Dynamics of the FOXO transcription factor network

FOXO 转录因子网络的动力学

• 批准号: 10547746
• 负责人: Andrew Luther Paek
• 金额: $ 31.68万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547746
• 关键词: AKT inhibition; Address; Affect; Apoptosis; Apoptotic; Autophagocytosis; CRISPR/Cas technology; Cell Cycle Arrest; Cell Death; Cell Death Induction; Cell Fate Control; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Cessation of life; Collaborations; Cytoplasm; Cytoprotection; DNA Damage; Data; Disparate; EGFR inhibition; Ensure; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; FOXO1A gene; Family; Family member; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Growth Factor; Health; Heterogeneity; Homeostasis; Human; Knowledge; Light; Link; Longevity; Measures; Microscopy; Nuclear; Nutrient; Oncogenes; Outcome; Output; Oxidative Stress; Pathway interactions; Pattern; Phase; Post-Translational Protein Processing; Protein Array; Protein Isoforms; Proteins; Reporter; Role; Serum; Signal Transduction; Signaling Protein; Specificity; Starvation; Stimulus; Stress; System; TP53 gene; Testing; Therapeutic; Time; Translating; Tumor Suppression; Variant; Work; cancer cell; cancer risk; chemotherapy; deprivation; environmental change; experimental study; inhibitor; insight; novel; optogenetics; response; single cell analysis; stem cell population; targeted treatment; transcription factor; transcriptome sequencing

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.

项目概要 FOXO 转录因子家族是进化上保守的体内平衡调节因子，其活性 与延长寿命和抑制肿瘤有关。与它们在维持细胞中的作用一致 体内平衡，FOXO 活性受到多种类型的细胞应激（包括营养/生长因子）的上调 剥夺、DNA 损伤和氧化应激。 FOXO 活性的控制主要通过后处理来实现 控制 FOXO 蛋白核质穿梭的翻译修饰。在细胞核中，FOXO 在多种常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 清除剂基因。细胞如何控制 FOXO 活性以确保其反应适合给定的情况 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 两种 FOXO 蛋白 Foxo1 和 Foxo3a 位于不同细胞系的内源基因座。我们用这些线来 检验 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 核/细胞质穿梭动力学决定不同的细胞反应的机制。 我们的假设受到 FOXO 通路和其他使用转录因子的相似性的启发 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 和的单细胞动力学 Foxo3a随着不同的刺激而穿梭变化。此外，对于相同的刺激，我们观察到不同的动态 对于每个亚型。在目标 1 中，我们探索 Foxo1 和 Foxo3a 响应血清的穿梭动力学 饥饿。我们结合反相蛋白阵列和 RNA-seq 来确定时间依赖性变化的方式 关键调控因子控制着每种亚型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 Foxo1 和 Foxo3a 穿梭的动态以及细胞死亡响应 EGFR 和 Akt 抑制剂。之前的实验表明 Foxo1 和 Foxo3a 都是细胞所必需的 EGFR 抑制剂导致死亡。我们确定与细胞死亡相关的每种亚型的动态 并开发转录报告基因以确定细胞如何在转录方面解码动态 输出。在目标 3 中，我们开发了一种光遗传学系统来控制 Foxo1 的光穿梭动力学。我们用这个 系统来确定 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 确定动态变化如何改变目标基因表达。进行的实验 这项研究将解决我们对 FOXO 动力学如何随着时间的推移进行控制以制定的知识中的一个关键差距 具体结果。更广泛地说，我们的工作将有助于阐明细胞信号传导电路如何感知和响应 不同的信号。