Dissecting the Role of RNA Pol II Pausing in Early Mammalian Development

项目摘要/摘要 转录调控是细胞类型和功能多样性的基础。转录过程本身 有多个步骤，可以单独进行监管。启动子-近端RNA Pol II暂停最近 被描述为广泛存在的转录限速步骤和可能的基因调节点。研究 破坏小鼠和小鼠胚胎干细胞中的临界暂停因子负延长因子-B 细胞(MES)强调其在发育中的重要作用，并与成纤维细胞生长因子/ERK信号转导相互作用。 然而，目前还不清楚停顿在哺乳动物发育中扮演着什么角色。此外，目前还不清楚如何 停顿可能会影响成纤维细胞生长因子/ERK信号的特定靶点，以及这种串扰是否与体内相关。 这项提案的长期目标是建立对基因调控方式的分子理解。 在转录过程中，在启动子-近端暂停的水平上实现。本提案的目标是 从机制上确定停顿在早期哺乳动物外胚层多能转化中的作用 发展。这一目标将通过对NELF-b-/-进行详细的表型和转录分析来实现。 分析几个早期发育阶段的胚胎，以分析多能性转变(目标1)。在此基础上进行扩展 结果和询问在高分子和时间分辨率下暂停，NELF-b-degron MES细胞系具有 被建立来测试MES细胞在不同信号下暂停丢失的即时和直接影响 条件。我将利用新兴的rna-seq技术和定向分化方法来构建 胚胎中暂停、成纤维细胞生长因子/ERK转录激活和多能性转换之间的机制联系 和MES细胞(目标2)。我的中心假设是，外胚层的多能性需要暂停。 通过直接调控成纤维细胞生长因子/ERK转录靶标实现小鼠发育的转变。 这个项目的预期结果是揭示停顿和信号之间的机械联系 转导，解释停顿在哺乳动物发育中的作用。它有望对基因产生新的见解 暂停层面的监管。停顿和信号之间的联系对其他生物来说是非常重要的 考虑到停顿的流行和信号转导执行基本操作的必要性的背景 在发育过程中，细胞功能超出了对成纤维细胞生长因子/ERK的反应。此外，鉴于成纤维细胞生长因子/ERK信号转导 是发育疾病和恶性肿瘤中最严重的失调途径之一，对 这一途径的分子机制将为治疗靶向提供新的见解。

PROJECT SUMMARY/ABSTRACT Transcription regulation underlies the diversity of cell types and functions. The process of transcription itself has multiple steps that may be regulated separately. Promoter-proximal RNA Pol II pausing has recently been described as a widespread rate-limiting step of transcription and a possible point of gene regulation. Studies disrupting the critical pausing factor Negative elongation factor-B (Nelf-b) in mouse and mouse embryonic stem cells (mES) highlighted its essential role in development and a cross-talk with FGF/ERK signaling transduction. However, it is unclear what role pausing plays in mammalian development. Furthermore, it is unclear how pausing may affect specific targets of FGF/ERK signaling, and whether this cross-talk is relevant in vivo. The long-term goal of this proposal is to establish a molecular understanding of how gene regulation can be achieved at the level of promoter-proximal pausing during transcription. The objective of the present proposal is to mechanistically determine the role of pausing during epiblast pluripotency transitions in early mammalian development. This objective will be achieved by a detailed phenotypic and transcriptomic analysis of Nelf-b-/- embryos at several early developmental stages to analyze pluripotency transitions (Aim 1). To expand on these results and interrogate pausing at a high molecular and temporal resolution, a Nelf-b-degron mES cell line has been established to test the immediate and direct effects of pausing-loss in mES cells under different signaling conditions. I will utilize nascent RNA-seq techniques and directed differentiation approaches to build a mechanistic link between pausing, FGF/ERK transcriptional activation, and pluripotency transitions in embryos and mES cells (Aim 2). My central hypothesis is that pausing is required for epiblast pluripotency transitions in mouse development via direct regulation of FGF/ERK transcriptional targets. The expected outcome of this project is to uncover the mechanistic link between pausing and signal transduction, explain the role of pausing in mammalian development. It promises to yield novel insights into gene regulation at the pausing level. The link between pausing and signaling is of high importance to other biological contexts considering the prevalence of pausing and necessity of signal transduction to perform fundamental cellular functions beyond responses to FGF/ERK in development. Furthermore, given that FGF/ERK signaling is amongst the top dysregulated pathways in developmental diseases and malignancies, insights into the molecular mechanisms of this pathway will yield novel insights to therapeutically target it.