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在RNA聚合酶II （pol II）转录中起一般和重要作用的蛋白质，并阐明这些蛋白质的作用机制。这项资助的重点是高度调控的转录延伸过程。虽然近年来已经鉴定了许多真核转录延伸因子，但对于这些蛋白质在染色质背景下如何促进或阻碍RNA合成的机制了解甚少。特异性目标1和2关注保守的Paf1复合物，该复合物与RNA pol II相互作用，并将染色质变化与转录伸长结合。特异性目的是确定Paf1复合体指导组蛋白修饰的机制。遗传抑制因子和生化筛选将用于鉴定与最近在Paf1复合体的Rtf1亚基中发现的组蛋白修饰域相互作用的蛋白质。实验将测试该结构域是否足以在没有持续转录的情况下建立组蛋白H2B泛素化和组蛋白H3赖氨酸4和79甲基化。Paf1复合体在调节活性基因上的另外两个组蛋白修饰（组蛋白H3赖氨酸36三甲基化和组蛋白乙酰化）中的作用将被研究。候选基因方法和遗传选择将揭示组蛋白乙酰转移酶(s)和组蛋白去乙酰化酶(s)与Paf1复合体合作，抑制编码区域内组蛋白乙酰化和虚假转录起始。特异性目的2是研究三种未知的Paf1复合物的转录功能。Paf1复合体在基因抑制中的功能将以ARG1为模式基因进行研究。Paf1复合体依赖性抑制的分子途径以及Paf1复合体对组蛋白修饰和核小体定位的影响将被研究。PAF1中特异性减轻ARG1抑制的突变将被发现，以揭示抑制的分子基础。我们最近的基因组拼接阵列研究揭示了Paf1复合物对基因表达的广泛影响。这些数据将进一步分析，并将详细研究两种特定的转录模式，这两种模式表明Paf1复合体在克服延伸阻滞和抑制隐转录起始方面是必需的。具体目标3是研究Rkr1的功能，Rkr1是一种新型的核泛素蛋白连接酶，与Paf1复合物和其他染色质功能所需的蛋白质具有强大的功能连接。本实验验证了Rkr1调控组蛋白变体的修饰和功能的假设，该组蛋白变体在转录中具有保守和重要的作用。人类Paf1复合物及其相互作用的SET结构域蛋白的活性或表达缺陷与多种癌症有关，包括胰腺癌、甲状旁腺功能亢进-颌肿瘤综合征和白血病。因此，拟议的研究结果有望深入了解几种折磨人类的癌症的原因。当基因突变导致细胞生长失控时，就会出现人类癌症。这些突变改变了对细胞分裂的适当调节至关重要的蛋白质的活性。改变Paf1复合体（本应用的主要焦点）活性的突变，以及与Paf1复合体相互作用的set结构域蛋白，导致参与细胞周期控制的基因的不正确表达，因此与多种类型的癌症相关，包括胰腺癌、白血病和甲状旁腺功能亢进颌肿瘤综合征。

DESCRIPTION (provided by applicant): The long-term objectives of the proposed research are to identify proteins that play general and important roles in transcription by RNA polymerase II (pol II) and to elucidate the mechanisms by which these proteins act. This grant focuses on the highly regulated process of transcription elongation. While a number of eukaryotic transcription elongation factors have been identified in recent years, there is little mechanistic understanding of how these proteins facilitate or impede RNA synthesis in the context of chromatin. Specific Aims 1 and 2 focus on the conserved Paf1 complex, which interacts with RNA pol II and couples chromatin changes to transcription elongation. Specific Aim1 is to determine the mechanism by which the Paf1 complex directs histone modifications. Genetic suppressor and biochemical screens will be used to identify proteins that interact with a recently identified histone modification domain in the Rtf1 subunit of the Paf1 complex. Experiments will be performed to test whether this domain is sufficient to establish histone H2B ubiquitylation and histone H3 lysine 4 and 79 methylation in the absence of ongoing transcription. The role of the Paf1 complex in regulating two additional histone modifications on active genes, histone H3 lysine 36 tri-methylation and histone acetylation, will be studied. Candidate gene approaches and genetic selections will be performed to reveal the histone acetyltransferase(s) and histone deacetylase(s) that collaborate with the Paf1 complex to repress histone acetylation and spurious transcription initiation within coding regions. Specific Aim 2 is to investigate three unexplored transcriptional functions of the Paf1 complex. The function of the Paf1 complex in gene repression will be studied using ARG1 as a model gene. The molecular pathway to Paf1 complex- dependent repression and the impact of the Paf1 complex on histone modification and nucleosome positioning will be investigated. Mutations in PAF1 that specifically alleviate ARG1 repression will be identified to expose the molecular basis of repression. Our recent genome tiling array studies revealed broad effects of the Paf1 complex on gene expression. These data will be analyzed further, and two specific transcriptional patterns, which indicate a requirement for the Paf1 complex in overcoming elongation blocks and in repressing cryptic transcription initiation, will be studied in detail. Specific Aim 3 is to investigate the function of Rkr1, a novel nuclear ubiquitin-protein ligase that exhibits strong functional connections to the Paf1 complex and other proteins required for proper chromatin function. Experiments in this Aim test the hypothesis that Rkr1 regulates the modification and function of a histone variant that has conserved and important roles in transcription. Defects in the activity or expression of the human Paf1 complex and the SET domain proteins with which it interacts are associated with multiple cancers, including pancreatic cancer, hyperparathyroidism- jaw tumor syndrome, and leukemia. Therefore, the results of the proposed studies are expected to yield insights into the causes of several types of cancer that afflict humans. PUBLIC HEALTH RELEVANCE Human cancers arise when genetic mutations lead to uncontrolled cell growth. These mutations alter the activities of proteins that are critical for the proper regulation of cell division. Mutations that change the activity of the Paf1 complex, the primary focus of this application, and the SET-domain proteins, which interact with the Paf1 complex, lead to the improper expression of genes involved in cell cycle control and are thus associated with multiple types of cancer, including pancreatic cancer, leukemia, and hyperparathyroidism-jaw tumor syndrome.