Author Summary Aneuploidy, the state of harboring an unbalanced number of chromosomes, has long been hypothesized to be at the basis of malignant transformation. Recent studies have also shown that aneuploidy is an important form of genome alteration underlying adaptive evolution of cells in response to harsh environments or genetic perturba- tions. In addition to the profound effect that aneuploidy has on gene expression and phenotype, another feature thought to contribute to aneuploidy’s role in cancer and cellular evolution is the heightened chromosome instabil- ity of aneuploid cells. Since chromosome instability is the condition of gaining and losing chromosomes at a high frequency, this could lead to a vicious cycle in which aneuploidy could lead to further enhanced genetic diversity. Given the ever-changing and heterogeneous aneuploid cell populations, and the difficulty of separating the effect of aneuploidy from other types of genetic aberrations, the molecular mechanisms underlying aneu- ploidy-driven chromosome instability have remained largely unexplored. Here we describe the first unbiased and systematic investigation of chromosome instability associated with aneuploid genomes in the budding yeast Saccharomyces cerevisiae. Our results revealed both genome-level and chromosome-specific determinants of chromosome instability in aneuploid yeast. Our findings potentially help explain the molecular mechanism under- lying a major source of genome instability in cancer. the haploid parent [18]. However, none of these studies investigated into the cellular mechanisms by which an aneuploid karyotype causes CIN. Insights into the mechanisms by which aneuploidy leads to CIN are important for understanding the dynamics of the cellular adaptation process and may ultimately enhance our ability to predict or modulate cancer progression. For example, as stable phenotypes are likely to require a certain degree of genetic stability, there may exist metastable aneuploid constellations amidst the genome chaos. If this was true, what may be the determinants underlying stable or unstable aneuploidy? Formally, aneuploidy could cause CIN through three conceptually distinct though not mutually exclusive mechanisms. First, as aneuploidy leads to varying degrees of growth impairment compared to euploids under standard culture conditions [14,15], CIN may be induced by the cellular stress present under such conditions. If this hypothesis were correct, then CIN would correlate with the level of growth impairment in specific aneuploid strains. Second, according to the genome chaos theory, the more chromosomes are in aneuploidy in a genome the more unstable that karyotype is expected to be [19]. If this hypothesis were true, then CIN would correlate with how far a strain deviates from the nearest euploid state. Third, it is possible that aneuploid chromosome stoichiom- etry leads to dosage imbalance for specific genes encoding structural or regulatory components that ensure chromosome stability. This possibility was previously proposed based on imbalance of mitotic spindle components directly involved in chromosome segregation [20]. If this were correct, then correla- tions might be found between CIN and the relative copy numbers of specific chromosomes or combinations thereof. In this paper we used an unbiased approach to examine the karyotypic features underlying CIN by generating random aneuploid karyotypes through triploid meiosis and following the dynamics of aneuploid populations with distinct original karyo- Karyotypic Determinants of Chromosome Instability types. Our results support a model in which CIN is promoted by both genome-level and chromosome/gene-specific determinants. Results Aneuploid yeast strains generated from triploid meiosis display varying degrees of chromosome instability Two methods were instrumental for the analysis explained below in this study. First, we used a high-throughput flow cytometry-based (FACS) assay to determine the overall genome content (referred to as ploidy) of a population of yeast cells. A non-integer ploidy revealed by FACS is likely to correspond to an aneuploid genome. However, FACS data is insufficient to reveal copy number for each chromosome. For this we used a recently established method for determining the relative copy number for each of the 16 yeast chromosomes that is based on quantitative polymerase chain reaction (qPCR) [15]. Combining the ploidy information revealed by FACS and chromosome stoichiometry revealed by qPCR allows determination of an aneuploid karyotype [15]. In our previous work [15], we generated isogenic aneuploid yeast strains with random chromosome stoichiometries as meiotic products from sporulated homozygous triploid or pentaploid strains of the S288c background. During meiosis I, chromosome segrega- tion of an odd number of homologs leads to highly frequent aneuploid spore progenies with random karyotypes [16]. In this work, we took a similar approach to generate fully isogenic aneuploid yeast strains, and consistent with previous studies [15,17], 45% of the aneuploid meiotic products were viable and gave rise to colonies (52 viable spores out of 116 expected from 29 tetrads). Unlike our previous study [15], however, where aneuploid strains with stable karyotypes were chosen for phenotypic comparison and gene expression analyses, in this study our goal was to follow karyotype changes for all (within our experimental limitations, see below) viable aneuploid spores resulting from triploid meiosis. Due to a lack of established methods for single-cell karyotyping in yeast, however, we devised a population-based approach (Figure 1A) that was predicated on the assumption that the modal karyotype of the population within a small colony reflects the karyotype of the cell that seeded the colony. As illustrated in Figure 1A, the colony grown from each of the 52 viable spores was picked in its entirety after the spore had undergone ,20 cell divisions, resuspended in liquid media and the actual cell number and thus the number of cell divisions was estimated (see Materials and Methods for details). As the aneuploid colonies grew at different rates, the colonies were picked at different times after tetrad dissection, and the time of colony picking was recorded. Each resulting culture at this time point was called generation 20 (g20) population sample. Due to contamination, only 47 g20 populations were obtained and further analyzed (see Figure S1). A small aliquot of the g20 population sample for each spore colony was used for FACS and qPCR karyotyping analysis, giving rise to the g20 population data (see below). If the initial aneuploid karyotype of a growing spore colony were unstable, karyotype heterogeneity would be expected within the g20 population. This karyotype heteroge- neity could in turn allow us to estimate the level of CIN of the initial aneuploid karyotype (see below). To observe it, ,200 cells from each g20 population sample were spread onto a YPD plate. As soon as the resulting colonies were visible, 11 colonies were randomly chosen from each plate (see Materials and Methods), harvested and frozen for prospective karyotype analysis by FACS and qPCR. These were referred to as the g20 colony samples, and a total of 47611 = 517 such samples were harvested and stored. Since some aneuploid karyotypes were more stable than others, to allow for more cell divisions that could give rise to karyotypic PLoS Genetics | www.plosgenetics.org 2 May 2012 | Volume 8 | Issue 5 | e1002719