Temporal coordination of the transcription factor response to H2O2 stress.

Oxidative stress from excess H2O2 activates transcription factors that restore redox balance and repair oxidative damage. Although many transcription factors are activated by H2O2, it is unclear whether they are activated at the same H2O2 concentration, or time. Dose-dependent activation is likely as oxidative stress is not a singular state and exhibits dose-dependent outcomes including cell-cycle arrest and cell death. Here, we show that transcription factor activation is both dose-dependent and coordinated over time. Low levels of H2O2 activate p53, NRF2 and JUN. Yet under high H2O2, these transcription factors are repressed, and FOXO1, NF-κB, and NFAT1 are activated. Time-lapse imaging revealed that the order in which these two groups of transcription factors are activated depends on whether H2O2 is administered acutely by bolus addition, or continuously through the glucose oxidase enzyme. Finally, we provide evidence that 2-Cys peroxiredoxins control which group of transcription factors are activated.

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

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 posttranslational modifications that control shuttling of FOXO proteins to the nucleus. In the nucleus, FOXOs up-regulate 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 is stimulus-dependent and corresponds with cell fate. H2O2 treatment leads to an all-or-none response where some cells show no nuclear FOXO accumulation, while other cells show a strong nuclear FOXO signal. The time that FOXO remains in the nucleus increases with the 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.

Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast.

We report here that a normal budding yeast chromosome (ChrVII) can undergo remarkable cycles of chromosome instability. The events associated with cycles of instability caused a distinctive "sectoring" of colonies on selective agar plates. We found that instability initiated at any of several sites on ChrVII, and was sharply increased by the disruption of DNA replication or by defects in checkpoint controls. We studied in detail the cycles of instability associated with one particular chromosomal site (the "403 site"). This site contained multiple tRNA genes known to stall replication forks, and when deleted, the overall frequency of sectoring was reduced. Instability of the 403 site involved multiple nonallelic recombination events that led to the formation of a monocentric translocation. This translocation remained unstable, frequently undergoing either loss or recombination events linked to the translocation junction. These results suggest a model in which instability initiates at specific chromosomal sites that stall replication forks. Forks not stabilized by checkpoint proteins break and undergo multiple rounds of nonallelic recombination to form translocations. Some translocations remain unstable because they join two "incompatible" chromosomal regions. Cycles of instability of this normal yeast chromosome may be relevant to chromosome instability of mammalian fragile sites and of chromosomes in cancer cells.