Determining feedback mechanisms between cell cycle and cell fate in pluripotent cells

项目摘要/摘要 胚胎干细胞(ESCs)在医学上有很大的希望，因为它们可以在虚拟环境中繁殖 数量不限，可以产生任何疾病相关的细胞类型。胚胎干细胞有三种独特的细胞生物学 使它们有别于体细胞谱系的特征：(I)一个多潜能转录网络， 促进自身活性；(Ii)非典型的快速细胞周期，具有短的G1期，缺乏典型的限制 点；(Iii)对丝裂原活化蛋白激酶(MAPK)活性的非典型反应1-3。虽然人们非常关注 一直专注于通过转录网络维持ESC的多能性，研究ESC细胞 自行车网络在很大程度上是描述性的，这两个网络之间的机械联系尚未得到 探索过了。此外，一个关键但知之甚少的过程是ESCs如何使用MAPK途径来控制 退出多能性。我的长期职业目标是发现使胚胎干细胞 在两个相互冲突的命运中做出选择，即自我更新和命运承诺。这其中的驱动假说 有人提出，胚胎干细胞特异的细胞周期与转录多能性网络在功能上是联系在一起的 由MAPK信号的上游活动调节的相互正反馈。为了检验这一假设，我们 建议用遗传学和分子生物学的方法研究磷酸化位点在多能性因子中的作用 生化方法(Aim1)。我们将使用单细胞定量成像来测量细胞周期的动力学 在胚胎干细胞中表达多能性和细胞周期的报告。测试多能因子是否直接促进细胞 周期进展，我们建议通过重新调整CRISPR/Cas9技术的用途来开发一种新的方法 检测体内特定转录因子结合位点(AIM2)的功能。要确定MAPK如何 信号调节退出多能性，我们将结合蛋白质工程和定量 磷酸蛋白质组学，以发现该途径的新靶点(Aim3)。完成这些目标将揭示 ESCs在对立的命运之间做出选择的机制，即自我更新和命运承诺。 在该奖项的培训阶段(K99)，我计划利用Skotheim实验室的量化洞察力来推动 在我的实验室里，一个跨学科的研究计划，研究多能细胞的细胞命运。为此，我已经确立了 与维尔尼希的实验室(斯坦福)、齐的实验室(斯坦福)和马切克的实验室(德国图宾根大学)合作 这将极大地促进我的研究项目的进展。为过渡到独立的 调查员，我将参加职业培训课程，如未来学院系列，由 斯坦福大学。除了代表着基础生物科学的重要进步之外，我们的 机制洞察力可能促进再生医学中胚胎干细胞的繁殖和谱系分化。

Project Summary/Abstract Embryonic stem cells (ESCs) hold great promise for medicine because they can be propagated to virtually unlimited numbers and can generate any disease relevant cell type. ESCs have three unique cell biological features that make them distinct from somatic cell lineages: (i) A pluripotency transcriptional network that promotes its own activity; (ii) An atypically rapid cell cycle with short G1 phase that lacks a typical restriction point; (iii) An atypical response to Mitogen Activated Protein Kinase (MAPK) activity1–3. While a lot of attention has focused on the maintenance of ESC pluripotency by a transcriptional network, research on the ESC cell cycle network has been largely descriptive, and the mechanistic links between the two networks have yet to be explored. In addition, a critical but poorly understood process is how ESCs use MAPK pathway to control the exit from pluripotency. My long-term career goal is to discover the molecular mechanisms that allows ESCs to choose between two conflicting fates, i.e, self-renewal vs. fate commitment. The driving hypothesis of this proposal is that the ESC-specific cell cycle is functionally linked with the transcriptional pluripotency network by mutual, positive feedback that is regulated by upstream activity of MAPK signaling. To test this hypothesis, we propose to investigate the function of phosphorylation sites on the pluripotency factors using both genetic and biochemical methods (Aim1). We will employ single cell quantitative imaging to measure dynamics of cell cycle in ESCs expressing reporters of pluripotency and cell cycle. To test if pluripotency factors directly promote cell cycle progression, we are proposing to develop a novel method by repurposing the CRISPR/Cas9 technology to examine the function of specific transcription factor binding sites in vivo (Aim2). To determine how MAPK signaling regulates exit from pluripotency, we will combine protein engineering and quantitative phosphoproteomics to uncover novel targets of this pathway (Aim3). Completion of these aims will reveal the mechanisms by which ESCs choose between opposing fates, i.e. self-renewal vs. fate commitment. During the training phase of this award (K99), I plan to leverage quantitative insight of Skotheim’s lab to advance an interdisciplinary research plan to study cell fate in pluripotent cells in my lab. To this end, I have established collaboration with Wernig’s lab (Stanford), Qi’s lab(Stanford) and Macek’s lab(Tubingen University, Germany) that will greatly facilitate the progress of my research project. To prepare for transition to an independent investigator, I will take part in career training courses such as The Future Faculty Series that are offered by Stanford University. In addition to representing an important advance in basic biological sciences, our mechanistic insight may facilitate propagation and lineage differentiation of ESCs for regenerative medicine.