Analysis of the Essential Transcription Factors Spt5 and Spn1/Iws1

必需转录因子 Spt5 和 Spn1/Iws1 的分析

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要该项目的长期目标是增加我们对真核转录的理解所提出的实验的重点是分析两个基本的转录延伸。 Spt5 和 Spn1（也称为 Iws1）这两个因子都与人类健康有关。 HIV 基因表达、NF-κB 激活基因表达以及 B 细胞中的免疫球蛋白类别转换与癌症有关。模型系统 S. pombe 中对 Spt5 的初步研究知之甚少。提供了强有力的证据表明 Spt5 是全基因组转录延伸正常水平所必需的， RNA 聚合酶 II 需要 Spt5 才能延伸穿过屏障。抑制一类新型反义转录物，该转录物在屏障处或附近启动并合成具体目标 1 中提出的实验涉及三个基因的 5' 端。相关领域，继续使用粟酒裂殖酵母作为模型系统。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 Spt5 是否控制染色质结构以及 (2) 绘制反义 RNA 的 5' 末端，作为定位反义启动子和屏障的方法该结果将为Spt5在核小体中的作用提供新的、全面的表征。目标 1.2 将重点关注具有三种可能功能的序列：延伸障碍、反义启动子和刺激延伸的可能位点。进行并测试以定义这些功能所需的序列，结果将提供新的见解。 Aim 1.3 深入研究了真核转录延伸的一个先前未研究的方面。本身，重点关注损害伸长的新 spt5 突变的分离。突变蛋白将揭示对 Spt5 蛋白功能的新认识。具体目标 2 提出了实验。目的 2.1 提出了两种方法：RNA-seq 和 RNA-seq。 NET-seq，用于表征 Spn1 耗尽细胞中转录的变化，以及 ChIP-nexus，一种高分辨率 ChIP-seq 方法，用于表征特定因素和组蛋白修饰后染色质的关联 Spn1 耗尽。目标 2.2 提出隔离不需要 Spn1 的突变，以了解初步研究中分离出的 Spn1 突变的要求已确定了所需的因素。这些研究将为 Spn1 在转录延伸中的功能提供新的见解。这些研究将极大地促进对 Spt5 和 Spn1 的理解。并增加对转录和共转录过程的理解。如果保守的话，研究这些模型系统所学到的知识将与人类生物学直接相关。