Analysis of conserved eukaryotic transcription elongation factors

项目摘要/摘要 这个项目的长期目标是增加我们对真核转录的理解。 延伸性，重点是组蛋白伴侣。组蛋白伴侣蛋白控制着细胞的组装和拆卸 核小体在转录、复制和修复过程中。拟议的实验解决了保守的 组蛋白伴侣Spt6，以酿酒酵母为模型系统。Spt6是保守的 它与发育控制和癌症有关联。以前的分析 Spt6已经证明它是酵母和酵母中广泛需要的转录和染色质结构所必需的。 哺乳动物细胞。虽然已经确定Spt6与组蛋白、RNA聚合酶II和其他 蛋白质，其发挥作用的机制尚不清楚。在具体目标1中提议的实验将 说明Spt6与另外两个基本和保守的组蛋白伴侣蛋白Spn1/Iws1和Spn1/Iws1的相互作用 事实。初步研究表明，spt6突变体spt6-yw会损害 Spt6和Spn1在生长、转录和染色质结构上都发生了变化。确定了其他研究 弥补spt6-yw突变缺陷的抑制子突变。其中几个抑制子突变 在保守的事实表面上造成聚集的变化。Aim 1.1检验SPT6-YW和其 抑制物通过改变转录延伸复合体来控制转录和染色质结构 和组蛋白修饰。这将通过一系列芯片序列实验进行分析。AIM 1.2研究了 弥补Spt6缺陷的事实上的变化。这些结果将为我们提供新的理解 组蛋白伴侣之间的功能关系，并为为什么它们中的许多是至关重要的提供了洞察力 在转录过程中。具体目标2集中在Spt6与组蛋白的结合，组蛋白是组蛋白的重要功能 监护人。这一目的是为了测试Spt6在其高度酸性和 无序的N-末端结构域。AIM 2.1将分离N-末端区域中存在缺陷的spt6突变体 功能。Aim 2.2将使用这些突变体在体外定义Spt6与组蛋白的结合。AIM 2.3将解决关键问题 关于Spt6-组蛋白在转录过程中的相互作用。总之，这些实验将阐明一种 Spt6的基本功能。具体目标3涉及Spt6在控制 基因组完整性，因为spt6突变体表现出基因组不稳定的表型。拟议中的实验将检验 Spt6是否是通过控制染色质结构、转录或分解来维持基因组稳定性所必需的 转录-复制冲突。实验将分析对基因组有贡献的rna：dna杂交体。 不稳定，双链DNA断裂，并将测试DNA复制也需要Spt6的模型 作为抄本。这些结果将提供对基因组稳定性控制的新理解，这是一个基本的 并保存对人类健康重要的过程。

PROJECT SUMMARY/ABSTRACT The long-term objectives of this project are to increase our understanding of eukaryotic transcription elongation, with a focus on histone chaperones. Histone chaperones control the assembly and disassembly of nucleosomes during transcription, replication, and repair. The proposed experiments address the conserved histone chaperone Spt6, using the yeast Saccharomyces cerevisiae, as a model system. Spt6 is conserved and its human counterpart has been implicated in developmental control and in cancer. Previous analysis of Spt6 has demonstrated that it is broadly required for transcription and chromatin structure in both yeast and mammalian cells. While it is established that Spt6 interacts with histones, RNA polymerase II, and other proteins, the mechanisms by which it functions are unknown. The proposed experiments in Specific Aim 1 will address the interactions of Spt6 with two other essential and conserved histone chaperones, Spn1/Iws1 and FACT. Preliminary studies have shown that an spt6 mutant, spt6-YW, that impairs the physical interaction of Spt6 with Spn1, has changes in growth, transcription and chromatin structure. Additional studies identified suppressor mutations that compensate for spt6-YW mutant defects. Several of these suppressor mutations cause clustered changes in a conserved surface of FACT. Aim 1.1 tests the model that spt6-YW and its suppressors control transcription and chromatin structure via alterations of the transcription elongation complex and histone modifications. This will be assayed by a set of ChIP-seq experiments. Aim 1.2 studies the changes in FACT that compensate for the Spt6 defect. These results will provide new understanding of the functional relationships among histone chaperones and provide insights into why so many of them are vital during transcription. Specific Aim 2 focuses on Spt6 binding to histones, an essential function for all of histone chaperones. This aim tests the model that Spt6 has multiple histone binding sites in its highly acidic and disordered N-terminal domain. Aim 2.1 will isolate spt6 mutants in the N-terminal region that are defective for function. Aim 2.2 will use these mutants to define Spt6-histone binding in vitro. Aim 2.3 will address key issues regarding Spt6-histone interactions during transcription. Together, these experiments will elucidate an essential function of Spt6. Specific Aim 3 addresses a related but distinct role for Spt6, in the control of genome integrity, as spt6 mutants display genome instability phenotypes. The proposed experiments will test whether Spt6 is required for genome stability by the control of chromatin structure, transcription, or resolving transcription-replication conflicts. Experiments will assay RNA:DNA hybrids, which contribute to genome instability, double-strand DNA breaks, and will test the model that Spt6 is required for DNA replication as well as transcription. The results will provide new understanding of the control of genome stability, a fundamental and conserved process important for human health.