Dynamics of the FOXO transcription factor network

项目摘要 转录因子的FOXO家族是进化上保守的稳态调节因子，其活性 与延长寿命和抑制肿瘤有关。这与它们在维持细胞 在体内平衡中，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。对FOXO活性的控制主要是通过后处理来实现的。 翻译修饰控制FOXO蛋白质的核质穿梭。在细胞核中，FOXO 在多个经常相互冲突的途径中上调基因，包括细胞周期停滞、凋亡、自噬和 ROS清除基因。细胞如何控制FOXO活性，以确保它们的反应适合给定的 压力是一个悬而未决问题。为了解决这个问题，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。我们用这些线 测试FOXO通路的输入/输出特异性是通过动态控制实现的假设 不同的FOXO细胞核/细胞质穿梭动力学决定了不同的细胞反应。 我们的假设受到FOXO通路和其他转录因子之间的相似性的启发， 包括p53和NF-ΚB B的输入/输出特异性的动态控制机制。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Foxo 1的单细胞动力学， Foxo 3a在不同刺激下穿梭变化。此外，对于相同的刺激，我们观察到不同的动力学 对于每种异构体。在目的1中，我们探索了Foxo 1和Foxo 3a响应血清的穿梭动力学。 饥饿我们将联合收割机反相蛋白质阵列和RNA-seq相结合， 在关键的调节器控制每个异构体的动力学，以及如何将其转化为不同的基因表达 模式.在目的2中，我们测量了Foxo 1和Foxo 3a穿梭的动力学以及细胞死亡对细胞凋亡的反应。 EGFR和Akt抑制剂。以前的实验表明，Foxo 1和Foxo 3a都是细胞生长所必需的。 对EGFR抑制剂有反应的死亡。我们确定与细胞死亡相关的每种亚型的动力学 并开发转录报告，以确定细胞如何在转录方面解码动态， 输出.在Aim 3中，我们开发了一种光遗传学系统来控制Foxo 1的穿梭动力学。我们用这个 确定Foxo 1穿梭的特定动态模式是否足以诱导细胞死亡的系统， 使用RNA-seq来确定动态变化如何改变靶基因表达。进行的实验 在这项研究中，将解决我们对FOXO动态如何随着时间的推移进行控制的知识中的一个关键空白， 具体成果。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。

Project Summary 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.