We are interested in understanding cellular signalling processes in budding yeast using functional genomics, high content screening and gene expression tools.
We use functional genomics tools to study pathways regulated by signalling and transcriptional enzymes through assessment of genetic redundancy and sensitivity to increased gene dosage.
Post translational modifications
Sara Sharifpoor, Wendy Liang, Harsha Garadi Suresh
We perform synthetic lethal or synthetic dosage lethal screens using Synthetic Genetic Array (SGA) technology to study genetic interactions of genes encoding a variety of post-translational modification enzymes (e.g. kinases and lysine deacetylases, also known as KDACs). Using colony size as a read-out, we assess increased or reduced fitness that results from a combination of null, hypomorphic or hyperactive alleles. To examine the effects of dosage, our lab has created a variety of yeast collections that can be used in the SGA platform. We use functional genomics to interrogate important signalling pathways, such as those involving phosphorylation, acetylation, and ubiquitylation. As such, we use a variety of computational and statistical methods to assess fitness using accurate measurements of colony size. Our ultimate goal is to unravel the complexity of complex signalling networks through large-scale analysis of genetic interactions.
Elena Kuzmin, Vincent Messier
In collaboration with Dr. Charlie Boone, our lab is also assessing combinatorial mutations (e.g. in double and triple mutants) to look for reduced or increased fitness of strains that can be attributed to perturbation of genes involved in similar processes; a phenomenon called genetic buffering. We are making all pair-wise double mutants using all 6000 genes, to assess the overall level of cellular buffering. We are also making triple mutants to fine-tune the degree of redundancy for highly buffered genes in the genome. In addition we are interested in understanding why a large fraction of genes have few or no genetic interactions. To answer this question we are now exploring conditional genetic interactions.
Our lab is interested in the underlying genome-wide changes that arise in cells under genetic or environmental conditions, an area of research called phenomics. We use high-throughput cell biological assays to look for either changes in localization and abundance of proteins or defects in subcellular morphology.
Dynamics of proteinlocalization and abundance
Mike Cox, Ben Grys,Oren Kraus, Helena Friesen, Harsha Garadi Suresh
High-throughput fluorescence microscopy has become a powerful tool in our lab to look for targets of enzymes (e.g. kinases, KDACs). To this end, we are currently screening for changes in the abundance and subcellular distribution of GFP-tagged proteins. We use Synthetic Genetic Array technology coupled with high-content screening (SGA-HCS) for genome-wide analysis of protein localization and abundance, under a variety of genetic, environmental and chemical perturbations. Changes in protein abundance and localization drive the cell cycle. To understand the dynamics of these properties, we are quantifying and analyzing the expressed yeast proteome in asynchronous and synchronized cell populations. Protein abundance and localization are often regulated by post-translational modifications. We are exploring how phosphorylation, acetylation and ubiquitylation control proteins by doing SGA-HCS in a varierty of genetic backgrounds.
Sub-cellular morphology - "The marker project"
Erin Styles, Adrian Verster, Dara Lo, Huan Lian
We are also using SGA-HCS to detect defects in subcellular morphology that are attributed to a genetic or environmental stimulation. The so called "marker project" aims to identify the subcellular morphology associated with mutation of every gene in yeast. In addtion, we are exploring the underlying cell biological causes of drastic phenotypic outcomes (e.g. lethality), as well as trying to understand the subtle intracellular changes that arise from a combination of genetic stresses.
Cell cycle-regulated transcription
In eukaryotic cells, cell division is primarily controlled in G1 phase of the cell cycle at a regulatory nexus called the restriction point or Start. The importance of understanding Start and other cell cycle transitions is underscored by the observation that perturbations of cell cycle regulators appear to be a universal feature of cancer cells. To get an overall picture of cell-cycle-dependent transcription we are screening for regulators of every class of cell-cycle- dependent transcript using Reporter-SGA (R-SGA) and identifying the mechanisms of action through directed experiments that monitor transcript levels at different stages of the cell cycle.
Our lab has developed a fluorescent reporter system called Reporter Synthetic Genetic Array (R-SGA) that exploits the tools available in budding yeast to assess consequences of genetic perturbations (e.g. gene deletion) on gene expression. In R-SGA, both a control promoter fused to RFP and a test promoter of interest fused to GFP, are introduced into an array of haploid deletion mutants producing an output array where both reporter genes are combined with each deletion mutant. The resulting panel of yeast deletion mutants is then assayed for enhanced or diminished promoter-GFP expression by scanning both fluorescence intensities directly from colonies arrayed from agar plates using a scanning fluoroimager. So far, our R-SGA project has focused largely on applications of the deletion mutant collection, but we are now extending this approach to overexpression collections.