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

项目总结/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 具有多个步骤，可以分别调节。启动子近端RNA Pol II暂停最近已被发现。 被描述为一个广泛的转录限速步骤和一个可能的基因调控点。研究 破坏小鼠和小鼠胚胎干细胞中的关键停顿因子负延伸因子-B（Nelf-b） 细胞（mES）的研究突出了其在发育中的重要作用以及与FGF/ERK信号转导的交叉作用。 然而，目前还不清楚停顿在哺乳动物发育中起什么作用。此外，尚不清楚如何 暂停可能会影响FGF/ERK信号传导的特定靶点，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一个分子的理解，基因调控如何可以 在转录过程中启动子近端暂停的水平上实现。本建议的目的是 为了机械地确定在早期哺乳动物中上胚层多能性转换期间暂停的作用， 发展这一目标将通过Nelf-b-/-的详细表型和转录组学分析来实现。 胚胎在几个早期发育阶段，以分析多能性转换（目标1）。为了扩展这些 结果和高分子和时间分辨率的询问暂停，Nelf-b-degron mES细胞系具有 建立了测试不同信号下mES细胞暂停丢失的即时和直接影响 条件我将利用新生RNA-seq技术和定向分化方法来建立一个 胚胎中停顿、FGF/ERK转录激活和多能性转换之间的机制联系 和mES细胞（Aim 2）。我的中心假设是停顿是外胚层多能性所必需的 通过直接调节FGF/ERK转录靶点在小鼠发育中的过渡。 这个项目的预期结果是揭示暂停和信号之间的机械联系 转导，解释暂停在哺乳动物发育中的作用。它有望产生新的见解基因 暂停级别的监管。停顿和信号之间的联系对其他生物学意义重大。 背景考虑暂停的流行和信号转导的必要性，以执行基本的 在发育过程中，细胞功能超出了对FGF/ERK的反应。此外，鉴于FGF/ERK信号传导 是发育性疾病和恶性肿瘤中最重要的失调途径之一， 这一途径的分子机制将产生新的见解，以治疗靶向它。

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.