O'Shea Lab > Projects

Systems Analysis

We seek to discover how the architecture of signaling pathways generates properties that are more complex than those of the constituent parts. Much of our effort is focused on homeostatic systems involved in the regulation of intracellular nutrient levels, including the phosphate-responsive signaling (Pho) pathway involved in phosphate homeostasis in budding yeast. Our approach involves studying signaling in single, living cells using microfluidic devices to control environmental conditions and fluorescent reporters to monitor pathway activity. Our studies have uncovered interesting properties in the phosphate homeostasis pathway, including buffering, differences in thresholds of expression of genes, bistability, and hysteresis. We are investigating the origins of these properties and how they relate to the ability of this system to maintain homeostasis.

Genetic, Cell Biological and Biochemical Analysis

The phosphate-responsive signaling pathway has been an excellent model system with which to investigate basic mechanisms of signal transduction and the regulation of transcription factor activity. Although this work has revealed much about the protein kinase Pho80–Pho85 and the regulation of its transcription factor substrate Pho4, we know little about how phosphate is sensed by cells and how changes in phosphate levels lead to changes in the kinase activity of Pho80–Pho85. We are taking two approaches to address these questions. First, to understand how cells sense phosphate levels, we are performing a novel, comprehensive screen to identify genes whose products are involved in phosphate sensing. Second, we are investigating connections between inositol polyphosphate metabolism and phosphate sensing. Our recent work with John York (HHMI, Duke University) indicates that an inositol pyrophosphate (IP7) plays a critical role in the regulation of Pho80–Pho85 by the cyclin-dependent kinase (CDK) inhibitor Pho81. We are exploring the mechanism of this regulation, and we will employ more general methods to globally investigate changes in metabolites in response to phosphate limitation.

Interaction with other Nutrient Response Pathways

Recent work indicates that there is communication between the phosphate-responsive signaling pathway and the adenine starvation response in budding yeast – starvation of cells for adenine activates expression of at least some phosphate starvation response genes, and starvation for phosphate activates expression of genes involved in adenine metabolism. We are interested in the mechanisms underlying this crosstalk. Is there a common mechanism for sensing phosphate and adenine limitation, or are the mechanisms of sensing distinct and these signals are integrated into both pathways?

Signal Processing by Promoters

We seek to understand how regulatory regions of genes transform information about transcription factor input into quantitative gene expression output. Our goal is to develop a quantitative model that describes how promoter sequence influences the threshold for gene activation, the maximum transcriptional output, and the sensitivity of the response. To achieve this goal we are analyzing the transcription factor input-gene expression output relationship in single cells using a model promoter from the phosphate-responsive signaling pathway. We quantify the input-output relationship in the wild-type promoter and in variants in which key features of promoter sequence have been altered and use these data to inform mechanistic models.

Transcriptional Regulatory Network Evolution

In collaboration with Aviv Regev, we are using the evolutionarily conserved HOG MAPK pathway and phosphate-responsive signaling pathways as models to investigate the evolution of transcriptional regulatory networks. This work is enabled by the genome sequences of a number of yeast species, by an orthology map generated in the Regev lab, and also by our development of methodology to construct quantitative maps of transcriptional networks from microarray data. We will combine experiments and computational approaches to characterize how these networks have evolved from Schizosaccharomyces pombe to S. cerevisiae and then explain the mechanisms allowing these regulatory changes.

Uncovering the Mechanism of Timekeeping in the Clock

Organisms exhibit oscillations in metabolism and behavior that are entrained by the environmental light-dark cycle. Circadian rhythms are present even in single-celled cyanobacteria, which use these oscillations to alternate photosynthesis with nitrogen fixation, two mutually exclusive biochemical processes. It is thought that circadian rhythms rely on an autoregulatory transcription and translation feedback cycle to generate oscillations. In the cyanobacterial circadian oscillator, genetic and biochemical studies have revealed that three proteins (KaiA, KaiB, and KaiC) play an important role in the oscillator and that KaiC undergoes changes in phosphorylation state with periodicity that corresponds to the circadian cycle. Remarkably, Takao Kondo's group (Nagoya University, Japan) has demonstrated that these three proteins (plus ATP) are sufficient to produce sustained oscillations in the phosphorylation state of KaiC in vitro. We have collaborated with Daniel Fisher (Stanford University) to combine experiments with modeling to uncover the basis of sustained oscillations. We are now interested in understanding other remarkable properties of this clock, observed in vivo and in vitro – the relative insensitivity of its period to changes in protein concentrations and temperature.

Studies of Clock Input and Output

Circadian clocks receive environmental input, and coordinate physiological responses, such as gene expression. We are interested in developing a biochemical understanding of these phenomena, building on the genetic studies from Kondo and Golden which have identified proteins involved in interactions with the core oscillator.