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

项目总结/摘要 胚胎干细胞（ESC）在医学上有很大的前景，因为它们可以繁殖到几乎所有的细胞。 无限数量，可以产生任何疾病相关的细胞类型。ESC具有三种独特的细胞生物学特性， 使它们不同于体细胞谱系的特征：（i）多能性转录网络， 促进自身活性;（ii）具有短G1期的快速细胞周期，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。虽然很多注意力 一直专注于通过转录网络维持ESC多能性，对ESC细胞的研究 循环网络在很大程度上是描述性的，两个网络之间的机械联系还有待进一步研究。 探讨了此外，一个关键但知之甚少的过程是ESCs如何使用MAPK途径来控制细胞的增殖， 退出多能性。我的长期职业目标是发现胚胎干细胞的分子机制 在自我更新与命运承诺这两种相互冲突的命运中做出选择。这个驱动假设 提出ESC特异性细胞周期通过以下方式与转录多能性网络功能性地连接： 相互的正反馈，由MAPK信号传导的上游活性调节。为了验证这个假设，我们 建议使用遗传和 生物化学方法（Aim 1）。我们将采用单细胞定量成像技术来测量细胞周期的动态变化 在表达多能性和细胞周期报告基因的ESC中。为了测试多能性因子是否直接促进细胞增殖， 我们建议通过重新利用CRISPR/Cas9技术来开发一种新方法， 检测体内特异性转录因子结合位点（Aim 2）的功能。为了确定MAPK如何 信号调节退出从多能性，我们将联合收割机蛋白质工程和定量 磷酸化蛋白质组学来揭示该途径的新靶点（Aim 3）。这些目标的实现将揭示 ESCs在相反命运之间选择的机制，即自我更新与命运承诺。 在此奖项的培训阶段（K99），我计划利用Skotheim实验室的定量洞察力， 一个跨学科的研究计划，在我的实验室里研究多能细胞的细胞命运。为此，我已建立了 与Wernig实验室（斯坦福大学）、Qi实验室（斯坦福大学）和Macek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。为过渡到独立国家做准备 调查员，我将参加职业培训课程，如未来教师系列，是由 斯坦福大学。除了代表基础生物科学的重要进展外，我们的 机械的洞察力可以促进再生医学的ESC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.