# Dynamics of the FOXO transcription factor network

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2020 · $316,775

## Abstract

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.

## Key facts

- **NIH application ID:** 9839635
- **Project number:** 5R01GM130864-02
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Andrew Luther Paek
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $316,775
- **Award type:** 5
- **Project period:** 2019-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9839635

## Citation

> US National Institutes of Health, RePORTER application 9839635, Dynamics of the FOXO transcription factor network (5R01GM130864-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9839635. Licensed CC0.

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