Defining the cellular functions of the conserved transcription complex Spt6/Iws1 in the control of gene expression

 描述(由申请人提供)：转录调控是细胞生长和分化的一个基本方面，也是细胞对环境变化的反应中的关键。在人类中，转录缺陷往往会导致疾病。因此，阐明转录机制对于理解人类生物学具有重要意义。这项建议重点关注两种蛋白质Spt6和Iws1，这两种蛋白质从酵母到人类都是保守的，对生存至关重要，并且与癌症有关。Spt6和Iws1作为全球转录调控因子，在多个水平上调控基因表达。尽管进行了深入的研究，但Spt6/Iws1调控转录的确切机制尚不清楚。为了研究Spt6和Iws1在基因表达中的作用，我们提出了以下目标：目的1.阐明Iws1蛋白的保守基序在体内的作用。使用保守残基的定点突变、条件致死突变体的分离和蛋白质耗竭的方法，将分析Iws1的功能。所提出的突变分析将揭示Iws1在转录、mRNA处理、蛋白质-蛋白质相互作用以及在整个基因组中招募关键因子到染色质中的功能。目的2.分离和鉴定spn1Δ和spt6Δ缺失等位基因的抑制子。将对iws1Δ和spt6Δ失活抑制物进行基因筛查，以发现新的细胞活动和途径，以弥补spt6和iws1的丢失。对已确定的抑制子的详细表征，包括在试点筛选中已经发现的几个突变，将有助于阐明这些突变体挽救Iws1缺失的机制，并将扩大我们对Spt6和Iws1在基因表达中的作用的理解。总之，拟议的研究将进一步加深我们目前对Spt6和Iws1在转录调控中的基本功能的理解。我们的长期目标是了解一系列事件以及每一项活动在基因表达控制中的贡献。相关性：基因表达的放松与肿瘤的发生密切相关。鉴于所研究因子的保守性及其在发育和肿瘤发生中的作用，我们以酵母为模式生物的研究将广泛适用于其他真核生物。这一研究项目对抗癌药物的发现具有长远的意义。

 DESCRIPTION (provided by applicant): Transcriptional regulation is an essential aspect of cell growth and differentiation, as well as critical in the response of cells to environmental changes. In humans, defects in transcription often lead to disease. Therefore, the elucidation of transcriptional mechanisms is important for understanding human biology. This proposal focuses on two proteins, Spt6 and Iws1, that are conserved from yeast to humans, that are essential for viability, and that have been implicated in cancer. Spt6 and Iws1 function as global transcriptional regulators and control gene expression at multiple levels. Despite intensive studies, the exact mechanisms by which Spt6/Iws1 regulates transcription is poorly understood. To investigate the roles that Spt6 and Iws1 play in gene expression the following specific aims are proposed: Aim 1. Elucidate the in vivo roles of the conserved motifs of the Iws1 protein. Using site- directed mutagenesis of the conserved residues, isolation of conditionally lethal mutants, and a protein depletion approach, the functions of Iws1 will be analyzed. The proposed mutant analysis will reveal the functions of Iws1 in transcription, mRNA processing, protein-protein interactions, and recruitment of critical factors to chromatin genome-wide. Aim 2. Isolate and characterize suppressors of spn1Δ and the spt6Δ null alleles. A genetic screen for suppressors of iws1Δ and spt6Δ inviability will be performed to discover novel cellular activitie and pathways that can compensate for loss of Spt6 and Iws1. Detailed characterization of the identified suppressors, including several mutations already identified in the pilot screen, will shed light on the mechanisms by which these mutants rescue absence of Iws1 and will expand our understanding on the roles of Spt6 and Iws1 in gene expression. Altogether, the proposed studies will further our current understanding of the essential functions of Spt6 and Iws1 in transcriptional regulation. Our long-term goal is to understand the cascade of events and the contribution of each activity in gene expression control. Relevance: Deregulation of gene expression is tightly linked to tumorigenesis. Given the conserved nature of the studied factors and their roles in development and oncogenesis, our studies using yeast as model organisms will be broadly applicable to other eukaryotes. This research project has a long-term implication in anticancer drug discovery.