Hydrogen peroxide induced loss of heterozygosity correlates with replicative lifespan and mitotic asymmetry in Saccharomyces cerevisiae

Yeast strains and H₂O₂ treatment

Strains with MET15^+/− have been described previously (Qin, Lu & Goldfarb, 2008). Yeast cells were grown overnight at 30 °C in Yeast extract Peptone Dextrose (YPD) media. On the next day, yeast cultures were diluted to OD₆₀₀ of 0.6 in fresh YPD and grown for an additional two hours to ensure that the optical density reached between 0.8 and 0.9, the mid-log phase. Cells were then harvested, washed with water to remove YPD, and sonicated for 4 min using a Fisher Scientific FS20D water-bath sonicator to disperse cell clumps.

Treatment of yeast cells with H₂O₂ was modified from other studies (Diezmann & Dietrich, 2009; Yu et al., 2012). Reactions were carried out by mixing 4 μl of yeast cells, 16 μl of water, and 20 μl of the 2-fold H₂O₂ working stocks. Tubes of cells in H₂O₂ treatment were incubated on a nutator for 3 h at 30 °C. For most strains, the final concentrations of H₂O₂ for cell treatment were typically in a group of ten: 0, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, and 0.3%. For strains that are more sensitive or more tolerant to H₂O₂, this concentration series was adjusted lower or higher, respectively. At the end of incubation, all reaction tubes were filled with 960 μl of water (a 50 fold dilution) and chilled on ice for 5 min. Cells were sonicated in a FS20D water-bath sonicator for 2 min. We spread 250 μl of each reaction mix onto 150 mm plates with the Met15 Lead Assay (MLA) media (Qin, Lu & Goldfarb, 2008). Plates were spread in triplicates for each H₂O₂ concentration. Plates were incubated at 30 °C for 2 days. Some plates were also left at room temperature for additional days for colony colors to develop a better contrast for LOH scoring. Colonies were visually assessed for color-sectoring patterns and tallied using a Bantex Colony Counter. We tallied the number of white, black, half-black, quarter-black, three-quarter black colonies and others. We ignored color-sector patterns that were less than one-eighth. For each experiment, viability at conditions with only water was deemed as 100%. Viability at a specific concentration of H₂O₂ was estimated from the average number of colony forming units (CFUs) at this concentration divided by the average number of CFUs at conditions with only water. Strains were assayed 2–5 times, for a total of 35 successful experiments performed with the 12 yeast strains under study.

Data and analysis codes

Fitting and analysis were conducted in the R statistical environment. Sample codes and data can be found on GitHub (https://github.com/hongqin/H2O2LOH_PeerJ2016). An R markdown script, “summary_analysis_LOHH2O2.Rmd,” is provided in the GitHub repository to reproduce key analyses and figures. A summary of the results in EXCEL format is provided in Table S1. The ratios of C_b/C_v were directly estimated from each experiment and then averaged for each strain. Data for replicative and CLS, and mitotic asymmetry measures were obtained from our previous studies (Qin & Lu, 2006; Qin, Lu & Goldfarb, 2008), and are also available in the same GitHub repository. Average replicative lifespan (ARLS) indicates the average RLS for each strain that was previously measured using micro-dissections (Qin & Lu, 2006). CLS in the dataset indicates average CLS for each strain that was previously measured using colony-forming units (Qin & Lu, 2006).

Overview of the experiment

To better understand the interconnection between oxidative stress, genomic instability, mitotic asymmetry, and CLS in S. cerevisiae, we used exogenous H₂O₂ to induce an oxidative stress response in yeast cells and quantified their H₂O₂ dose-response curves of LOH and viability. LOH is an effective method for gauging genome integrity in yeast cells (McMurray & Gottschling, 2003; McMurray & Gottschling, 2004). We previously introduced heterozygosity on the MET15 locus (MET15^+/−) by knocking out one copy of the wild-type allele using a kanamycin-resistance marker (Qin, Lu & Goldfarb, 2008). LOH can be monitored when MET15^+/− strains are converted into homozygous MET15^−/− strains that can generate black colors on lead-containing medium (Fig. 1). In other words, we can observe half of the LOH events at the MET15^+/− locus.

Quantitative measurement of H₂O₂–dose dependent changes of viability and LOH

The exogenous H₂O₂ dose-dependent changes of viability, black colonies, and half-black colonies are generally sigmoid in the studied yeast strains (Figs. 2 and S1). Half-black colonies indicated LOH occurred after cells divided on MLA plates (Fig. 1).

As shown in Fig. 2, we quantified viability s as a function of hydrogen peroxide concentration, [H₂O₂], using a sigmoid mathematical function, similar to our previous study (Qin, Lu & Goldfarb, 2008): $s([{\text{H}}_{\text{2}}{\text{O}}_{\text{2}}])=\frac{1}{1+{\left(\frac{[{\text{H}}_{\text{2}}{\text{O}}_{\text{2}}]}{C_v}\right)}^{w_v}},$ where C_v is the H₂O₂ concentration at the midpoint of viability, and w_v is a weight parameter.

We quantified the percentage of black colonies, b([H₂O₂]), as a function of [H₂O₂]: ${\text{b([H}}_2{\text{O}}_2{\text{]) = b}}_{\max }-\frac{{\text{b}}_{\max }-{\text{b}}_{\min }}{1+{\left(\frac{{\text{[H}}_2{\text{O}}_2\text{]}}{C_b}\right)}^{w_b}}$ where C_b is the H₂O₂ concentration at the midpoint of black colony change, b_max and b_min is the maximum and minimum of b([H₂O₂]), and w_b is a weight parameter. Likewise, the change of half-black colonies was quantified: $b_{0.5}{\text{([H}}_2{\text{O}}_2{\text{]) = b}}_{0.5\max }-\frac{{\text{b}}_{0.5\max }-{\text{b}}_{0.5\min }}{1+{\left(\frac{{\text{[H}}_2{\text{O}}_2\text{]}}{C_{b0.5}}\right)}^{w_{b0.5}}}$ where C_b0.5 is the H₂O₂ concentration at the midpoint of half-black colony change.

