ABSTRACT Aneuploidy, the state in which cells carry an incorrect number of chromosomes, is a major problem for human health. Aneuploidy is toxic during mammalian development and a leading cause of pregnancy loss. Down syndrome (DS) due to trisomy 21 is one of the few viable aneuploid syndromes, but affected individuals have life-long problems including premature aging. Despite intense study, the reasons for aneuploidy toxicity are still incompletely understood, presenting challenges for understanding DS. In contrast, aneuploidy is very common in human cancers, where most tumors tolerate and may even benefit from extra chromosomes. It is unclear how cancer cells overcome the stress of aneuploidy, because we don’t fully understand how aneuploidy affects cells in the first place. This proposal will utilize an extremely powerful and unique system to study the consequence of aneuploidy in an important model system, wild strains of budding yeast Saccharomyces cerevisiae. Yeast is a powerful model for dissecting cellular biology, because many of the mechanisms and defense strategies are conserved in humans. We recently made an exciting discovery that chromosome duplication in healthy yeast strains disrupts nutrient responses and quiescence, a conserved cellular program important for growth control and cell maintenance and renewal. Strains of multiple genetic background and carrying different chromosome amplifications display shared phenotypes, including incomplete cell-cycle arrest upon nutrient depletion, metabolic aberrations, defects in quiescence-induced silencing, and ultimately reduced chronological life span. This is remarkable, because defects in similar markers of quiescence are seen in both DS and many cancers – if disruption of quiescence is a conserved response to aneuploidy, it could have transformative impacts for future studies. This grant will elucidate how aneuploidy disrupts quiescence in an important eukaryotic model system. Aim 1 will use dynamic transcriptomics and single-cell microscopy to characterize the temporal order of defects, test several initial hypotheses, and implicate upstream regulators. It will also distinguish common versus chromosome-specific effects. Aim 2 will use a barcoded plasmid over-expression library to identify genes that complement aneuploid defects along the progression to quiescence. Integrating Aim 1 and 2 results will define a temporal map of genes and processes defective in aneuploid yeast strains and involved in quiescence. It will also point to the upstream defect(s) directly caused by chromosome duplication, whose further study will expand our understanding of aneuplodiy Aim 3 will use genomic, proteomic, single-cell and single-molecule analysis to define and characterize the “Ssd1 Q granule”, a phase separated granule containing the RNA-binding protein Ssd1, which we previously showed is fundamental for aneuploidy tolerance in healthy yeast. Since many mechanisms in yeast are conserved in higher or...