Execution of cell fate choice by cooperative gene activities

更广泛的研究背景：我们对小鼠胚胎干细胞从naïve到形成多能性身份的研究表明，这种模型细胞命运的决定是由多个合作和部分冗余的过程控制的。没有单因素突变，而只有双重信号抑制或多因素的遗传密码缺失足以锁定naïve身份，这一事实突出了这种冗余性。因此，专注于单个基因的遗传方法肯定会错过控制细胞命运的重要冗余连接功能。目的：该项目的中心目标是通过完成两个中心目标，为冗余遗传控制下的过程提供基本见解，这些过程对于适当的发育进展至关重要：目的1：使用计算和新型组合筛选方法确定naïve多能性退出过程中的冗余和合作基因活动。目的2：研究协同细胞功能如何使和执行naïve多能性退出的分子机制。方法：我们开发了一种创新的高通量兼容3D细胞聚集屏幕，以识别以合作方式起作用的相互作用基因/过程对。这种设置解决了划分复杂突变谱和表型的问题，同时允许高通量。分子机制将通过应用遗传学、生物化学和网络分析方法来揭示。这些方法将功能遗传学数据与敲除的高维分子谱和分化时间过程信息相结合。创新水平：从概念上讲，两个合作且部分冗余的基因的组合活性单独只导致弱表型，但如果共同耗尽导致完全分化阻断，则在功能上比单个基因的个体活性更重要，单个基因在KO时表现出强烈的退出延迟。因此，我们有很多东西要发现，只有开始剖析复杂的基因相互作用，我们才能真正理解细胞命运的决定。我们建议我们的项目可以揭示完全未知的分化过程的调控控制，这是由于单因素遗传方法的冗余而无法访问的。我们设想这个项目为执行组合遗传学设定一个标准。

Wider research context:Our work on the exit of mouse embryonic stem cells from a naïve to a formative pluripotent identity showed that this model cell-fate decision is controlled by multiple cooperative and partially redundant processes. This redundancy is highlighted by the fact that no single factor mutation, but only dual signalling inhibition or genetic codeletion of multiple factors is sufficient to lock in naïve identity. Hence, genetic approaches focusing on single genes, will certainly miss important redundantly wired functions that control cell fate.Objectives:The central goal of this project is to provide fundamental insights into the processes under redundant genetic control that are crucial for proper developmental progression by completion of two central Aims:Aim 1: To identify redundant and cooperative gene activities during the exit from naïve pluripotency using computational and novel combinatorial screening approaches.Aim 2: To investigate the molecular mechanisms of how cooperative cellular functions enable and execute the exit from naïve pluripotency.Approach:We have developed an innovative high-throughput compatible 3D cell-aggregate screen to identify pairs of interacting genes/processes that act in a cooperative manner. This setup solves the problem of compartmentalizing complex mutation profiles and phenotype, while allowing high throughput. Molecular mechanisms will be revealed through application of genetic, biochemical and network-analysis approaches. These methods will integrate functional genetics data with high-dimensional molecular profiling of knockouts and differentiation time-course information.Level of innovation:Conceptually, the combined activity of two cooperating and partially redundant genes that individually result in only weak phenotypes, but if co-depleted cause a complete differentiation block, is functionally more important than the individual activity of a single gene that exhibits a strong exit-delay upon KO. Hence, we have much to discover and only by starting to dissect complex genetic interactions, we will truly understand cell fate decisions. We propose that our project can reveal entirely unknown regulatory control of differentiation processes which are not accessible due to redundancy in single-factor based genetic approaches. We envision this project to set a standard for performing combinatorial genetics.