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.

项目摘要/摘要 该项目的长期目标是增加我们对真核转录的理解 伸长。提出的实验的重点是对两个必需转录伸长的分析 名为SPT5和SPN1的因素（也称为IWS1）。这两个因素都在人类健康中暗示。 SPT5 是HIV基因表达，NF-B激活基因的表达所必需的，这是必需的 B细胞中的免疫球蛋白类切换。 SPN1在癌症中隐含。对于这两个因素，都有 几乎没有理解的几个重要领域。 SPT5在模型系统中的初步研究，S。Pombe， 已经提供了有力的证据，表明正常的转录伸长基因组水平需要SPT5， RNA聚合酶II所需的SPPT5以使屏障延伸。这些研究还表明SPT5 压抑一类新型的反义转录本，该转录本在屏障处或附近启动并合成 在大多数基因的5'端。特定目标1中的拟议实验3地址 相关领域，继续使用S. Pombe作为模型系统。在AIM 1.1中，两种全基因组方法 （MNase-Seq和TSS-Seq），将雇用（1）测试SPT5是否控制染色质结构和（2） 将反义RNA的5末端映射为与尊重的反义启动子和障碍物定位的一种方式 到核小体位置。结果将为SPT5在 转录和染色质结构。 AIM 1.2将专注于具有三个可能功能的序列： 伸长的障碍，反义启动子以及刺激伸长的可能位点。构造将 进行和测试以定义这些功能所需的序列。结果将提供新的见解 进入真核转录伸长的先前未研究的方面。 AIM 1.3解决SPT5蛋白 本身，重点是隔离损害延伸的新SPT5突变。位置和缺陷 突变蛋白将揭示对SPT5蛋白功能的新理解。具体目标2提案实验 在酿酒酵母中了解转录因子SPN1。 AIM 2.1提案两种方法RNA-Seq和 net-seq，以表征SPN1耗尽细胞中转录的变化，以及高分辨率的Chip-Nexus chip-seq方法，以表征特定因素和Hisstone修饰的染色质关联 SPN1消耗。 AIM 2.2提出绕过SPN1需求的突变的隔离，以了解 SPN1在体内的要求。试点研究中孤立的突变已经确定了所需的因素 转录伸长。这些研究将提供有关SPN1功能的新见解 转录伸长复合物。这些研究将共同​​提高对SPT5和SPN1的理解 从而增加对转录和共转录​​过程的了解。因为SPT5和SPN1是 保守的，研究这些模型系统的知识将与人类生物学直接相关。