Assessing the contribution of low-copy-limit DSGs to the proliferation defects of aneuploid cells

All disomic yeast strains exhibit proliferation defects (Torres et al. 2007), but only one disomy, disomy VI, is lethal, as this chromosome harbors the β-tubulin-encoding gene TUB2 (Neff et al. 1983; Katz et al. 1990; Liu et al. 1992). The reasons why disomes proliferate more slowly than euploid cells under standard growth condition are unknown. To determine whether the slow proliferation of disomes is caused by two copies of DSGs[5], we compared the doubling times of yeast strains harboring a specific disomy with wild-type strains containing CEN plasmids carrying DSGs[5] encoded by the disomic chromosome. For instance, chromosome XI encodes two DSGs[5]: SPC42 and TPK3 (Table 1). Introduction of a CEN plasmid carrying these two genes into wild-type cells did not significantly lengthen cell cycle time (Fig. 1C) even though expression of the two genes was comparable with that of disome XI cells (Supplemental Fig. 2N). Similar results were obtained for DSGs[5] encoded on chromosomes IX (PRK1) (Fig. 1D; Supplemental Fig. 2J), VIII (SFB3 and STE12) (Fig. 2A; Supplemental Fig. 2H), X (BFA1, KAR2, IRC8, and TPK1) (Fig. 2B; Supplemental Fig. 2L,M), and XV (EFT1 and SLG1) (Fig. 2C; Supplemental Fig. 2R). Introducing DSGs[5] encoded on chromosomes IV (ARF1 and SAC6) (Fig. 1E; Supplemental Fig. 2F) and XIV (KSH1, PUB1, and SSN8) (Fig. 1F; Supplemental Fig. 2P) into wild-type cells did not slow proliferation either; however, in these two cases, expression of one of the DSGs[5] (SAC6 or PUB1) (Supplemental Fig. 2F,P) from the CEN plasmid did not reach expression levels observed in the disomes.

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Figure 2.

Consequences of an additional copy of genes with copy number limits of <10 (limit 0–10, henceforth DSGs[10]) on cell proliferation. (A–F) Cells were grown as described in Figure 1. Genes were grouped into graphs according to chromosomal location. Dark-gray bars represent control strains carrying empty plasmids, light-gray bars represent the corresponding disomic strain (Dis), and striped bars represent DSG plasmid-containing strains. Empty vector CEN plasmid controls were EP1-TRP1, EP2-HIS3, and EP3-LEU2. SD is shown. (E) (**) P = 0.001. (F) (***) P = 0.0002; Student's t-test. The strains, in order, were A22361, A27036, A35953, and A36930 (A); A22361, A27093, A36953, and A36936 (B); A22361, A29730, A36950, and A36943 (C); A22361, A13975, A36954, and A36933 (D); A22361, A12685, A36950, and A36923 (E); and A22361, A28344, A36953, and A36942 (F).

Only three DSGs[5] led to a significant proliferation defect when expressed from a CEN plasmid. GLN3, the sole DSG[5] encoded on chromosome V, slowed the doubling time of wild-type cells by 5.1 min (±1.3 min) (Fig. 1G; Supplemental Fig. 2G). For comparison, an additional copy of chromosome V increases doubling time by 33 min (±3.1 min). DSGs[5] encoded on chromosome XVI also slowed proliferation. Chromosome XVI encodes 10 DSGs[5] (Table 1). Cells disomic for chromosome XVI proliferate 25 min (±2.3 min) slower than wild type (Fig. 1H,I). We examined the effects of six chromosome XVI DSGs[5] individually and in combination. A plasmid containing TEF1 and VPS4 slowed doubling time by 6.3 min (±0.7 min) (Fig. 1H; Supplemental Fig. 2U). Additionally, when combined with a plasmid harboring the DSGs[5] KES1 and PEP4, time of doubling increased by 14.6 min (±1.8 min) compared with empty vector controls (Fig. 1I). The addition of HAA1 and TPK2 with TEF1 and VPS4 did not slow proliferation more than TEF1 and VPS4 alone (Fig. 1I).

To determine how an additional copy of KES1, PEP4, TEF1, and VPS4 interferes with cell proliferation, we analyzed progression through the cell cycle in cultures synchronized by centrifugal elutriation. Previous cell cycle studies of disome XVI cells revealed a substantial G1 delay, largely due to a growth defect (Thorburn et al. 2013) and a metaphase delay (Torres et al. 2007). Wild-type cells expressing an extra copy of KES1, PEP4, TEF1, and VPS4 exhibited a minor G1 delay, as judged by a subtle delay in bud formation (Supplemental Fig. 3A) and a 15-min-long metaphase delay (Supplemental Fig. 3B). TEF1 overexpression was previously proposed to interfere with bud formation, which could explain the slight delay in bud emergence (Munshi et al. 2001). We do not yet understand why expressing this gene combination causes a metaphase delay. Overall, however, we conclude that DSGs present in low copy number cannot account for the proliferation defect observed in disomic yeast strains.

