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重要方面，并且对于细胞对环境变化的反应至关重要。在人类中，转录缺陷通常会导致疾病。因此，阐明转录机制对于理解人类生物学非常重要。该提案重点关注两种蛋白质：Spt6 和 Iws1，它们从酵母到人类都是保守的，对于生存至关重要，并且与癌症有关。 Spt6 和 Iws1 作为全局转录调节因子，在多个水平上控制基因表达。尽管进行了大量研究，但 Spt6/Iws1 调节转录的确切机制仍知之甚少。为了研究 Spt6 和 Iws1 在基因表达中发挥的作用，提出了以下具体目标： 目标 1. 阐明 Iws1 蛋白保守基序的体内作用。使用保守残基的定点诱变、条件致死突变体的分离以及蛋白质消耗方法，将分析Iws1的功能。拟议的突变体分析将揭示 Iws1 在转录、mRNA 加工、蛋白质-蛋白质相互作用以及在全基因组染色质中招募关键因子方面的功能。目标 2. 分离并表征 spn1Δ 和 spt6Δ 无效等位基因的抑制子。将进行 iws1Δ 和 spt6Δ 抑制因子的遗传筛选，以发现可以补偿 Spt6 和 Iws1 损失的新细胞活性和途径。已识别抑制因子的详细表征，包括在试点筛选中已识别的几个突变，将揭示这些突变体挽救 Iws1 缺失的机制，并将扩大我们对 Spt6 和 Iws1 在基因表达中作用的理解。总而言之，拟议的研究将进一步加深我们目前对 Spt6 和 Iws1 在转录调控中基本功能的理解。我们的长期目标是了解事件的级联以及每个活动在基因表达控制中的贡献。相关性：基因表达失调与肿瘤发生密切相关。鉴于所研究因素的保守性及其在发育和肿瘤发生中的作用，我们使用酵母作为模式生物的研究将广泛适用于其他真核生物。该研究项目对抗癌药物的发现具有长期影响。

 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.