Heat shock stimulates genomic instability in S. cerevisiae JSC25-1
To determine the effect of heat shock on chromosomal stability in S. cerevisiae, a sectored colony assay system was first used. S. cerevisiae JSC25-1 is homozygous for ade2-1, which displays a red phenotype because of a red pigment accumulation (a precursor of adenine) (St Charles and Petes 2013). In this strain, the ochre-suppressing tRNA mutant gene SUP4-o was inserted in the right end of the YJM789-derived chr IV (St Charles and Petes 2013). Since only one SUP4-o copy could partly suppress the ochre mutation of ade2-1, JSC25-1 formed pink colonies on the solid medium. Increased (two) and reduced (zero) SUP4-o copies in JSC25-1-derived isolates produce white and red colonies, respectively. Figure 1a shows a crossover event initiated by a double-strand break (DSB) on the right arm of chr IV in the first cell cycle after plating results in the white/red-sectored JSC25-1 colonies. Break-induced replication (BIR) would produce red/pink- or white/pink-sectored colonies (Fig. 1a). This means that the frequency of sectored colonies determines the degree of chromosomal instability. Our results showed that the cell viability of JSC25-1 was reduced to 86%, 28%, and 1.2% after heat treatment (52 °C) duration of 3 min, 3.5 min, and 4 min, respectively (Fig. 1b). Without heat shock, the frequency of white/red JSC25-1 colonies was 3 × 10–5 (Fig. 1c), and this rate was elevated 2, 4, and 11 times after heat shock for 3 min, 3.5 min, and 4 min, respectively (Fig. 1c). Using the PCR diagnosis described in the materials and methods section, we found that 2 of the 25 red parts of the sectored colonies came from the loss of YJM789-derived chr IV. By contrast, no chromosome loss was detected in the 25 spontaneous sectored colonies. These results suggest that heat shock can stimulate mitotic recombination and chromosome instability in yeast.
Chr IV–specific SNP microarray analysis of sectored colonies
To map the selected crossover events in the white/red-sectored colonies, genomic DNA was extracted from the white and red sectors, respectively. The sample DNA and control DNA were competitively hybridized on a customized SNP microarray specific to chr IV (1.1 Mb, accounting for up to 10% of yeast genome) (St Charles and Petes 2013). This array includes 2,300 SNPs across the right arm of chr IV, allowing for the mapping of crossover events to about 0.5 kb resolution (Additional file 1: Fig. S1). In addition, other genomic regions were represented by sparse SNPs that allows for the detection of all chromosomes’ aneuploidy events (Additional file 1: Fig. S1). Figure 2 shows one example of the SNP microarray results. Since a single mismatch (SNP site) is sufficient to destabilize short duplexes, the genomic DNA homozygous for the W303-1A-derived SNPs hybridizes better with the W303-1A-derived probes than with the YJM789-derived SNPs and vice versa. The blue and red lines/points in Fig. 2 indicate the hybridization level of the DNA sample from W303-1A-derived and YJM789-derived SNPs, respectively. The hybridization ratio (HR) for each oligonucleotide was normalized to the Cy5/Cy3 ratio of all the oligonucleotides on the microarray. HR values close to 1.5, 1, and 0.2 indicate 2, 1, and 0 copies of a homolog, respectively. In the white sector, the hybridization signal was transferred from heterozygosity to homozygosity (YJM789-derived homolog) between coordinates 749,244 and 750,033 (Fig. 2a, b). In the red sector, a signal transition was identified between 753,192 and 754,057 (Fig. 2c, d). For this crossover event, the region from 749,244 to 754,057 bp was identified as the crossover-associated gene conversion tract, which should include the initial recombinational lesion (Fig. 2e). Considering both sectors within the gene conversion tract, the blue SNPs were represented thrice, and the red SNPs were represented once (Fig. 2e), defined as a 3:1 pattern. As discussed in previous studies (St Charles and Petes 2013; Yin and Petes 2013; Zhang et al. 2019), such a pattern indicates that the initial recombinational lesion is likely to be a DSB occurring at one sister chromatid in the S/G2 phase (Additional file 1: Fig. S2A). If a DSB took place in the G1 phase, both sister chromatids would harbor DSBs at the same location after DNA replication (Additional file 1: Fig. S2B). DSB repair on both sister chromatids led to a 4:0 region within the gene conversion tract (Additional file 1: Fig. S2B). Of the 16 analyzed crossover events, 4 have no detectable conversion tracts, 7 have 3:1 pattern tracts, and 5 have complex patterns (containing 4:0 region). These results indicate that DSBs in the S/G2 phase initiated more than half of the heat-shock-induced crossover events. It should be noted that the 4 crossover events with no detectable conversion tracts were not included in this calculation, because the phases of their initial DSBs were not identified.
