Transcription factor mutants of yeast

拟议研究的长期目标是确定调节蛋白质和机制， 在真核细胞的染色质环境中，RNA聚合酶II（Pol II）的转录。这项建议 着重于Pol II的转录延伸以及转录延伸与co- 转录事件。所提出的实验解决了与以下修饰相关的基本问题： 转录染色质上的组蛋白和从延伸到转录终止的过渡。一个主要重点 其中最重要的是高度保守的多功能Paf 1复合物（Paf 1C）。Paf 1C是一家全球性的 一种转录延伸因子，在真核生物中的大多数（如果不是全部）活性基因上与Pol II相关 基因组除了调节许多基因的转录外，Paf 1C还需要用于建立 转录延长期间的组蛋白修饰和促进适当的转录终止。 特定目的1和2研究Paf 1C在建立组蛋白H2 B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，因为它是一个 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， 从而确保基因组的正确表达。具体目标1研究了 Paf 1C刺激H2 B K123泛素化。生物化学、遗传学和蛋白质交联方法将成为 用于阐明Paf 1C与泛素缀合酶Rad 6和 核小体靶点具体目标2探讨了Paf 1C在决定H2 B基因组模式中的重要性 通过分析破坏Paf 1C两种不同途径的酵母突变株的K123泛素化 招聘基因组实验将用于绘制Paf 1C占用和H2 B K123的模式 在缺乏Paf 1C结构域的细胞中泛素化，该结构域将其拴系到Pol II延伸机制的组分。 转录终止标志着延伸过程的结束，先前的研究表明Paf 1C 以及其依赖的组蛋白修饰在终止调节中的作用。因此，第二个主要重点是 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， Paf 1C和建立在最近鉴定的一类特定的组蛋白突变体，损害转录 终止非编码RNA。这项工作将在酵母中进行，以利用强大的遗传工具 在这个系统中可用的，包括用突变体版本精确替换染色体基因的能力， 通过全面的基因筛选来确定新的调节因子。大规模保护 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理解，特别是多种形式的癌症， 当Paf 1C或染色质的表观遗传修饰被改变时出现。

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.