Dynamics of the FOXO transcription factor network

项目摘要 FOXO转录因子家族是进化上保守的动态平衡调节器，其活性 既与延长寿命有关，也与肿瘤抑制有关。与他们在维持细胞 动态平衡，FOXO活性被包括营养/生长因子在内的不同类型的细胞应激上调 剥夺、DNA损伤和氧化应激。FOXO活性的控制主要是通过后处理实现的 控制FOXO蛋白核质穿梭的翻译修饰。在原子核中，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、细胞凋亡、自噬和 ROS清道夫基因。细胞如何控制FOXO活动，以确保它们的反应适合给定的 压力是一个悬而未决的问题。为了解决这个问题，我们使用CRISPR/Cas9基因编辑来荧光标记 两个FOXO蛋白，Foxo1和Foxo3a，位于不同细胞系的内源位点。我们使用这些线路来 检验FOXO通路的输入/输出专一性是通过动态控制实现的假设 不同的FOXO核质穿梭动力学决定不同的细胞反应的机制。 我们的假设是受到FOXO途径和其他转录因子之间的相似之处的启发 输入/输出特异性的动态控制机制包括p53和NF-ΚB。此外，我们的初步研究 数据支持FOXO动力学在控制细胞命运方面的作用。我们发现了Foxo1的单细胞动力学 FOXO3a穿梭随不同刺激而变化。此外，对于相同的刺激，我们观察到不同的动态 对于每一种异构体。在目标1中，我们探索了Foxo1和Foxo3a对血清的响应的穿梭动力学 饿死了。我们结合反相蛋白质阵列和rna-seq来确定依赖时间的变化。 关键调控因子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穿梭的动力学以及细胞对 EGFR和Akt抑制剂。以往的实验表明，细胞同时需要Foxo1和Foxo3a 死亡是对EGFR抑制剂的反应。我们确定了与细胞死亡相关的每种异构体的动力学 并开发转录记者来确定动态是如何被细胞从转录方面解码的 输出。在目标3中，我们开发了一个光遗传系统来控制Foxo1的光穿梭动力学。我们用这个 确定Foxo1穿梭的特定动态模式是否足以诱导细胞死亡和 使用RNA-seq来确定动态变化如何改变目标基因的表达。所进行的实验 在这项研究中，我们将解决我们关于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缺口 具体结果。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.