Besides the crossover events on the right arm of chr IV, we also observed 8 chromosomal aneuploidy events, demonstrating the tendency of heat shock to cause chromosome aberration (Additional file 1: Table S1). We found 3 monosomic chromosomes (chr III, chr IX, and chr VI) and 5 trisomic chromosomes (chr III, chr IX, chr VI, chr VIII, and chr XVI) (Additional file 1: Table S1). As shown in Additional file 1: Table S1, we observed the monosomy of YJM789-derived chr III in the red sector and the trisomy of YJM789-derived chr III in the white sector. Such a paired event took place in three sectored colonies. These results indicate that heat shock would interrupt the normal segregation of sister chromatids and cause chromosomal nondisjunction.
Whole-genome SNP microarray analysis of JSC25-1-derived isolates after heat shock
To explore how heat shock affects chromosomal stability at the whole-genome level, JSC25-1 cells (2 OD600) were heated at 52 °C for 4 min and plated on YPD plates to form colonies. A total of 21 colonies (named JP1–JP21) were randomly selected for SNP microarray analysis.
Mitotic recombination events and loss of heterozygosity (LOH)
In yeast, mitotic recombination is the main pathway to repair DSBs during vegetative growth, which is crucial to cell viability in the presence of DNA damage agents but inevitably leads to LOH (Symington et al. 2014). Thus, detection of LOH events in the heat-shock-treated yeast cells allows the determination of heat-shock-induced DSBs and mitotic recombination. As shown in Fig. 3a, we observed an increase in the signal of YJM789-derived SNPs and a decrease in the signal derived from the W303-1A homolog near 240 kb of chr XII. This result indicates an internal LOH (gene conversion) on the chr XII, which may be explained by the repair of a DSB around 240 kb on the W303-1A-derived chr XII using the YJM789-derived homolog as a template. In addition, we also observed that the region from 625 kb to the right end was homozygous for the YJM789-derived sequence. Such terminal LOH might be due to a reciprocal crossover or a BIR event, as shown in Fig. 1a. In summary, we found 17 genomic alterations, including 11 gene conversions, 5 terminal LOH events, and 1 internal deletion in the 21 JSC25-1-derived isolates after heat shock (Fig. 3b and Additional file 1: Table S2). The rate of mitotic recombination in heat-shock-treated cells was calculated at about 3 × 10–2 events per genome per cell division (17 events/21 isolates/25 cell divisions) during the growth from a single cell to a colony on the YPD plate. Figure 3b shows the patterns of all detected genetic events, and Fig. 3c presents the distribution of genetic events across 16 chromosomes. All these genetic events have unique genomic locations, indicating that none of these events took place before heat shock. Previously, O’Connell et al. (2015) detected 10 LOH events in 10 diploid yeast isolates (isogenic to JSC25-1) that underwent 5,000 cell divisions, which means that the spontaneous rate of mitotic recombination in a wild type strain was about 2 × 10–3 per genome per division. Our results showed that heat shock (52 °C for 4 min) elevated the mitotic recombination rate by at least one order of magnitude at the whole-genome level.
Aneuploidy events detected in the JSC25-1-derived isolates
Besides mitotic recombination, aneuploidy events occurred frequently in the 21 heat-shock-treated, JSC25-1-derived isolates. Figure 4a, b show examples of chromosome loss (monosomy) and duplication (trisomy), respectively. In some cases, the HR values of one chromosome were reduced to 0.2 while those of the other were increased to 1.5 (Fig. 4c). This pattern indicates a uniparental disomy (UPD) event. In Fig. 4d, we showed chromosome copy number changes in the 21 isolates. There were 26 trisomic chromosomes, 4 uniparental chromosomes, 2 monosomic chromosomes, 1 pentasomic chromosome, and 1 tetrasomic chromosome. The aneuploidy event frequency was about 6.5 × 10–2 (34 events/21 isolates/25 cell divisions) in the heat-shock-treated cells. At a horizontal level, the isolates with similar aneuploidy events were clustered into groups. We found that 11 of the 21 isolates had at least one aneuploidy event. In addition, trisomic chromosomes occurred at a significantly higher rate than monosomic chromosomes in the heat-shock-treated yeast cells, which may be because chromosome loss is strongly and dominantly deleterious. Zhu et al. (2014) identified 29 trisomy and 2 monosomy events in 145 wild-type diploid isolates that underwent ~ 311,000 cell divisions, indicating that the spontaneous rate of aneuploidy is about 1 × 10–4 events per diploid genome per generation. Thus, our results showed an elevated chromosomal aberration rate of two orders of magnitude by heat shock.
