Transcription Factor Mutants of Yeast

描述（由申请人提供）： 拟议研究的长期目标是确定蛋白质和机制，特别是在染色质的背景下，通过RNA聚合酶II（pol II）调节转录。该提案的重点是全球作用的多功能Paf 1复合物（Paf 1C）。Paf 1C与RNA聚合酶II在转录延伸，耦合关键事件的RNA合成。这些事件包括组蛋白修饰和有效的转录终止以及RNA 3 '端形成。Paf 1C对基因表达的广泛影响凸显了该研究计划的重要性。具体目标1是揭示组蛋白H2 B K123单泛素化中需要Paf 1C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。生物化学和遗传学研究将用于鉴定与Paf 1C结构域相互作用的蛋白质，该结构域对于H2 B K123泛素化既必要又足够。酶和基因组实验将测试Paf 1C在体外和体内对H2 B K123泛素化的影响。此外，暴露的核小体表面在调节H2 B K123泛素化和下游甲基化标记H3的作用将被探测。具体目标2是确定在延伸过程中将Paf 1C偶联到RNA pol II的机制。这种偶联的缺乏导致严重的突变表型，表明广泛的转录中断。蛋白质相互作用和遗传学研究将被用来确定的蛋白质系Paf 1C的RNA聚合酶II延伸机制和调节这种相互作用。将利用突变体破坏Paf 1C-RNA pol II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 pol II磷酸化和转录物合成的影响。设计用于干扰Paf 1C从RNA pol II解离的突变体将用于测试Paf 1C在基因上的保守定位模式的重要性。具体目标3是阐明Paf 1C在调节转录终止中的作用， 非编码RNA的合成，包括snoRNA和隐蔽的不稳定转录物。分子实验将确定Paf 1C的染色质相关功能影响snoRNA基因终止效率的机制。平铺阵列的研究将被用来确定参与的范围Paf 1C，其活动的相对贡献，在调节合成的snoRNA和隐蔽的不稳定的成绩单。最后，将进行全面的遗传筛选，以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Paf 1复合物之间存在广泛的保守性。Paf 1C基因失调与多种人类癌症有关，包括胰腺癌、乳腺癌、子宫癌和甲状腺癌。因此，拟议研究的结果预计将对理解癌症的原因产生重要而广泛的影响。

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.