PROJECT SUMMARY/ABSTRACT The long-term goal of this research program is to understand the mechanistic and evolutionary causes of variation in complex traits. The primary experimental approach is to perform large-scale analyses of single-cell traits of the budding yeast, Saccharomyces cerevisiae, and follows two major lines of work. One line of work aims to understand how interaction between genes (epistasis) contributes to natural trait variation. Understanding the sources of variation in complex traits is a major goal in biomedical research because this knowledge impinges directly on the prospect of personalized medicine, for example the prediction of disease risk from an individual’s genotype. If not taken into account, epistasis can confound such predictions. Epistasis is also important because it can constrain evolutionary adaptation to follow particular paths, making adaptation more predictable. This predictability could be valuable in the treatment of diseases that have a strong evolutionary component, such as microbial infections and cancer. Although epistasis has been well studied using lab- derived mutations, as well as in some cases of viruses or microbes under strong pressures to evolve, its role in determining how traits vary in natural populations is poorly understood. Key goals of this research program are to perform experiments with dramatically increased power to detect interactions, and to expand the range of traits that are studied to include cell shape and size, which are important in many disease processes. These studies will leverage recent progress in using high-throughput, microscopy-based methods to quantify many independent cellular features, and they will create and use strains of S. cerevisiae that make searching for epistasis much more powerful. The other line of work aims to understand molecular mechanisms that allow clonal cell populations to generate heterogeneity that might be beneficial in the face of environmental uncertainty. Such heterogeneity is seen in the responses of pathogenic microbes and tumor cells to drugs, and therefore has major clinical implications, yet there is very little known about how heterogeneity is regulated and how it can be altered. Recent work has shown that clonal populations of S. cerevisiae contain fast-growing cells that are susceptible to acute stress and slow-growing cells that are tolerant of acute stress, and that these differences are mediated by variable activity of the conserved Ras/cyclic AMP/protein kinase A pathway. The role of this kinase in tuning growth rates and stress tolerances will be probed using chemical-genetic manipulation. The goal is to better understand the mechanistic basis of adaptive heterogeneity in this model system, and ultimately to advance treatment of persistent pathogens and cancers.