Analysis of the Essential Transcription Factors Spt5 and Spn1/Iws1

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。 伸长率。拟议的实验的重点是分析两个基本的转录延伸 名为Spt5和Spn1(也称为Iws1)的因子。这两个因素都与人类健康有关。SPT5 是艾滋病毒基因表达所必需的，是表达NF-B激活的基因所必需的，并且它是 B细胞中免疫球蛋白类的转换。Spn1被认为与癌症有关。对于这两个因素，都有 几个人们知之甚少的重要领域。Spt5基因在模型体系中的初步研究 已经提供了强有力的证据表明，Spt5是全基因组正常水平的转录延伸所必需的， RNA聚合酶II需要Spt5才能延长通过屏障。这些研究还表明，Spt5 抑制一类新的反义转录物，该反义转录物在屏障处或附近启动，并被合成 在大多数基因的5‘端。在具体目标1中提出的实验涉及三个问题 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)，将用于(1)测试Spt5是否控制染色质结构和(2) 绘制反义RNA的5‘端的图谱作为一种尊重地定位反义启动子和障碍的方法 转到核小体位置。这些结果将为Spt5在人类免疫系统中的作用提供新的、全面的表征 转录和染色质结构。AIM 1.2将重点介绍具有三种可能功能的序列： 伸长的障碍，反义启动子，以及刺激伸长的可能位置。构造将 进行制作和测试，以确定这些功能所需的序列。研究结果将提供新的见解 变成了以前从未研究过的真核转录延伸的一个方面。AIM 1.3解决了Spt5蛋白 本身，专注于分离损害伸长的新spt5突变。中的位置和缺陷 突变蛋白将揭示Spt5蛋白功能的新认识。具体目标2建议进行实验 了解酿酒酵母中转录因子Spn1。Aim 2.1提出了两种方法，RNA-seq和 Net-seq，以表征Spn1缺失细胞中转录的变化，以及芯片连接，一种高分辨率 ChIP-SEQ方法，以表征特定因子和组蛋白修饰后的染色质关联 Spn1耗尽。Aim 2.2建议分离绕过Spn1的突变，以了解 体内对Spn1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Spn1在体内的功能提供新的见解。 转录延伸复合体。总之，这些研究将极大地促进对Spt5和Spn1的理解 从而增加对转录和共转录过程的理解。因为Spt5和Spn1是 保守地说，在研究这些模型系统中学到的东西将与人类生物学直接相关。

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.