O'Shea Lab > Projects

A novel mechanism of metabolite-dependent kinase regulation. Signaling pathways play crucial roles in the ability of cells to sense and respond to changes in the availability of essential nutrients. We have used the phosphate-responsive signaling pathway in budding yeast as a model system with which to investigate basic mechanisms of signal transduction and the regulation of transcription factor activity. A central player in this pathway is the cyclin–cyclin-dependent protein kinase (CDK) Pho80-Pho85, whose function is to phosphorylate and regulate the activity of the transcription factor Pho4, controlling expression of phosphate-responsive genes. The activity of Pho80-Pho85 is regulated in response to nutrient availability by the CDK inhibitor Pho81. In collaboration with John York's lab (HHMI, Duke University), we discovered that inhibition of Pho80-Pho85 by Pho81 requires an evolutionarily conserved secondary metabolite, the inositol pyrophosphate IP7. Levels of this metabolite are regulated in response to phosphate availability. Our current work is focused on identifying the mechanism underlying phosphate-dependent changes in inositol pyrophospate levels and defining the mechanism of inhibition of Pho80-Pho85 by Pho81 and IP7.

Sensing nutrient availability. Although we know much about the function and regulation of the phosphate-responsive signaling pathway downstream of the kinase Pho80-Pho85, we know less about how phosphate is sensed by cells and how changes in phosphate levels lead to changes in the kinase activity of Pho80-Pho85. To identify proteins that function upstream of this kinase we are performing a genome-wide screen to identify genes whose products are involved in phosphate sensing. This screen may reveal new connections between phosphate metabolism and other nutrient-sensing and metabolic 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, maximum transcriptional output, and the sensitivity of the response. To achieve this goal, we are using model promoters to analyze the relationship between transcription factor input and gene expression output in single cells. 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 we use these data to inform mechanistic models.

Encoding and decoding information in transcription factor dynamics. Many transcription factors undergo dynamic changes in subcellular localization in response to alterations in signaling. We are interested in whether different patterns of transcription factor dynamics are generated in response to different environmental changes, and whether different dynamic patterns produce distinct patterns of gene expression. To investigate these issues we are using microarrays and single-cell experiments to study the budding yeast transcription factor Msn2, which is activated in response to numerous stress conditions and controls expression of ~100 genes.

Transcriptional regulatory network evolution. In collaboration with Aviv Regev (Massachusetts Institute of Technology/Broad Institute), 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 are combining 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. Organisms exhibit oscillations in metabolism and behavior that are coordinated with 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) make up the timekeeping mechanism 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—thus, the oscillator in cyanobacteria is post-translational. 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—the relative insensitivity of its period to changes in protein concentrations and temperature—which are observed in vivo and in vitro.

Studies of clock input. Circadian clocks receive environmental input, which allows them to adjust the phase of their internal oscillator to match cycles in the environment. We are developing a mechanistic understanding of how changes in light availability and temperature can shift the phase of the oscillator. These studies build on pioneering genetic work of Kondo and Susan Golden (University of California, San Diego), who have identified proteins involved in interactions with the core oscillator. Our recent work explores the influence of metabolism on the phase shift.

Studies of clock output. The cyanobacterial circadian clock coordinates cell physiology with the environmental light-day cycle, in part by controlling gene expression. Genetic studies have identified several proteins that are required for circadian gene expression. Additionally, it has been shown that the state of supercoiling of endogenous plasmids and the state of chromosome compaction vary with circadian time. Our recent work has shown that changes in supercoiling correlate with circadian gene expression and that perturbation of supercoiling can elicit gene expression changes similar to those in the circadian cycle. We are now focused on determining how the core oscillator controls supercoiling and on defining what determines how promoters respond to changes in superhelical status. We are also investigating the role of proteins required to generate circadian oscillations in gene expression.