ABSTRACT: The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.
Project description:The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes is not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features - thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.
Project description:Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input-output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to ?-radiation. We further found that while ?-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus-dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.
Project description:The cell stress responsive transcription factor p53 influences the expression of its target genes and subsequent cellular responses, based in part on its dynamics (changes in level over time). The mechanisms decoding p53 dynamics into subsequent target mRNA and protein dynamics remain unclear. We systematically quantified p53 target mRNA and protein expression over time under two p53 dynamical regimes, oscillatory and sustained, using RNA-sequencing and TMT mass-spectrometry. Oscillatory dynamics allowed for a greater variety of dynamical patterns for both mRNAs and proteins. Mathematical modeling of empirical data revealed three distinct mechanisms that decode p53 dynamics. Specific combinations of these mechanisms at the transcriptional and post-transcriptional levels enabled exclusive induction of proteins under particular dynamics. In addition, sustained induction of p53 led to higher induction of proteins regardless of their functional class, including proteins promoting arrest of proliferation, the primary cellular outcome under sustained p53. Our results highlight the diverse mechanisms cells employ to distinguish complex transcription factor dynamics to regulate gene expression.
Project description:Information about environmental stimuli is often transmitted using common signaling molecules, but the mechanisms that ensure signaling specificity are not entirely known. Here we show that the identities and intensities of different stresses are transmitted by modulation of the amplitude, duration or frequency of nuclear translocation of the Saccharomyces cerevisiae general stress response transcription factor Msn2. Through artificial control of the dynamics of Msn2 translocation, we reveal how distinct dynamical schemes differentially affect reporter gene expression. Using a simple model, we predict stress-induced reporter gene expression from single-cell translocation dynamics. We then demonstrate that the response of natural target genes to dynamical modulation of Msn2 translocation is influenced by differences in the kinetics of promoter transitions and transcription factor binding properties. Thus, multiple environmental signals can trigger qualitatively different dynamics of a single transcription factor and influence gene expression patterns.
Project description:The tumor suppressor p53 protein shows various dynamic responses depending on the types and extent of cellular stresses. In particular, in response to DNA damage induced by γ-irradiation, cells generate a series of p53 pulses. Recent research has shown the importance of sustaining repeated p53 pulses for recovery from DNA damage. However, far too little attention has been paid to understanding how cells can sustain p53 pulses given the complexities of genetic heterogeneity and intrinsic noise. Here, we explore potential molecular mechanisms that enhance the sustainability of p53 pulses by developing a new mathematical model of the p53 regulatory system. This model can reproduce many experimental results that describe the dynamics of p53 pulses. By simulating the model both deterministically and stochastically, we found three potential mechanisms that improve the sustainability of p53 pulses: 1) the recently identified positive feedback loop between p53 and Rorα allows cells to sustain p53 pulses with high amplitude over a wide range of conditions, 2) intrinsic noise can often prevent the dampening of p53 pulses even after mutations, and 3) coupling of p53 pulses in neighboring cells via cytochrome-c significantly reduces the chance of failure in sustaining p53 pulses in the presence of heterogeneity among cells. Finally, in light of these results, we propose testable experiments that can reveal important mechanisms underlying p53 dynamics.
Project description:In response to DNA damage, the transcription factor p53 accumulates in a series of pulses. While p53 dynamics play a critical role in regulating stress responses, how p53 pulsing affects target protein expression is not well understood. Recently, we showed that p53 pulses generate diversity in target mRNA expression dynamics; however, given that mRNA and protein expression are not necessarily well correlated, it remains to be determined how p53 pulses impact target protein expression. Using computational and experimental approaches, we show that target protein decay rates filter p53 pulses: Distinct target protein expression dynamics are generated depending on the relationship between p53 pulse frequency and target mRNA and protein stability. Furthermore, by mutating the targets MDM2 and PUMA to alter their stabilities, we show that downstream pathways are sensitive to target protein decay rates. This study delineates the mechanisms by which p53 dynamics play a crucial role in orchestrating the timing of events in the DNA damage response network.