We detected a strong association between C_v and C_b with R² = 0.77 and p-value = 0.00019 using the averaged values of each strain (Fig. S1), indicating that hydrogen peroxide induced LOH and loss of viability co-vary, demonstrating the consistency of our approach.

Genomic tolerance to oxidative stress correlates with replicative lifespan

C_b is lower than C_v in seven out of the 12 strains under study (Figs. 2 and S2; Table S1). The median and mean values of C_b/C_v are 0.60 and 0.92, respectively, indicating that lower doses of H₂O₂ are needed to trigger genomic instability than to trigger the drop of viability. In other words, genome integrity is more sensitive to oxidative stress than cellular viability during exogenous H₂O₂ treatment. Strains with higher C_b/C_v values, such as YPS128 in Fig. 3A, indicates their genome integrity is more tolerant of hydrogen peroxide. Strains with lower C_b/C_v values, such as M32 in Fig. 3A, indicate their genome integrity is more sensitive to hydrogen peroxide. Linear regression analysis reveals that average RLS, ARLS, is positively correlated with C_b/C_v with p-value = 0.039 and R² = 0.36, suggesting that genomic tolerance to oxidative stress is associated with replicative longevity.

Trade-off between genomic tolerance to oxidative stress and mitotic asymmetry in mother cells

We previously measured an inverse proxy for the mitotic asymmetry, L₀, of these yeast strains during normal growth conditions (Qin, Lu & Goldfarb, 2008). This inverse proxy for mitotic asymmetry, L₀, is the ratio of daughter-cell LOH versus mother-cell LOH in YPD. Small L₀ values suggest mother cells can more effectively keep genotoxic stress factors to themselves and thereby provide their daughter cells with healthier lives (Qin, Lu & Goldfarb, 2008). Regression analysis revealed that C_b/C_v significantly correlates with L₀, with p-value = 0.0079 and R² = 0.56 (Fig. 3B). Based on the positive correlation between C_b/C_v and L₀, yeast cells with better mitotic asymmetry, such as M1–2 and M13, are more sensitive to genotoxic stress because relatively lower H₂O₂ can trigger LOH in their genomes. Hence, better abilities of mother cells to prevent genotoxic stress factors from leaking to daughter cells also render mothers’ own genomes more sensitive to elevated oxidative stress.

Concerted increase of genomic instability

We observed a large number of multiple LOH events where H₂O₂ induced LOH occurred in both mother and daughter cells. We focused on the three-quarter black colonies to illustrate the nature of concerted multiple LOH events. Based on parsimonious reasoning, a three-quarter black LOH can be considered as a joint outcome of a half-black and a quarter-black LOH. The null hypothesis here is that half-black LOH and quarter-black LOH occur independently, which means that the probability of three-quarter black colonies is the product of the probabilities of half- and quarter-blacks, i.e. random expectation. The alternative hypothesis is that events of half-black LOH and quarter-black LOH are associated with and lead to an increased occurrence of three-quarter black LOH. We performed one-tailed Fisher’s exact tests on experimental observations with sufficient events of half-, quarter-, and three-quarter black LOH events, and applied Bonferroni correction to adjust for multiple tests. Of the 35 experiments, 21 experiments show that three-quarter LOH events occurred at significantly higher frequencies in all H₂O₂ conditions with sufficient number of LOH events (Table S2). In nine experiments, three-quarter LOH events occurred at significantly higher frequencies in all but one H₂O₂ condition. Overall, 163 out of 185 measurements show significantly higher three-quarter LOH events than the null hypothesis (Table S2). Hence, the null hypothesis can generally be rejected. We concluded that the high occurrence of three-quarter black colonies is the result of multiple concerted LOH events.

Contrasting LOH onsets between hydrogen peroxide treatment and chronological aging suggest cells’ ability to suppress endogenous oxidative stress during aging

In our previous study on LOH during chronological aging, we found that the onset of LOH often lagged behind the drop of viability. We used T_g to represent the mid-point for the increase of LOH, and T_c as the midpoint for the drop of viability during chronological aging (Fig. 4) (Qin, Lu & Goldfarb, 2008). T_g values are often larger than T_c values in the collection of yeast strains that we studied (Fig. 4) (Qin, Lu & Goldfarb, 2008).

In this study, onset of exogenous H₂O₂ induced LOH often occurred earlier than the drop of viability, as C_b is often smaller than C_v (Fig. 4). For most natural isolates, this C_b/C_v ratio is lower than 1.0 (Fig. 4). If we assume that a similar level of oxidative stress corresponds to the 50% viability in each strain, this contrast suggests that during chronological aging, the genotoxic effect of endogenous oxidative stress is shielded even when viability has dropped during chronological aging. Hence, the contrast between LOH onsets is a proxy for the extent to how much cells can suppress the genotoxic effect of endogenous oxidative stress on genome integrity. Based on this reasoning, the ratio of $\frac{T_g/T_c}{C_b/C_v}$ indicates the ability of yeast cells to suppress the level of endogenous oxidative stress during the normal chronological aging process. Remarkably, we found a trade-off between mitotic asymmetry and ability of cells to suppress endogenous oxidative stress during chronological aging, as shown by a negative linear correlation between mitotic asymmetry L₀ and $\frac{T_g/T_c}{C_b/C_v}$ (Fig. 4D). This finding corroborates the tradeoff observed in Fig. 3B.