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.

Temporal Coordination of the Transcription Factor Response to H2O2 stress.

The p53 and FOXO transcription factors (TFs) share many similarities despite their distinct evolutionary origins. Both TFs are activated by a variety of cellular stresses and upregulate genes in similar pathways including cell-cycle arrest and apoptosis. Oxidative stress from excess H2O2 activates both FOXO1 and p53, yet whether they are activated at the same time is unclear. Here we found that cells respond to high H2O2 levels in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 levels remain low. In the second phase FOXO1 exits the nucleus and p53 levels rise. We found that other oxidative stress induced TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN) but not both following H2O2 stress. The two TF phases result in large differences in gene expression patterns. Finally, we provide evidence that 2-Cys peroxiredoxins control the timing of the TF phases in response to H2O2.

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.

Feedback in the ß-catenin destruction complex imparts bistability and cellular memory.

Wnt ligands are considered classical morphogens, for which the strength of the cellular response is proportional to the concentration of the ligand. Herein, we show an emergent property of bistability arising from feedback among the Wnt destruction complex proteins that target the key transcriptional co-activator ß-catenin for degradation. Using biochemical reconstitution, we identified positive feedback between the scaffold protein Axin and the kinase glycogen synthase kinase 3 (GSK3). Theoretical modeling of this feedback between Axin and GSK3 suggested that the activity of the destruction complex exhibits bistable behavior. We experimentally confirmed these predictions by demonstrating that cellular cytoplasmic ß-catenin concentrations exhibit an "all-or-none" response with sustained memory (hysteresis) of the signaling input. This bistable behavior was transformed into a graded response and memory was lost through inhibition of GSK3. These findings provide a mechanism for establishing decisive, switch-like cellular response and memory upon Wnt pathway stimulation.

Live-cell imaging in human colonic monolayers reveals ERK waves limit the stem cell compartment to maintain epithelial homeostasis.

The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here, we developed a human 2D patient derived organoid screening platform to study tissue patterning and kinase pathway dynamics in single cells. Using this system, we discovered that waves of ERK signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If ERK is activated acutely across all cells instead of in wave-like patterns, then tissue patterning and stem cells are lost. Conversely, if ERK activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated ERK signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how ERK can antagonize stem cells while supporting cell replacement and the function of the gut.

Designing combination therapies with modeling chaperoned machine learning.

Chemotherapy resistance is a major challenge to the effective treatment of cancer. Thus, a systematic pipeline for the efficient identification of effective combination treatments could bring huge biomedical benefit. In order to facilitate rational design of combination therapies, we developed a comprehensive computational model that incorporates the available biological knowledge and relevant experimental data on the life-and-death response of individual cancer cells to cisplatin or cisplatin combined with the TNF-related apoptosis-inducing ligand (TRAIL). The model's predictions, that a combination treatment of cisplatin and TRAIL would enhance cancer cell death and exhibit a "two-wave killing" temporal pattern, was validated by measuring the dynamics of p53 accumulation, cell fate, and cell death in single cells. The validated model was then subjected to a systematic analysis with an ensemble of diverse machine learning methods. Though each method is characterized by a different algorithm, they collectively identified several molecular players that can sensitize tumor cells to cisplatin-induced apoptosis (sensitizers). The identified sensitizers are consistent with previous experimental observations. Overall, we have illustrated that machine learning analysis of an experimentally validated mechanistic model can convert our available knowledge into the identity of biologically meaningful sensitizers. This knowledge can then be leveraged to design treatment strategies that could improve the efficacy of chemotherapy.

Hidden heterogeneity and circadian-controlled cell fate inferred from single cell lineages.

The origin of lineage correlations among single cells and the extent of heterogeneity in their intermitotic times (IMT) and apoptosis times (AT) remain incompletely understood. Here we developed single cell lineage-tracking experiments and computational algorithms to uncover correlations and heterogeneity in the IMT and AT of a colon cancer cell line before and during cisplatin treatment. These correlations could not be explained using simple protein production/degradation models. Sister cell fates were similar regardless of whether they divided before or after cisplatin administration and did not arise from proximity-related factors, suggesting fate determination early in a cell's lifetime. Based on these findings, we developed a theoretical model explaining how the observed correlation structure can arise from oscillatory mechanisms underlying cell fate control. Our model recapitulated the data only with very specific oscillation periods that fit measured circadian rhythms, thereby suggesting an important role of the circadian clock in controlling cellular fates.

Drug-induced aneuploidy and polyploidy is a mechanism of disease relapse in MYC/BCL2-addicted diffuse large B-cell lymphoma.

