Transcription Factor Mutants of Yeast

描述(由申请人提供)： 这项拟议研究的长期目标是确定调节RNA聚合酶II(PolII)转录的蛋白质和机制，特别是在染色质的背景下。这项提案的重点是全球代理的多功能Paf1复合体(Paf1C)。Paf1C在转录延伸过程中与RNA PolII结合，将关键事件与RNA合成偶联。这些事件包括组蛋白修饰、有效的转录终止和RNA3‘端的形成。Paf1C对基因表达的广泛影响突显了该研究计划的重要性。具体目的1是揭示组蛋白H2 B K123单泛素化过程中需要Paf1C的机制基础。这种修饰标记活性基因，调节转录，并控制染色质上的其他关键表观遗传标记。生化和遗传学研究将被用来识别与Paf1C结构域相互作用的蛋白质，Paf1C结构域是H2B K123泛素化所必需的，也是充分的。酶和基因组实验将在体外和体内测试Paf1C对H2B K123泛素化的影响。此外，还将探讨暴露的核小体表面在调节H3上的H2 B K123泛素化和下游甲基化标记中的作用。特定目标2是确定在伸长过程中将Paf1C连接到RNA PolII的机制。缺乏这种偶联会导致严重的突变表型，这表明转录的广泛中断。蛋白质相互作用和遗传学研究将被用来识别将Paf1C拴在RNA PolII延伸机制上的蛋白质，并调节这种相互作用。将利用突变体来破坏Paf1C-RNA polII的相互作用，并将使用靶向和基因组策略评估这些突变体对组蛋白修饰、RNA PolII磷酸化和转录本合成的影响。旨在干扰Paf1C从RNA PolII解离的突变体将被用来测试Paf1C在基因上保守定位模式的重要性。具体目的3是阐明Paf1C在调节转录终止和 非编码RNA的合成，包括snoRNA和隐蔽的不稳定转录本。分子实验将确定Paf1C染色质相关功能影响snoRNA基因终止效率的机制。平铺阵列研究将被用来确定Paf1C参与的范围以及其活性在调节snoRNAs和隐蔽的不稳定转录本的合成中的相对贡献。最后，将进行全面的遗传筛查，系统地询问组蛋白在转录终止中的作用。这项工作将在酵母中进行，以利用该系统中可用的强大遗传工具，因为酵母和人类Paf1复合体之间存在广泛的保守性。Paf1C基因紊乱与多种类型的人类癌症有关，包括胰腺癌、乳腺癌、子宫癌和甲状腺癌。因此，拟议的研究结果有望对了解癌症的原因产生重要和广泛的影响。

DESCRIPTION (provided by applicant): The long-term objectives of the proposed research are to identify the proteins and mechanisms that regulate transcription by RNA polymerase II (pol II) particularly within the context of chromatin. The focus of this proposal is on the globally acting, multi-functional Paf1 complex (Paf1C). Paf1C associates with RNA pol II during transcription elongation, coupling critical events to RNA synthesis. These events include histone modification and efficient transcription termination and RNA 3'-end formation. The broad impact of Paf1C on gene expression highlights the significance of the research plan. Specific Aim 1 is to uncover the mechanistic basis for the requirement of Paf1C in histone H2B K123 mono-ubiquitylation. This modification marks active genes, regulates transcription, and controls other key epigenetic marks on chromatin. Biochemical and genetic studies will be used to identify proteins that interact with a domain of Paf1C that is both necessary and sufficient for H2B K123 ubiquitylation. Enzymatic and genomic experiments will test the impact of Paf1C on H2B K123 ubiquitylation in vitro and in vivo. In addition, the role of an exposed nucleosomal surface in regulating H2B K123 ubiquitylation and downstream methylation marks on H3 will be probed. Specific Aim 2 is to determine the mechanisms that couple Paf1C to RNA pol II during elongation. Absence of this coupling leads to severe mutant phenotypes, indicative of broad disruptions in transcription. Protein interaction and genetic studies will be used to identify the proteins that tether Paf1C to the RNA pol II elongation machinery and regulate this interaction. Mutants will be exploited to disrupt the Paf1C-RNA pol II interaction, and the consequences of these mutants on histone modification, RNA pol II phosphorylation, and transcript synthesis will be assessed using targeted and genomic strategies. Mutants designed to interfere with Paf1C dissociation from RNA pol II will be used to test the importance of the conserved localization pattern of Paf1C on genes. Specific Aim 3 is to elucidate the role of Paf1C in regulating transcription termination and synthesis of noncoding RNAs, including snoRNAs and cryptic unstable transcripts. Molecular experiments will determine the mechanisms by which the chromatin-related functions of Paf1C impact termination efficiency at snoRNA genes. Tiling array studies will be used to determine the scope of the involvement of Paf1C, and relative contributions of its activities, in regulating synthesis of snoRNAs and cryptic unstable transcripts. Finally, a comprehensive genetic screen will be performed to systematically interrogate the role of histones in transcription termination. The work will be performed in yeast to exploit the powerful genetic tools available in this system and because extensive conservation exists between the yeast and human Paf1 complexes. Genetic perturbations that deregulate Paf1C are associated with multiple types of human cancers, including those of pancreatic, breast, uterine, and thyroid origin. The results of the proposed studies are therefore expected to have important and broad consequences for understanding the causes of cancer.