Dynamics of the FOXO transcription factor network

资助父母奖摘要 转录因子的FOXO家族是进化上保守的稳态调节因子，其 活性与寿命延长和肿瘤抑制有关。与其在 维持细胞内稳态，FOXO活性被多种类型的细胞应激上调，包括 营养/生长因子剥夺、DNA损伤和氧化应激。FOXO活性的控制是 主要通过翻译后修饰来控制核质穿梭 FOXO蛋白质在细胞核中，FOXO上调多个经常相互冲突的途径中的基因 包括细胞周期停滞、凋亡、自噬和ROS清除剂基因。细胞如何控制FOXO 活动，以确保他们的反应是适当的一个给定的压力是一个悬而未决的问题。为了解决这个 我们使用CRISPR/Cas9基因编辑来荧光标记两种FOXO蛋白，Foxo 1和Foxo 3a， 在不同细胞系的内源基因座上。我们用这些线来检验输入/输出 FOXO途径的特异性是通过动态控制机制实现的， 细胞核/细胞质穿梭动力学决定了单独的细胞反应。我们的假设是受到 FOXO途径和其他使用动态控制的转录因子之间的相似性 包括p53和NF-ΚB B的输入/输出特异性机制。此外，我们的初步数据显示， 支持FOXO动力学在控制细胞命运中的作用。我们发现Foxo 1的单细胞动力学 而Foxo 3a则随刺激的不同而变化。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。在目的1中，我们探索了Foxo 1和Foxo 3a在应答中的穿梭动力学。 到血清饥饿我们将联合收割机反相蛋白质阵列和RNA-seq结合起来，以确定时间- 关键调控因子的依赖性变化控制着每种亚型的动力学，以及这种动力学如何转化为 不同的基因表达模式。在目标2中，我们测量Foxo 1和Foxo 3a穿梭的动力学， 以及对EGFR和Akt抑制剂应答的细胞死亡。先前的实验表明， Foxo 1和Foxo 3a是EGFR抑制剂导致细胞死亡所必需的。我们决定动力学 与细胞死亡相关的每种亚型，并开发转录报告，以确定如何 动态由细胞根据转录输出解码。在目标3中，我们开发了一种光遗传学方法， 系统，以控制Foxo 1穿梭动力学与光。我们用这个系统来确定 Foxo 1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 动力学的变化改变靶基因的表达。本研究中进行的实验将解决 我们对如何随着时间的推移控制FOXO动态以产生特定结果的知识存在重大差距。 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。 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.