Double-hit (DH) or double-expresser (DE) lymphomas are high-grade diffuse large B-cell lymphomas (DLBCL) that are mostly incurable with standard chemo-immunotherapy due to treatment resistance. The generation of drug-induced aneuploid/polyploid (DIAP) cells is a common effect of anti-DLBCL therapies (e.g. vincristine, doxorubicin). DIAP cells are thought to be responsible for treatment resistance, as they are capable of re-entering the cell cycle during off-therapy periods. Previously we have shown that combination of alisertib plus ibrutinib plus rituximab can partially abrogate DIAP cells and induce cell death. Here, we provide evidence that DIAP cells can re-enter the cell cycle and escape cell death during anti-DLBCL treatment. We also discuss MYC/BCL2 mediated molecular mechanism that underlie treatment resistance. We isolated aneuploid/polyploid populations of DH/DE-DLBCL cells after treatment with the aurora kinase (AK) inhibitor alisertib. Time-lapse microscopy of single polyploid cells revealed that following drug removal, a subset of these DIAP cells divide and proliferate by reductive cell divisions, including multipolar mitosis, meiosis-like nuclear fission and budding. Genomic, proteomic, and kinomic profiling demonstrated that alisertib-induced aneuploid/polyploid cells up-regulate DNA damage, DNA replication and immune evasion pathways. In addition, we identified amplified receptor tyrosine kinase and T-cell receptor signaling, as well as MYC-mediated dysregulation of the spindle assembly checkpoints RanGAP1, TPX2 and KPNA2. We infer that these factors contribute to treatment resistance of DIAP cells. These findings provide opportunities to develop novel DH/DE-DLBCL therapies, specifically targeting DIAP cells. KEY POINTS: ● MYC mediated upregulation of TPX2, KPNA2 and RanGAP1 dysregulate the spindle assembly checkpoint in drug-induced polyploid cells.● Drug-induced polyploid cells re-enter the cell cycle via multipolar mitosis, fission or budding, a mechanism of disease relapse.

A dynamical framework for complex fractional killing.

When chemotherapy drugs are applied to tumor cells with the same or similar genotypes, some cells are killed, while others survive. This fractional killing contributes to drug resistance in cancer. Through an incoherent feedforward loop, chemotherapy drugs not only activate p53 to induce cell death, but also promote the expression of apoptosis inhibitors which inhibit cell death. Consequently, cells in which p53 is activated early undergo apoptosis while cells in which p53 is activated late survive. The incoherent feedforward loop and the essential role of p53 activation timing makes fractional killing a complex dynamical challenge, which is hard to understand with intuition alone. To better understand this process, we have constructed a representative model by integrating the control of apoptosis with the relevant signaling pathways. After the model was trained to recapture the observed properties of fractional killing, it was analyzed with nonlinear dynamical tools. The analysis suggested a simple dynamical framework for fractional killing, which predicts that cell fate can be altered in three possible ways: alteration of bifurcation geometry, alteration of cell trajectories, or both. These predicted categories can explain existing strategies known to combat fractional killing and facilitate the design of novel strategies.

Cell-to-Cell Variation in p53 Dynamics Leads to Fractional Killing.

Many chemotherapeutic drugs kill only a fraction of cancer cells, limiting their efficacy. We used live-cell imaging to investigate the role of p53 dynamics in fractional killing of colon cancer cells in response to chemotherapy. We found that both surviving and dying cells reach similar levels of p53, indicating that cell death is not determined by a fixed p53 threshold. Instead, a cell's probability of death depends on the time and levels of p53. Cells must reach a threshold level of p53 to execute apoptosis, and this threshold increases with time. The increase in p53 apoptotic threshold is due to drug-dependent induction of anti-apoptotic genes, predominantly in the inhibitors of apoptosis (IAP) family. Our study underlines the importance of measuring the dynamics of key players in response to chemotherapy to determine mechanisms of resistance and optimize the timing of combination therapy.

Draft Genome Sequence of the Ale-Fermenting Saccharomyces cerevisiae Strain GSY2239.

Saccharomyces cerevisiae strain GSY2239 is derived from an industrial yeast strain used to ferment ale-style beer. We present here the 11.5-Mb draft genome sequence for this organism.

DNA replication: failures and inverted fusions.

DNA replication normally follows the rules passed down from Watson and Crick: the chromosome duplicates as dictated by its antiparallel strands, base-pairing and leading and lagging strand differences. Real-life replication is more complicated, fraught with perils posed by chromosome damage for one, and by transcription of genes and by other perils that disrupt progress of the DNA replication machinery. Understanding the replication fork, including DNA structures, associated replisome and its regulators, is key to understanding how cells overcome perils and minimize error. Replication fork error leads to genome rearrangements and, potentially, cell death. Interest in the replication fork and its errors has recently gained added interest by the results of deep sequencing studies of human genomes. Several pathologies are associated with sometimes-bizarre genome rearrangements suggestive of elaborate replication fork failures. To try and understand the links between the replication fork, its failure and genome rearrangements, we discuss here phases of fork behavior (stall, collapse, restart and fork failures leading to rearrangements) and analyze two examples of instability from our own studies; one in fission yeast and the other in budding yeast.

Choreography of the 9-1-1 checkpoint complex: DDK puts a check on the checkpoints.

Checkpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.

The role of replication bypass pathways in dicentric chromosome formation in budding yeast.

Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20-200 bp of homology, separated by ∼1-5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.

Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast.

Large-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term "faulty template switching"). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.