Many disease states result from gene overexpression, often in a specific genetic context. To explore gene overexpression phenotypes systematically, we assembled an array of 5280 yeast strains, each containing an inducible copy of an S. cerevisiae gene, covering >80% of the genome. Approximately 15% of the overexpressed genes (769) reduced growth rate. This gene set was enriched for cell cycle-regulated genes, signaling molecules, and transcription factors. Overexpression of most toxic genes resulted in phenotypes different from known deletion mutant phenotypes, suggesting that overexpression phenotypes usually reflect a specific regulatory imbalance rather than disruption of protein complex stoichiometry. Global overexpression effects were also assayed in the context of a cyclin-dependent kinase mutant (pho85Delta). The resultant gene set was enriched for Pho85p targets and identified the yeast calcineurin-responsive transcription factor Crz1p as a substrate. Large-scale application of this approach should provide a strategy for identifying target molecules regulated by specific signaling pathways.

Summary Odorant receptors (ORs) in the olfactory epithelium bind to volatile small molecules leading to the perception of smell. ORs are expressed in many tissues but their functions are largely unknown. We show multiple ORs display distinct mRNA expression patterns during myogenesis in vitro and muscle regeneration in vivo. Mouse OR23 (MOR23) expression is induced during muscle regeneration when muscle cells are extensively fusing and plays a key role in regulating migration and adhesion of muscle cells in vitro, two processes common during tissue repair. A soluble ligand for MOR23 is secreted by muscle cells in vitro and muscle tissue in vivo. MOR23 is necessary for proper skeletal muscle regeneration as loss of MOR23 leads to increased myofiber branching, commonly associated with muscular dystrophy. Together these data identify a functional role for an OR outside of the nose and suggest a larger role for ORs during tissue repair.

Calcineurin is a Ca

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase required for Saccharomyces cerevisiae to adapt to a variety of environmental stresses. Once activated, calcineurin dephosphorylates the Zn-finger transcription factor Crz1p/Tcn1p, causing it to accumulate in the nucleus where it activates gene expression. Here we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates and negatively regulates Crz1p activity by inhibiting its nuclear import. Activation of PKA in vivo decreases Crz1p-dependent transcription. PKA phosphorylates Crz1p in vitro, and we identify specific residues required for this phosphorylation, all of which reside in or adjacent to the nuclear localization signal. Mutation of these residues to alanine results in increased nuclear import of Crz1p and results in higher levels of both basal and Ca2+-induced Crz1p transcriptional activity. PKA regulates the general stress response in yeast and coordinates this response with nutrient availability. In contrast, calcineurin regulates the cellular response to a restricted set of environmental insults. Thus, these studies identify a specific biochemical mechanism through which the activities of multiple stress-activated signaling pathways are integrated in vivo.

Sca-1 (Stem Cell Antigen-1) is a member of the Ly-6 family proteins that functions in cell growth, differentiation, and self-renewal in multiple tissues. In skeletal muscle Sca-1 negatively regulates myoblast proliferation and differentiation, and may function in the maintenance of progenitor cells. We investigated the role of Sca-1 in skeletal muscle regeneration and show here that Sca-1 expression is upregulated in a subset of myogenic cells upon muscle injury. We demonstrate that extract from crushed muscle upregulates Sca-1 expression in myoblasts in vitro, and that this effect is reversible and independent of cell proliferation. Sca-1-/- mice exhibit defects in muscle regeneration, with the development of fibrosis following injury. Sca-1-/- muscle displays reduced activity of matrix metalloproteinases, critical regulators of extracellular matrix remodeling. Interestingly, we show that the number of satellite cells is similar in wild-type and Sca-1-/- muscle, suggesting that in satellite cells Sca-1 does not play a role in self-renewal. We hypothesize that Sca-1 upregulates, directly or indirectly, the activity of matrix metalloproteinases, leading to matrix breakdown and efficient muscle regeneration. Further elucidation of the role of Sca-1 in matrix remodeling may aid in the development of novel therapeutic strategies for the treatment of fibrotic diseases.