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(PolII)转录中发挥普遍和重要作用的蛋白质，并阐明这些蛋白质的作用机制。这项拨款的重点是转录延伸的高度调控过程。虽然近年来已经发现了一些真核转录延长因子，但对于这些蛋白质如何在染色质背景下促进或阻碍RNA合成的机制了解很少。特异性目标1和2集中在保守的Paf1复合体上，该复合体与RNA PolII相互作用，并将染色质的变化与转录延伸结合在一起。特异的Aim1是为了确定Paf1复合体引导组蛋白修饰的机制。遗传抑制和生化筛选将用于识别与最近在Paf1复合体的Rtf1亚单位中发现的组蛋白修饰结构域相互作用的蛋白质。将进行实验，以测试该结构域是否足以在没有正在进行的转录的情况下建立组蛋白H2 B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方面的作用将被研究。候选基因方法和遗传选择将揭示组蛋白乙酰化转移酶(S)和组蛋白去乙酰化酶(S)与Paf1复合体合作，在编码区内抑制组蛋白乙酰化和虚假转录启动。具体目标2是研究Paf1复合体的三个未被探索的转录功能。以ARG1为模型基因，研究Paf1复合体在基因抑制中的作用。Paf1复合体依赖于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中特定减轻ARG1抑制的突变将被确定，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复合体对基因表达的广泛影响。这些数据将被进一步分析，并将详细研究两种特定的转录模式，这两种模式表明Paf1复合体在克服伸长障碍和抑制隐秘转录起始方面是必需的。具体目的3是研究Rkr1的功能，Rkr1是一种新型的核泛素蛋白连接酶，它与Paf1复合体和其他正常染色质功能所需的蛋白质具有很强的功能联系。这一目的的实验验证了Rkr1调节组蛋白变体的修饰和功能的假设，组蛋白变体在转录中具有保守和重要的作用。人类Paf1复合体及其相互作用的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、甲状旁腺功能亢进症-颌骨肿瘤综合征和白血病。因此，拟议中的研究结果有望对几种折磨人类的癌症的原因有深入的了解。当基因突变导致细胞生长失控时，人类就会患上癌症。这些突变改变了蛋白质的活性，而蛋白质对细胞分裂的适当调控至关重要。改变Paf1复合体活性的突变是这一应用的主要焦点，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当表达，从而与多种类型的癌症有关，包括胰腺癌、白血病和甲状旁腺功能亢进症-颌骨肿瘤综合征。

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.