Different chromosomal instability patterns caused by heat shock and carbendazim
We hypothesized that heat-shock-induced aneuploidy could be ascribed to the suppression of microtubule assembly dynamic. To test this hypothesis, chromosomal instability patterns caused by heat shock and carbendazim (microtubule inhibitor) were compared. Forty JSC25-1-derived isolates (MT1–MT40) were treated with 25 mg/L carbendazim for 2 h and then plated on YPD plates. For each isolate, only one colony was randomly selected for chr IV–specific SNP microarray analysis. In the 40 selected mutants, we observed 10 terminal LOH events (2 on chr II, 3 on chr IV, 3 on chr VII, 1 on chr X, and 1 on chr XVI). Figure 5a shows an example of these events. In the mutant MT39, we detected a deletion of the right arm of the W303-1A-derived chr III (Fig. 5b) and a duplication of the left arm of the YJM789-derived chr XIV (Fig. 5c). Such paired event is likely to reflect the DSB repair occurring on the right arm of chr III using the left arm of chr XIV through the BIR pathway. In Fig. 5c, we showed at least one aneuploidy event in 18 mutants. In the 40 analyzed mutants, there were 28 monosomic chromosomes, 9 trisomic chromosomes, 4 tetrasomic chromosomes, and 27 UPD (Fig. 5d). Interestingly, 25 of these 27 UPD events occurred in two mutants (MT3 and MT12). The peculiar karyotypes of these two isolates suggest that carbendazim treatment readily causes UPD. Except for MT3 and MT12, other isolates tended to lose chromosomes (Fig. 5c). This result was consistent with those of previous studies showing that carbendazim exposure causes frequent chromosome loss in both yeast and mammalian cells (Wood 1982; Zuelke and Perreault 1995; Zheng et al. 2017). Using Fisher’s exact test, we found that the monosomy–trisomy ratio in carbendazim-treated cells was significantly higher than that of heat-shock-treated cells. This result indicated various mechanisms underlying the carbendazim- and heat-shock-induced aneuploidy events.
Heat shock drives phenotypic diversification in JSC25-1
While chromosome aneuploidy and large-scale chromosomal rearrangements are always detrimental to mammalian cells, these genetic events enhance the adaptability of yeast under certain conditions (Gilchrist and Stelkens 2019). To examine whether and to what extent heat shock can promote phenotypic changes in yeast, we compared untreated and heat-shock-treated cells in terms of the frequencies of resistance mutants that can appear on the YPD plates with stressors. Compared with untreated cells, the cells treated with heat shock showed at least 10 times more resistant colonies on the YPD plate containing 120 g/L ethanol, 0.1 g/L fluconazole, 1.2 g/L vanillin, or 4 mg/L tunicamycin (Fig. 6a). Five independent isolates from different stressor-containing plates were purified on the YPD plates (from single cells to colonies). All the purified isolates from the YPD plates still showed better tolerance than that of the parental strain JSC25-1 under the test conditions (Fig. 6b), showing that heat-shock-induced phenotypic variations were not caused by a transient transcriptional or post-transcriptional state. Since ethanol-resistant strains have promising applications in bioethanol production, the five mutants selected from ethanol-containing plates were analyzed using whole-genome SNP microarray. Additional file 1: Table S3 shows the genetic events on the genomes of the five mutants. Interestingly, 5 of the 7 genetic events occurred on chr IV, including 1 UPD and 4 terminal LOH (Additional file 1: Table S3). As shown in Additional file 1: Table S3, all the five genetic events led to the homozygosity of large regions on the right arm of chr IV. A possible explanation for the improved ethanol tolerance of these five selected isolates will be discussed below.