Project description:Transcription factors (TFs) can relay signals through temporal alterations in their levels. The cell stress responsive TF p53 exhibits different dynamics depending on the type of stress, which influence the magnitude and dynamics of expression of its target genes and subsequent cellular outcomes. The mechanisms decoding p53 dynamics into levels and dynamics of RNA and protein of its targets remain unclear. We systematically quantified p53 target mRNA and protein levels over time under two p53 dynamical regimes – oscillatory and sustained – using RNA-seq and quantitative mass spectrometry. We categorized the mRNA dynamical patterns arising from oscillatory or sustained p53 expression, as well as the corresponding protein dynamics. We found that in both cases oscillatory dynamics allowed for the greatest variety of dynamical patterns of the downstream species. Target proteins from a wide range of functional categories were induced under both p53 dynamic conditions, with induction levels being overall higher under sustained dynamics. Mathematical modeling combined with analysis of empirical data revealed three mechanisms of decoding p53 dynamics: adjustment of mRNA and protein degradation rates, adjustment of activation thresholds for expression of mRNA and corresponding protein levels, and usage of coherent and incoherent feed-forward loop motifs in generating exclusive responses to p53 oscillatory or sustained dynamics, respectively. Our results highlight the diversity of mechanisms that decode complex TF dynamics into dynamical patterns of mRNA and proteins.
Project description:Males in numerous animal species use mating songs to attract females and intimidate competitors. We demonstrate that modulations in song amplitude are behaviourally relevant in the fruit fly Drosophila We show that Drosophilamelanogaster females prefer amplitude modulations that are typical of melanogaster song over other modulations, which suggests that amplitude modulations are processed auditorily by D. melanogaster Our work demonstrates that receivers can decode messages in amplitude modulations, complementing the recent finding that male flies actively control song amplitude. To describe amplitude modulations, we propose the concept of song amplitude structure (SAS) and discuss similarities and differences to amplitude modulation with distance (AMD).This article has an associated First Person interview with the first author of the paper.
Project description:Recent studies have shown that many cell-signaling networks contain interactions and feedback loops that give rise to complex dynamics. Synthetic biology has allowed researchers to construct and analyze well-defined signaling circuits exhibiting behavior that can be predicted and quantitatively understood. Combining these approaches--wiring natural network components together with engineered interactions--has the potential to precisely modulate the dynamics of endogenous signaling processes and control the cell decisions they influence. Here, we focus on the p53 signaling pathway as a template for constructing a tunable oscillator comprised of both natural and synthetic components in mammalian cells. We find that a reduced p53 circuit implementing a single feedback loop preserves some features of the full network's dynamics, exhibiting pulses of p53 with tightly controlled timing. However, in contrast to the full natural p53 network, these pulses are damped in individual cells, with amplitude that depends on the input strength. Guided by a computational model of the reduced circuit, we constructed and analyzed circuit variants supplemented with synthetic positive and negative feedback loops and subjected to chemical perturbation. Our work demonstrates that three important features of oscillator dynamics--amplitude, period, and the rate of damping--can be controlled by manipulating stimulus level, interaction strength, and feedback topology. The approaches taken here may be useful for the rational design of synthetic networks with defined dynamics, and for identifying perturbations that control dynamics in natural biological circuits for research or therapeutic purposes.
Project description:p53 is a major sensor of cellular stresses, and its activation influences cell fate decisions. We identified SUV39H1, a histone code 'writer' responsible for the histone H3 Lys9 trimethylation (H3K9me3) mark for 'closed' chromatin conformation, as a target of p53 repression. SUV39H1 downregulation was mediated transcriptionally by p21 and post-translationally by MDM2. The H3K9me3 repression mark was found to be associated with promoters of representative p53 target genes and was decreased upon p53 activation. Overexpression of SUV39H1 maintained higher levels of the H3K9me3 mark on these promoters and was associated with decreased p53 promoter occupancy and decreased transcriptional induction in response to p53. Conversely, SUV39H1 pre-silencing decreased H3K9me3 levels on these promoters and enhanced the p53 apoptotic response. These findings uncover a new layer of p53-mediated chromatin regulation through modulation of histone methylation at p53 target promoters.