Karyotypic Determinants of Chromosome Instability Figure 4. Frequency of aneuploid chromosomes in the different CIN classes. The graph shows the frequency at which each of the 16 chromosomes was found in aneuploidy (either monosomic in a diploid background or disomic in a haploid background) in the analyzed aneuploid strains. The frequency histogram is stratified across the three different CIN classes. Red: HU strains; blue: MU strains; green: S strains. P-value refers to the global association between the 16 chromosomes and the three CIN classes according to a Fisher’s exact test. doi:10.1371/journal.pgen.1002719.g004 The effect of ChrVII and ChrX imbalance on CIN may be explained by a dosage imbalance between MAD1 and MAD2 genes A recent study found that heterozygosity of MAD2, located on ChrX and encoding a key component of the spindle assembly checkpoint (SAC) [27], leads to partial SAC inactivation and elevated CIN in a diploid background [28]. This effect can be rescued by restoring a 1:1 stoichiometry for the gene copy number of MAD2 vs. MAD1, located on ChrVII and encoding another SAC component that physically interacts with Mad2 protein [28]. This finding implies a ratio of 0.5 for the copy numbers of MAD2:MAD1 to be sufficient to induce CIN and led us to hypothesize that the association of ChrX and ChrVII imbalance with CIN may be attributed to an imbalanced MAD2:MAD1 ratio of 0.5. To increase the statistical power for testing this hypothesis over the data from the 27 karyotyped strain populations, we isolated another 56 fresh aneuploid strains as the meiotic products of the same triploid strain used before. These aneuploid strains were again collected ,20 cell divisions after tetrad dissection and an aliquot of each population was used for analysis of the relative gene copy number of MAD1 vs. MAD2 by qPCR on genomic DNA. Each g20 population was also plated to single colonies and after three days of growth at 23uC, 11 colonies were randomly picked from each and analyzed by FACS to determine ploidy variation (Figure 5B). This analysis found 44 of the 55 aneuploid strains to display unstable ploidies with obviously divergent ploidy profiles between the 11 colonies or Coefficient of Variation (CV) of the G1 peak positions much larger than that of the control haploid sample, whereas 11 strains were found to be ploidy-stable where the 11 picked colonies showed identical G1 peak position and CV similar to or smaller than that of the haploid control (Table S3). Genomic qPCR analysis of the g20 population samples using probes against MAD1 and MAD2 gene sequences found that: (i) strains with different MAD2:MAD1 ratios were not uniformly distributed across the stable and unstable strains (P = 0.027, Figure 5C); and (ii) 18 aneuploid strains with a MAD2:MAD1 ratio of 0.5 all fell into the ‘ploidy unstable’ category, whereas all 11 ‘ploidy stable’ aneuploid strains had a MAD2:MAD1 ratio of 1 or higher, indicating a highly significant association of a MAD2:- MAD1 ratio of 0.5 with CIN (P = 0.01, Figure 5D). We note that 25 strains with unstable ploidy did not have the MAD2:MAD1 ratio to be 0.5, indicating that a MAD2:MAD1 ratio of 0.5 was sufficient but not required to cause elevated CIN. qPCR analysis using probes against the ChrVII or ChrX arm opposite to the MAD1 or MAD2 locus, respectively, indicated that the MAD2:MAD1 gene copy number imbalance was indeed due to copy number imbalance between these two chromosomes (Table S3). These results demonstrate a specific case where gene dosage imbalance affecting two components of the mitotic system underlies the association between chromosome imbalance and CIN. Discussion The results described above support the notion that aneuploid genomes are in general less stable than euploid genomes and prone to further karyotype changes. These findings in yeast are in agreement with recent observations that chromosomally stable pseudo-diploid human cells that accumulate aneuploid chromo- somes frequently become chromosomally unstable [29]. However, our results also indicate that different aneuploid karyotypes can exhibit different degrees of CIN, with some being more stable than others, suggesting that CIN is not a necessary outcome of aneuploidy. In other words, CIN does not appear to be caused PLoS Genetics | www.plosgenetics.org 8 May 2012 | Volume 8 | Issue 5 | e1002719