Mechanisms that coordinate transcriptional programs in response to specific stimuli are central to understanding normal development and homeostasis. The pheromone-induced transition of budding yeast to either a chemotrophic or mating competent form is a model for dissecting the molecular basis of this regulation. A single mitogen activated protein kinase (MAPK) cascade mediates both transitions. This pathway utilizes two MAPKs, Fus3 and Kss1, that both negatively and positively regulate activity of the Ste12 and Tec1 transcriptional regulators. Ste12 is essential for the changes in gene expression that underlie establishment of both fates while Tec1 is important only for the chemotrophic fate. The genetic program for the chemotrophic fate transition induced by low pheromone concentration is still undefined. Fus3 and Kss1 activation profiles are differentially affected at high vs. low pheromone. We hypothesize that the dose-dependent differences in their activation and their antagonistic regulatory roles control Ste12 and Tec1 activity and degradation in a manner that prepares cells for one or the other differentiation program. We propose a multidisciplinary approach to compare the regulatory networks for the two fates and the molecular basis of the developmental switch:
Aim 1. Define the global expression program for chemotrophic growth using microarray technology and compare it to that for mating differentiation. Bioinformatic approaches are proposed to delineate the physiological and phenotypic signatures and regulatory networks that distinguish the two programs.
Aim 2. a and b) Combine experimental analyses with computational modeling to quantify the relative contributions from positive and negative regulation by Fus3 and Kss1 and the temporal control of transcription factor degradation on mediating the switch between alternative programs. Empirical measurements of transcription factor abundance and representative mRNAs in wild-type and mutant backgrounds that perturb regulation will be used to test the underlying hypotheses of the model. c) Time-lapse imaging under different pheromone induction regimes will be used to test whether a pheromone gradient reinforces feedback loops that regulate fluctuations and thereby reduce variability in pathway activity and fate determination. Our understanding of the pheromone induced MAPK pathway and the ease of genetic manipulations available with yeast allow discernment of how differences in the amplitude and timing of MAPK activation translate into different transcriptional patterns. Because of the conservation of MAPK pathways, the regulatory paradigms defined here will apply to different MAPK mediated signaling pathways in humans that are at the root of the pathogenesis of hormone dependent cancers, inflammatory diseases, and metabolic disorders.
Mitogen activated protein kinase (MAPK) pathways control normal cellular development and function. Differences in the amplitude and timing of MAPK activation are known to affect whether cells divide and multiply or develop into a specialized cell types, such as liver, bone, or brain cells. This proposal exploits advantages of studies with the model genetic organism, S. cerevisiae, to define the molecular mechanisms that translate different patterns of MAPK activation into different developmental fates. The findings will give us a better understanding of how aberrant regulation of these pathways in humans leads to hormone dependent cancers, inflammatory diseases, and metabolic disorders such as Type II diabetes.
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