducible genes led us to characterize this complex, as it appears to play a role in regulating DNA damage stress responses. Over the last funding period we established it as a bona-fide elongation factor that directly activates arrested RNA polymerase II (RNAPII). Interestingly, Ccr4-Not appears to function via a mechanism unique from other elongation factors characterized to date; thus, analyzing its function has the potential to reveal novel insights into how factors regulate RNAPII. We have a developed a number of powerful assays to fully exploit the biochemistry, powerful genetics and genomics of yeast to characterize its mechanism of action and the role it plays in gene expression in vivo. The goals of the studies described in this proposal are to investigate the structure of arrested elongation complexes bound by Ccr4-Not to gain a greater understanding of how it promotes elongation; to explore its collaboration with other elongation factors to regulate RNAPII activity; to analyze the physiological functions of the complex in vivo by mapping changes in RNAPII arrest across the genome in Ccr4-Not mutants. The long term goals of this project are to understand how this multi-functional complex regulates multiple steps in gene regulation to maintain normal development and the integrity of the genome in eukaryotic cells.