Project SummaryThe FOXO family of transcription factors are evolutionarily conserved regulators of homeostasis whose activitiesare linked to both increased lifespan and tumor suppression. Consistent with their role in maintaining cellularhomeostasis FOXO activity is upregulated by diverse types of cellular stress including nutrient/growth factordeprivation 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 FOXOsupregulate genes in multiple often conflicting pathways including cell-cycle arrest apoptosis autophagy andROS scavenger genes. How cells control FOXO activity to ensure that their response is appropriate for a givenstress is an open question. To address this question we used CRISPR/Cas9 gene editing to fluorescently tagtwo FOXO proteins Foxo1 and Foxo3a at the endogenous locus of different cell lines. We use these lines totest the hypothesis that input/output specificity of the FOXO pathway is achieved through a dynamic controlmechanism 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 usedynamic control mechanisms for input/output specificity including p53 and NF-B. In addition our preliminarydata supports a role for FOXO dynamics in controlling cell fate. We found the single-cell dynamics of Foxo1 andFoxo3a shuttling change with different stimuli. Moreover for the same stimulus we observed different dynamicsfor each isoform. In Aim 1 we explore the shuttling dynamics of Foxo1 and Foxo3a in response to serumstarvation. We combine reverse phase protein arrays and RNA-seq to determine how time-dependent changesin key regulators control the dynamics of each isoform and how this is translated into different gene expressionpatterns. In Aim 2 we measure the dynamics of Foxo1 and Foxo3a shuttling as well as cell death in response toEGFR and Akt inhibitors. Previous experiments have shown that both Foxo1 and Foxo3a are required for celldeath in response to EGFR inhibitors. We determine the dynamics of each isoform associated with cell deathand develop transcriptional reporters to determine how dynamics are decoded by cells in terms of transcriptionaloutput. In Aim 3 we develop an optogenetic system to control Foxo1 shuttling dynamics with light. We use thissystem to determine whether specific dynamic patterns of Foxo1 shuttling are sufficient to induce cell death anduse RNA-seq to determine how changes in dynamics alter target gene expression. The experiments performedin this study will address a critical gap in our knowledge of how FOXO dynamics are controlled over time to enactspecific outcomes. More broadly our work will help elucidate how cell signaling circuits sense and respond todifferent signals.