Ten-copy-limit DSGs cause a minor cell proliferation defect

Next, we investigated the possibility that synergistic effects of genes with slightly higher copy number limits account for the proliferation defects of disomes. We examined the consequences of expressing genes with copy number limits of <10 (limit 0–10, henceforth DSGs[10]) from CEN plasmids. Chromosome VIII encodes six DSGs[10] (Table 1). Expression of five of these genes (DMA1, MYO1, OPI1, SFB3, and STE12) together did not interfere with cell proliferation (Fig. 2A; Supplemental Fig. 2H,I). Similar results were obtained for DSGs[10] encoded on chromosomes X (BFA1, KAR2, HSP150, IRC8, and TPK1) (Fig. 2B; Supplemental Fig. 2L,M), XV (EFT1, RPS12, and SLG1) (Fig. 2C; Supplemental Fig. 2R), and IX (AXL2, PRK1, and TED1) (Fig. 2D; Supplemental Fig. 2J,K). Coexpression of DSGs[10] from either chromosome II or XIV conferred a measurable proliferation defect. APE3, OM14, PET9, and TEF2 expressed from a CEN plasmid lengthened doubling time by 5.8 min (±1.4 min) compared with control cells (Fig. 2E; Supplemental Fig. 2D). Cells with plasmids containing the six genes with limits from 0 to 10 on chromosome XIV exhibited doubling times 15 min (±3.2 min) slower than control cells (KSH1, MKS1, POR1, PUB1, SSN8, and TOM70) (Fig. 2F; Supplemental Fig. 2P,Q). For comparison, the doubling time of disome XIV cells is 32 min (±5.2 min) slower than that of wild-type cells (Fig. 2F).

Furthermore, for chromosomes I, II, and XI, we determined the effects of expressing all genes with upper limits of ≤20 copies (DSGs[20]; for chromosome XI, we were only able to analyze three of four DSGs[20]). Proliferation was not significantly slowed when DSGs[20] encoded on chromosome I or XI were expressed from CEN plasmids (Fig. 3A,B; Supplemental Fig. 2B,C,N,O). Coexpression of all DSGs[20] located on chromosome II led to an increase in doubling time by 6.6 min (±1.4 min) (Fig. 3C; Supplemental Fig. 2D,E) compared with cells containing empty vectors, which is a slightly slower rate of doubling than seen in cells harboring a plasmid containing chromosome II DSGs[10] alone (Fig. 2E).

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Figure 3.

Effects of DSGs[20] on cell proliferation. (A–C) Cells were grown as described in Figure 1. Genes were grouped into graphs according to their chromosomal location. Dark-gray bars represent control strains carrying empty plasmids, light-gray bars represent the corresponding disomic strain (Dis), and striped bars represent DSG plasmid-containing strains. Empty vector CEN plasmid controls were EP1-TRP1, EP2-HIS3, and EP3-LEU2. SD is shown. (****) P < 0.0001; Student's t-test. The strains used, in order, were A22361, A12683, A36954, and A36922 (A); A22361, A28266, A36953, and A36939 (B); and A22361, A12685, A35954, and A36925 (C).

Additionally, we note that an extra copy of DSGs did not cause other phenotypes characteristic of disomic yeast strains such as sensitivity to high temperature, cycloheximide, or phleomycin (Supplemental Fig. 4). This was not unexpected given that gTOW DSGs were identified based on their proliferation defect, not sensitivity to adverse growth conditions.

Our results indicate that a doubling in gene dosage of DSGs[5] is partly responsible for the proliferation defect of only two out of nine disomic yeast strains analyzed (disomes V and XVI). A portion of the proliferation defect of an additional two disomies (disomes II and XIV) can be explained by a doubling in gene dosage of DSGs[10]. Nevertheless, it is important to state that no plasmids containing DSGs slowed doubling times to rates seen in cells expressing an extra copy of the corresponding whole chromosome. These results support the conclusion that the proliferation defect observed in disomic yeast strains is caused by increased expression of genes that, altered individually, have little or no observable effect rather than a few proliferation antagonists at altered copy number. We suspect that this concept applies to other phenotypes widespread among aneuploid cells. For instance, in other work, we were not able to identify a single gene responsible for the hydroxyurea sensitivity of disome VIII strains (Blank et al. 2015). Additionally, Chen et al. (2015) were unable to ascribe the hygromycin sensitivity of disome XV cells to a specific gene combination.

