Transcription factor mutants of yeast

这项拟议研究的长期目标是确定调节蛋白质和机制 真核细胞染色质环境中RNA聚合酶II(POL II)的转录。这项建议 重点是POL II的转录延伸以及转录延伸与辅酶-2的协调。 转录事件。拟议的实验解决了与修改 转录染色质上的组蛋白以及从延伸到转录终止的转变。一个主要的焦点 该提案的一部分是高度保守的多功能Paf1复合体(Paf1C)。Paf1C是一家全球代理 真核生物中大多数(如果不是全部)活性基因上与POL II相关的转录延伸因子 基因组。除了调节许多基因的转录外，Paf1C还是建立 组蛋白在转录延伸过程中的修饰，并促进适当的转录终止。 特异性目标1和2研究Paf1C在建立组蛋白H_2B基因组模式中的重要性 赖氨酸123单泛素化。这一修改具有特殊的意义，因为它是 保守的组蛋白修饰级联导致随后的组蛋白甲基化和乙酰化事件， 这反过来又确保了基因组的正确表达。具体目标1研究了 Paf1C刺激H_2B_K123泛素化。生物化学、遗传和蛋白质交联法将是 用于阐明Paf1C与泛素结合酶RAD6的物理和功能相互作用 核小体靶标。特定目标2探索Paf1C在决定H_2B基因组模式中的重要性 通过分析破坏Paf1C两条不同途径的酵母突变株的K123泛素化 招聘。基因组实验将被用来绘制Paf1C占有率和H2B K123的模式 在缺乏Paf1C结构域的细胞中泛素化，Paf1C结构域将其连接到POL II延伸机制的组件上。 转录终止标志着延伸过程的结束，之前的研究已经表明Paf1C 及其依赖的组蛋白修饰在终止调节中的作用。因此，第二个主要焦点是 建议是染色质在转录终止中的作用。具体目标3超越了以前的研究 Paf1C，并建立在最近发现的一类损害转录的特定组蛋白突变体的基础上 非编码RNA的终止。这项工作将在酵母中进行，以利用强大的遗传工具 在这个系统中可用，包括用突变版本精确替换染色体基因的能力 通过全面的基因筛查识别新的调控因素。广泛的保护 酵母和人类中的转录因子和组蛋白修饰强烈表明 研究将促进对许多人类疾病的了解，特别是多种形式的癌症，这些疾病 当Paf1C或染色质的表观遗传修饰改变时发生。

The long-term objectives of the proposed research are to identify the proteins and mechanisms that regulate transcription by RNA polymerase II (Pol II) within the chromatin environment of eukaryotic cells. This proposal focuses on transcription elongation by Pol II and the coordination of transcription elongation with co- transcriptional events. The proposed experiments address fundamental questions related to the modification of histones on transcribed chromatin and the transition from elongation to transcription termination. A major focus of the proposal is the highly conserved, multifunctional Paf1 complex (Paf1C). Paf1C is a globally acting transcription elongation factor that associates with Pol II on most, if not all, active genes in the eukaryotic genome. In addition to regulating transcription of many genes, Paf1C is required for the establishment of histone modifications during transcription elongation and for promoting proper transcription termination. Specific Aims 1 and 2 investigate the importance of Paf1C in establishing genomic patterns of histone H2B lysine 123 mono-ubiquitylation. This modification is of special significance because it is the first step in a conserved histone modification cascade that leads to subsequent histone methylation and acetylation events, which in turn ensure proper expression of the genome. Specific Aim 1 investigates the mechanism by which Paf1C stimulates H2B K123 ubiquitylation. Biochemical, genetic, and protein-crosslinking approaches will be used to elucidate the physical and functional interactions of Paf1C with the ubiquitin conjugase Rad6 and the nucleosome target. Specific Aim 2 explores the importance of Paf1C in dictating genomic patterns of H2B K123 ubiquitylation through an analysis of yeast mutant strains that disrupt two distinct pathways for Paf1C recruitment. Genomic experiments will be used to map the patterns of Paf1C occupancy and H2B K123 ubiquitylation in cells lacking Paf1C domains that tether it to components of the Pol II elongation machinery. Transcription termination marks the end of the elongation process and previous studies have implicated Paf1C and its dependent histone modifications in the regulation of termination. Therefore, a second major focus of the proposal is the role of chromatin in transcription termination. Specific Aim 3 extends beyond previous studies of Paf1C and builds on the recent identification of a specific class of histone mutants that impair transcription termination of noncoding RNAs. The work will be performed in yeast to exploit the powerful genetic tools available in this system, including the ability to precisely replace chromosomal genes with mutant versions and to identify new regulatory factors through comprehensive genetic screens. The extensive conservation of transcription factors and histone modifications in yeast and humans strongly suggests that the outcome of the research will advance understanding of numerous human diseases, particularly multiple forms of cancer, which arise when Paf1C or the epigenetic modification of chromatin is altered.