FOXO nuclear shuttling dynamics are stimulus-dependent and correspond with cell fate

Abstract

FOXO transcription factors are regulators of cellular homeostasis linked to increased lifespan and tumor suppression. FOXOs are activated by diverse cell stresses including serum starvation and oxidative stress. FOXO activity is regulated through post-translational modifications that control shuttling of FOXO proteins to the nucleus. In the nucleus, FOXOs upregulate genes in multiple, often conflicting pathways including cell-cycle arrest and apoptosis. How cells control FOXO activity to ensure the proper response for a given stress is an open question. Using quantitative immunofluorescence and live-cell imaging we found that the dynamics of FOXO nuclear shuttling are stimulus dependent and correspond with cell fate. H2O2 treatment leads to an all-or-none response where some cells show no nuclear FOXO accumulation, while other cells show strong nuclear FOXO signal. The time that FOXO remains in the nucleus increases with dose and is linked with cell death. In contrast, serum starvation causes low amplitude pulses of nuclear FOXO and predominantly results in cell-cycle arrest. The accumulation of FOXO in the nucleus is linked with low AKT activity for both H2O2 and serum starvation. Our findings suggest the dynamics of FOXO nuclear shuttling is one way in which the FOXO pathway dictates different cellular outcomes. [Media: see text] [Media: see text] [Media: see text]

Temporal Coordination of the Transcription Factor Response to H2O2stress

“…In addition, autocorrelation analysis revealed oscillations in p53 activation in surviving cells but not in dying cells (Figure 2G). Dying cells also showed an increase in the duration of FOXO1 activation as compared to surviving cells, in agreement with our previous study 39 (Figure S2E,F). Together these data show that higher concentrations of H 2 O 2 cause prolonged activation of FOXO1, a delayed yet stronger p53 response, and an increase in cell death.…”

“…We used a previously developed MCF7 cell line where CRISPR/Cas9 was used to tag the endogenous locus of FOXO1 at the C-terminus with the mVenus fluorescent protein. This cell line also contains a H2B-CFP tag for tracking nuclei 39 . For p53, we added an exogenously expressed p53-mCherry reporter used and validated in previous studies 40 .…”

“…This could ensure adequate induction of genes in the first TF phase, either to ensure proper repair, or alternatively for ensuring cell death if the initial TF phase is prolonged. Indeed, prolonged FOXO activation following H 2 O 2 treatment is associated with increased cell death 39 . There is also increased expression of genes in the pentose phosphate pathway and nucleotide biosynthesis in the second transcription factor phase.…”

Temporal Coordination of the Transcription Factor Response to H2O2 stress

“…2G ). Dying cells also showed an increase in the duration of FOXO1 activation as compared to surviving cells, in agreement with our previous study 36 (Figure S2E,F). Together these data show that higher concentrations of H 2 O 2 cause prolonged activation of FOXO1, a delayed yet stronger p53 response, and an increase in cell death.…”

“…We used a previously developed MCF7 cell line where CRISPR/Cas9 was used to tag the endogenous locus of FOXO1 at the C-terminus with the mVenus fluorescent protein. This cell line also contains a H2B-CFP tag for tracking nuclei 36 . For p53, we added an exogenously expressed p53-mCherry reporter used and validated in previous studies 37 .…”