Molecular Regulation of Stem Cell Quiescence

项目摘要 干细胞负责体内的稳态和修复身体中的大多数组织。许多人口 的干细胞持续处于静止状态，直到被刺激进入细胞周期，增殖并分化为 特定组织的功能细胞。近年来，我们小组和其他人的工作引起了人们的注意， 静态肌肉干细胞（MuSC）的几个意想不到的特征，其中许多是由 其他静止的干细胞群体。这些包括积极维持细胞静止，独特的 静止和活化干细胞中的代谢和能量机制，以及大量干细胞的存在， 没有检测到蛋白质产物的转录物。后一种观察提出了三个主要问题， 1）MuSC（或任何其他干细胞群体）的转录谱是否与细胞的分化有关？ 与通过荧光激活细胞分选（FACS）分离和纯化的细胞相似？ 2)什么是转录后机制，负责缺乏蛋白质产品时， 转录本存在于静止细胞中3)这些蛋白质积累的后果是什么 产品在静止的细胞，有必要的机制，以防止这种积累？ 为了解决这些问题，本提案分为以下三个具体目标。目标1：研究 静态和激活MuSC转录组的动力学。我们将使用标记新生RNA的方法， 体内（使用4-硫尿嘧啶（TU）和5-乙炔基尿苷（EU）），随后进行标记的转录物纯化和RNA- seq以评估体内和离体MuSC转录动力学。我们还将使用RNA-seq分析转录本， 固定MuSC以评估体内转录物丰度。目的2：研究小鼠骨髓基质干细胞的翻译组和蛋白质组 体内和离体。我们将分离核糖体相关转录物（使用RiboTag小鼠），然后 RNA-seq和OP-嘌呤霉素标记标记的蛋白质，然后进行质谱分析以评估转录物 与多聚核糖体相关并在体内静止MuSC中翻译成可检测的蛋白质 和离体。我们还将评估MuSC在体内和离体过程中的蛋白质翻译 activation.目的3：了解MyoD翻译的调控和异常MyoD的后果 在静止MuSC中的蛋白表达。根据初步数据，我们将检验Staufen 1的假设 抑制静止细胞中MyoD转录本的翻译，我们将分析其功能性。 通过抑制这些抑制机制， 基因上和使用siRNA方法。这些研究的总体目标是提供一个更准确的 评估静止干细胞的体内状态，并更详细地了解其分子生物学特性。 维持干细胞静止并同时引发细胞活化的机制， 分化

PROJECT SUMMARY Stem cells are responsible for homeostasis and repair of most of the tissues in the body. Many populations of stem cells persist in a quiescent state until stimulated to enter the cell cycle, proliferate, and differentiate into functional cells of the particular tissue. In recent years, work from our group and others has drawn attention to several unexpected characteristics of quiescent muscle stem cells (MuSCs), many of which are shared by other quiescent stem cell populations. These include the active maintenance of cellular quiescence, unique metabolic and energetic mechanism in quiescent and activating stem cells, and the presence of large numbers of transcripts for which no protein product is detected. This latter observation raises three major questions that are the focus of this proposal: 1) Is the transcriptional profile of MuSCs (or any other stem cell population) in vivo similar to that of cells that have been isolated and purified by fluorescence-activated cell sorting (FACS)? 2) What are the post-transcriptional mechanisms that are responsible for the absence of protein products when transcripts are present in the quiescent cells? 3) What are consequences of accumulation of those protein products in quiescent cells that necessitate mechanisms to prevent such an accumulation? To address these issues, this proposal is divided into three Specific Aims as follows. Aim 1: To study the dynamics of the quiescent and activating MuSC transcriptome. We will use methods to label nascent RNA in vivo (using 4-thiouracil (TU) and 5-ethynyl uridine (EU)) followed by labelled transcript purification and RNA- seq to assess MuSC transcript dynamics in vivo and ex vivo. We will also profile transcripts using RNA-seq of fixed MuSCs to assess transcript abundance in vivo. Aim 2: To study the translatome and proteome of MuSCs in vivo and ex vivo. We will isolate ribosome-associated transcripts (using the RiboTag mouse) followed by RNA-seq and OP-puromycin labelling of labelled proteins followed by mass spectrometry to assess transcripts that are associated with the polyribosome and translated into detectable proteins in quiescent MuSCs in vivo and ex vivo. We will also assess protein translation in MuSCs in vivo and ex vivo during the process of MuSC activation. Aim 3: To understand the regulation of MyoD translation and the consequences of aberrant MyoD protein expression in quiescent MuSCs. Based on Preliminary Data, we will test the hypothesis that Staufen1 suppresses the translation of the MyoD transcript in quiescent cells, and we will analyze the functional consequences of MyoD expression in quiescent MuSCs by inhibiting those suppressive mechanisms genetically and using an siRNA approach. The overall goals of these studies are to provide a more accurate assessment of the in vivo state of the quiescent stem cell and to understand in greater detail the molecular mechanisms that maintain stem cell quiescence and at the same time prime the cell for activation and differentiation.