Analysis of the Essential Transcription Factors Spt5 and Spn1/Iws1

必需转录因子 Spt5 和 Spn1/Iws1 的分析

• 批准号: 9754185
• 负责人: FRED M. WINSTON
• 金额: $ 48.95万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754185
• 关键词: Address; Antisense RNA; Area; B-Lymphocytes; Binding; Biological Assay; Biological Models; Bypass; Cell Survival; Cells; ChIP-seq; Chromatin; Chromatin Structure; Complex; DNA; Defect; EAF1 gene; Elongation Factor; Essential Genes; Fission Yeast; Gap Junctions; Gene Expression; Genes; Genetic Transcription; Genetic study; Genomic approach; Growth and Development function; HIV; Health; Histones; Human; Human Biology; Immunoglobulin Class Switching; Impairment; Learning; Malignant Neoplasms; Maps; Methods; Molecular Chaperones; Mutation; Names; Nucleosomes; Organism; Pilot Projects; Play; Positioning Attribute; Process; Proteins; RNA; RNA Polymerase II; RNA Splicing; Regulation; Repression; Resolution; Role; Saccharomyces cerevisiae; Site; Structure; Study models; Suppressor Mutations; Testing; Transcript; Transcription Elongation; Transcription Initiation Site; Transcription Process; Transcriptional Elongation Factors; Yeasts; chimeric gene; experimental study; genetic approach; genetic information; genome-wide; histone methylation; histone modification; human disease; improved; in vivo; insight; mRNA capping; mutant; novel; promoter; protein function; transcription factor; transcription factor S-II; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT The long-term objectives of this project are to increase our understanding of eukaryotic transcription elongation. The focus of the proposed experiments is the analysis of two essential transcription elongation factors named Spt5 and Spn1 (also known as Iws1). Both factors have been implicated in human health. Spt5 is required for HIV gene expression, expression of NF-B-activated genes, and it is required for immunoglobulin class switching in B cells. Spn1 has been implicated in cancer. For both factors, there are several important areas where little is understood. Preliminary studies of Spt5 in the model system, S. pombe, have provided strong evidence that Spt5 is required for normal levels of transcription elongation genome-wide, with Spt5 required for RNA polymerase II to elongate past a barrier. These studies have also shown that Spt5 represses a novel class of antisense transcript that initiates at or near the barrier and that is synthesized across the 5' ends of the majority of genes. The proposed experiments in Specific Aim 1 address three related areas, continuing to use S. pombe as a model system. In Aim 1.1, two genome-wide approaches (MNase-seq and TSS-seq), will be employed to (1) test whether Spt5 controls chromatin structure and (2) to map the 5' ends of the antisense RNAs as a way to localize the antisense promoters and barriers with respect to nucleosome position. The results will provide new and comprehensive characterization of the role of Spt5 in transcription and chromatin structure. Aim 1.2 will focus on the sequences that have three possible functions: the barrier to elongation, the antisense promoter, and a possible site that stimulates elongation. Constructs will be made and tested to define the sequences required for these functions. The results will provide new insights into a previously unstudied aspect of eukaryotic transcription elongation. Aim 1.3 addresses the Spt5 protein itself, focusing on the isolation of new spt5 mutations that impair elongation. The positions and defects in the mutant proteins will reveal new understanding of Spt5 protein function. Specific Aim 2 proposes experiments in S. cerevisiae to understand the transcription factor Spn1. Aim 2.1 proposes two methods, RNA-seq and NET-seq, to characterize changes in transcription in Spn1-depleted cells, and ChIP-nexus, a high-resolution ChIP-seq method, to characterize chromatin association of specific factors and histone modifications after Spn1 depletion. Aim 2.2 proposes the isolation of mutations that bypass the need for Spn1, to understand the requirements for Spn1 in vivo. Mutations isolated in a pilot study have identified factors required for transcription elongation. These studies will provide new insights into the function of Spn1 within the transcription elongation complex. Together, these studies will greatly advance understanding of Spt5 and Spn1 and thereby increase understanding of transcription and co-transcriptional processes. As Spt5 and Spn1 are conserved, what is learned in studying these model systems will be directly relevant to human biology.

项目总结/摘要 这个项目的长期目标是增加我们对真核生物转录的理解 伸长率实验的重点是分析两个重要的转录延伸 Spt 5和Spn 1（也称为Iws 1）。这两个因素都与人类健康有关。Spt5 是HIV基因表达、NF-κ B激活基因表达所必需的， B细胞中的免疫球蛋白类别转换。Spn 1与癌症有关。对于这两个因素， 几个重要的领域，很少有人了解。Spt 5在模式系统中的初步研究，S.粟酒， 提供了强有力的证据表明Spt 5是全基因组转录延伸正常水平所必需的， 其中Spt 5是RNA聚合酶II伸长通过屏障所需的。这些研究还表明，Spt 5 抑制一类新的反义转录物，该转录物起始于屏障处或屏障附近， 在大多数基因的5'末端。具体目标1中拟议的实验涉及三个方面： 相关领域，继续使用S. pombe作为一个模型系统。在目标1.1中，两种全基因组方法 （MNase-seq和TSS-seq）将用于（1）测试Spt 5是否控制染色质结构和（2） 绘制反义RNA的5'末端，作为定位反义启动子和屏障的一种方式， 到核小体位置。这些结果将为Spt 5在以下方面的作用提供新的全面表征： 转录和染色质结构。目标1.2将重点关注具有三种可能功能的序列： 延伸屏障、反义启动子和刺激延伸的可能位点。构建体将 并进行测试，以确定这些功能所需的序列。结果将提供新的见解 真核生物转录延伸的一个以前未被研究的方面。目的1.3针对Spt 5蛋白 本身，集中在分离新的spt 5突变，损害延伸。的立场和缺陷， 突变蛋白将揭示Spt 5蛋白功能的新认识。具体目标2提出实验 in S.酿酒酵母，以了解转录因子Spn 1。目标2.1提出了两种方法，RNA-seq和 NET-seq，以表征Spn 1缺失细胞中转录的变化，ChIP-nexus，一种高分辨率的 ChIP-seq方法，以表征染色质缔合后的特定因子和组蛋白修饰， Spn 1耗竭。目的2.2提出了绕过Spn 1的突变的分离，以了解Spn 1的突变。 Spn 1在体内的要求。在一项初步研究中分离出的突变已经确定了 转录延伸这些研究将为Spn 1在细胞内的功能提供新的见解。 转录延伸复合体总之，这些研究将大大促进对Spt 5和Spn 1的理解。 从而增加对转录和共转录过程的理解。由于Spt 5和Spn 1是 因此，在研究这些模型系统中所学到的东西将与人类生物学直接相关。