Positive-Feedback Loops as a Flexible Biological Module

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Current Biology (Impact Factor: 9.57). 04/2007; 17(8):668-77. DOI: 10.1016/j.cub.2007.03.016
Source: PubMed


Bistability in genetic networks allows cells to remember past events and to make discrete decisions in response to graded signals. Bistable behavior can result from positive feedback, but feedback loops can have other roles in signal transduction as well.
We introduced positive feedback into the budding-yeast pheromone response to convert it into a bistable system. In the presence of feedback, transient induction with high pheromone levels caused persistent pathway activation, whereas at lower levels a fraction of cells became persistently active but the rest inactivated completely. We also generated mutations that quantitatively tuned the basal and induced expression levels of the feedback promoter and showed that they qualitatively changed the behavior of the system. Finally, we developed a simple stochastic model of our positive-feedback system and showed the agreement between our simulations and experimental results.
The positive-feedback loop can display several different behaviors, including bistability, and can switch between them as a result of simple mutations.

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    • "Engineering signalling pathways has significant potential to provide novel and complementary information that cannot be achieved by traditional genetic approaches such as gene knockout and overexpression1112. For instance, rewiring signalling components between MAPK pathways1314, introducing synthetic negative or positive feedback loops1516, tethering signalling components with specific localization motifs17, assembling or recombining modular signalling domains1819 and reconstitution of a heterologous MAPK cascade20 are highly informative for understanding the design principles of MAPK pathways and enable generating novel signalling properties. In the present study, we reconstituted osmoadaptation in hog1Δ cells by rewiring osmostress signalling through the MAPK network. "
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    ABSTRACT: Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data.
    Full-text · Article · Apr 2014 · Scientific Reports
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    • "All GAL1 promoter induction data is from the same yeast strain yJHK312 in which transcription of SUC2 is driven by the GAL1 promoter. Galactokinase (GAL1) is deleted from this strain so that galactose acts as an inducer and not as a carbon source, and the Gal regulon has been engineered to produce a graded rather than a bistable response to increased galactose concentrations by overexpressing GAL3 from the ACT1 promoter (Ingolia and Murray, 2007). (B) Sucrose import allows growth from a single cell in low sucrose concentrations. "
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    ABSTRACT: We do not know how or why multicellularity evolved. We used the budding yeast, Saccharomyces cerevisiae, to ask whether nutrients that must be digested extracellularly select for the evolution of undifferentiated multicellularity. Because yeast use invertase to hydrolyze sucrose extracellularly and import the resulting monosaccharides, single cells cannot grow at low cell and sucrose concentrations. Three engineered strategies overcame this problem: forming multicellular clumps, importing sucrose before hydrolysis, and increasing invertase expression. We evolved populations in low sucrose to ask which strategy they would adopt. Of 12 successful clones, 11 formed multicellular clumps through incomplete cell separation, 10 increased invertase expression, none imported sucrose, and 11 increased hexose transporter expression, a strategy we had not engineered. Identifying causal mutations revealed genes and pathways, which frequently contributed to the evolved phenotype. Our study shows that combining rational design with experimental evolution can help evaluate hypotheses about evolutionary strategies. DOI:
    Full-text · Article · Apr 2013 · eLife Sciences
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    • "Construction of synthetic bistability by engineering graded-response MAPK systems can help understanding how bistability is regulated . Ingolia and Murray (2007) introduced synthetic positive-feedback loops (constitutive active components under the pathway-dependent FUS1 promoter) into the yeast mating MAPK pathway converting it into a bistable system: in the presence of the positive feedback, the pathway was persistently activated even by transient pheromone treatment. Implementing this positive feedback loop and using FUS1 promoter variants revealed several features required for bistability such as low-basal and high-induced expression of the positive feedback. "
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    ABSTRACT: All living cells respond to external stimuli and execute specific physiological responses through signal transduction pathways. Understanding the mechanisms controlling signalling pathways is important for diagnosing and treating diseases and for reprogramming cells with desired functions. Although many of the signalling components in the budding yeast Saccharomyces cerevisiae have been identified by genetic studies, many features concerning the dynamic control of pathway activity, cross-talk, cell-to-cell variability or robustness against perturbation are still incompletely understood. Comparing the behaviour of engineered and natural signalling pathways offers insight complementary to that achievable with standard genetic and molecular studies. Here, we review studies that aim at a deeper understanding of signalling design principles and generation of novel signalling properties by engineering the yeast mitogen-activated protein kinase (MAPK) pathways. The underlying approaches can be applied to other organisms including mammalian cells and offer opportunities for building synthetic pathways and functionalities useful in medicine and biotechnology.
    Full-text · Article · Mar 2013 · Molecular Microbiology
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