Dynamics of the FOXO transcription factor network

受资助家长奖摘要 FOXO转录因子家族是进化上保守的动态平衡调节器，其 活动与延长寿命和抑制肿瘤有关。与他们在以下方面的角色一致 维持细胞内稳态，FOXO活性被不同类型的细胞应激上调，包括 营养/生长因子缺乏、DNA损伤和氧化应激。FOXO活动的控制是 主要通过控制核质穿梭的翻译后修饰实现 FOXO蛋白质。在细胞核中，FOXO通过多条经常相互冲突的途径上调基因 包括细胞周期停滞、细胞凋亡、自噬和ROS清道夫基因。细胞如何控制FOXO 确保他们的反应对给定的压力是适当的是一个悬而未决的问题。要解决这个问题 问题：我们使用CRISPR/Cas9基因编辑对两个FOXO蛋白Foxo1和Foxo3a进行了荧光标记， 在不同细胞系的内源性基因座。我们使用这些行来检验输入/输出的假设 FOXO途径的特异性是通过一种动态控制机制实现的，其中不同的FOXO 核质穿梭动力学决定了不同的细胞反应。我们的假设是受 FOXO途径与其他使用动态调控的转录因子的相似性 输入/输出特异性的机制包括P53和NF-ΚB。此外，我们的初步数据 支持FOXO动力学在控制细胞命运中的作用。我们发现了Foxo1的单细胞动力学 Foxo3a穿梭随刺激强度的不同而变化。此外，对于相同的刺激，我们观察到不同的 每种异构体的动力学。在目标1中，我们探索了Foxo1和Foxo3a作为响应的穿梭动力学 导致血清饥饿。我们结合反相蛋白质阵列和RNA-SEQ来确定时间- 关键调控因子的依赖变化控制着每种异构体的动态，以及如何将其转化为 不同的基因表达模式。在目标2中，我们测量了Foxo1和Foxo3a作为 以及EGFR和Akt抑制剂对细胞死亡的影响。此前的实验表明，两者都 Foxo1和Foxo3a是EGFR抑制剂引起细胞死亡所必需的。我们确定了动态 每一种与细胞死亡相关的亚型，并开发转录报告以确定如何 动力学是由细胞根据转录输出进行解码的。在目标3中，我们开发了一种光遗传学 用光控制Foxo1穿梭动力学的系统。我们使用这个系统来确定特定的 Foxo1穿梭的动态模式足以诱导细胞死亡，并使用RNA-seq来确定如何 动态的变化会改变目标基因的表达。这项研究中进行的实验将解决一个 在我们关于如何随着时间的推移控制FOXO动态以制定特定结果的知识方面存在严重差距。 更广泛地说，我们的工作将有助于阐明细胞信号电路如何感知和响应不同的 信号。

Summary of funded parent award The FOXO family of transcription factors are evolutionarily conserved regulators of homeostasis whose activities are linked to both increased lifespan and tumor suppression. Consistent with their role in maintaining cellular homeostasis, FOXO activity is upregulated by diverse types of cellular stress including nutrient/growth factor deprivation, DNA damage and oxidative stress. Control of FOXO activity is predominantly achieved through post-translational modifications that control nuclear-cytoplasmic shuttling of FOXO proteins. In the nucleus, FOXOs upregulate genes in multiple, often conflicting pathways including cell-cycle arrest, apoptosis, autophagy and ROS scavenger genes. How cells control FOXO activity to ensure that their response is appropriate for a given stress is an open question. To address this question we used CRISPR/Cas9 gene editing to fluorescently tag two FOXO proteins, Foxo1 and Foxo3a, at the endogenous locus of different cell lines. We use these lines to test the hypothesis that input/output specificity of the FOXO pathway is achieved through a dynamic control mechanism where different FOXO nuclear/cytoplasmic shuttling dynamics dictate separate cellular responses. Our hypothesis is inspired by similarities between the FOXO pathway and other transcription factors that use dynamic control mechanisms for input/output specificity including p53 and NF-ΚB. In addition, our preliminary data supports a role for FOXO dynamics in controlling cell fate. We found the single-cell dynamics of Foxo1 and Foxo3a shuttling change with different stimuli. Moreover, for the same stimulus we observed different dynamics for each isoform. In Aim 1 we explore the shuttling dynamics of Foxo1 and Foxo3a in response to serum starvation. We combine reverse phase protein arrays and RNA-seq to determine how time- dependent changes in key regulators control the dynamics of each isoform and how this is translated into different gene expression patterns. In Aim 2 we measure the dynamics of Foxo1 and Foxo3a shuttling as well as cell death in response to EGFR and Akt inhibitors. Previous experiments have shown that both Foxo1 and Foxo3a are required for cell death in response to EGFR inhibitors. We determine the dynamics of each isoform associated with cell death and develop transcriptional reporters to determine how dynamics are decoded by cells in terms of transcriptional output. In Aim 3 we develop an optogenetic system to control Foxo1 shuttling dynamics with light. We use this system to determine whether specific dynamic patterns of Foxo1 shuttling are sufficient to induce cell death and use RNA-seq to determine how changes in dynamics alter target gene expression. The experiments performed in this study will address a critical gap in our knowledge of how FOXO dynamics are controlled over time to enact specific outcomes. More broadly, our work will help elucidate how cell signaling circuits sense and respond to different signals.