Deletion of DSGs from disomic strains does not improve cellular fitness

Our results reveal that an additional copy of some DSGs is sufficient to explain a small portion of the proliferation delay in disomic cells, but are DSGs necessary for the proliferation defects of disomic yeast strains? To address this question, we investigated the consequences of deleting one copy of GLN3 in cells disomic for chromosome V. Introduction of an extra copy of GLN3 into wild-type cells subtly impaired proliferation (Fig. 1G). However, disome V cells lacking one copy of GLN3 did not proliferate faster than cells disomic for a wild-type copy of chromosome V (Fig. 4A). Similar results were obtained for TEF1 and VPS4. Expression of these DSGs slows proliferation (Fig. 1H), but deleting one copy in disome XVI cells did not improve proliferation (Fig. 4B). Deletion of one copy of other chromosome XVI DSGs[5] (TPK2 or KES1) did not improve proliferation rate either (Fig. 4B).

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Figure 4.

Deletion of one copy of DSGs from disomic yeast strains does not improve cellular fitness. (A) Cells harboring an additional copy of chromosome V (A28265) or an additional copy of chromosome V lacking GLN3 (A36470) were grown as described in Figure 1 to measure their doubling time. (B) Cells harboring an additional copy of chromosome XVI (A34149) or an additional copy of chromosome XVI lacking the indicated genes (A36585, A36650, A36689, and A36859) were grown as described in Figure 1 to measure their doubling time. (C, left graph) Cells with an additional copy of chromosome IX (A13975) or an additional copy of chromosome IX lacking the indicated genes (A36233, A36215, and A36069) were grown as described in Figure 1 to measure their doubling time. The middle and right graphs show competition experiments in which equal numbers of disome IX cells harboring GFP (A36219) and disome IX cells lacking the indicated genes (A36233 and A36215) were coinoculated in –His G418 medium. The percentages of GFP-positive and GFP-negative cells were monitored at the indicated times. (D, left graph) Cells with an additional copy of chromosome XI (A28266) or an additional copy of chromosome XI lacking the indicated genes (A36919 and A36336) were grown as described in Figure 1 to measure their doubling time. The middle and right graphs show competition experiments in which equal numbers of disome XI cells expressing GFP (A36222) and GFP⁻ disome XI cells lacking the indicated genes (A36919 and A36336) were coinoculated in −His G418 medium. The percentages of GFP-positive and GFP-negative cells were monitored at the indicated times.

Similarly, we asked whether low-copy-limit genes might be necessary for disomic proliferative defects even if they were not sufficient. We deleted one copy of the DSG[5] PRK1, the DSGs[10] TED1 and AXL2, and the DSG[20] GVP36 in disome IX cells. Doubling time measurements did not reveal either a decrease in cell cycle duration or a fitness advantage when competed against the parental disome IX strain (Fig. 4C). The parental and DSG deletion disomic strains maintained an approximately equal share of the population following serial dilutions for 48 h. Similar results were obtained for DSGs encoded by chromosome XI (Fig. 4D). These data demonstrate that individual genes whose alteration in copy number is most toxic are not solely responsible for the proliferation defect of cells harboring an additional copy of the entire chromosome.

Doubling time analysis

Strains were grown overnight at room temperature in medium to select for maintenance of plasmids. The following morning strains were diluted to OD₆₀₀ = 0.1 in yeast extract/peptone medium containing 2% glucose (YEPD). The growth rate was measured in triplicate using a Biotek plate reader at 25°C. OD₆₀₀ measurements were taken every 15 min for 24 h. Only the period of exponential growth was used to determine doubling time. Two to four transformants per plasmid were tested on at least two different days and compared with a wild-type strain carrying the relevant empty vector plasmid. Data were accumulated using Gen5 software. Statistics were performed using GraphPad Prism software.

Competition experiments

Approximately equal amounts of cells with and without integrated PGK1-GFP were mixed in selective medium (−His +G418) at OD₆₀₀ = 0.15 and maintained in exponential growth phase at room temperature. Samples were taken every 12 h for 48 h. Each sample was fixed for 5 min in 3.7% formaldehyde in KPi (pH = 6.4). Relative cell populations in the cultures were measured by quantifying the percentage of GFP-positive versus GFP-negative cells via flow cytometry.

Real-time RT–PCR

Analysis of gene expression was performed as previously described (Thorburn et al. 2013).

Elutriation

Elutriation performed as described in Thorburn et al. (2013) with the exception that elutriated cells were resuspended in YEPD at 25°C.

We thank Frank Solomon and members of the Amon laboratory for suggestions and critical reading of this manuscript. This work was supported by the National Institutes of Health (GM056800 to A.A). A.A is also an investigator of the Howard Hughes Medical Institute.