Fig 1.
Yeast cells exhibit high noise levels upon experimental evolution.
(A) Schematic of our evolution experiment. Cells were treated with 2.8% EMS, regrown for 12 h and then selected for the top 5% (or bottom 5%) of the population in terms of GFP intensity. Alternating selection enriched for mutant cells that presented higher expression noise in terms of GFP intensity. (B) Most of the evolved clones exhibit increased expression noise. Single clones were isolated from eight evolved cultures, and their reporter gene expression was measured. The x- and y-axes represent fold-change in noise and mean expression, respectively, after evolving. The median and range for 4–5 replicates for each evolved clone are indicated by circles and error bars, respectively. The red lines indicate values for the ancestral line. Data associated with this figure can be found in the supplemental data file (S1 Data). EMS, ethyl methanesulfonate; GFP, green fluorescent protein.
Fig 2.
Cells with different levels of Tdh2-GFP exhibit different physiological states.
(A) Stationary-phase cells with high Tdh2-GFP levels (red circles) survive better than those with low levels (blue squares) after growing in H2O2-containing medium (Fisher’s exact test, p = 0.1099 for untreated samples, p < 2.2 × 10−16 for H2O2-treated samples). Single cells with high or low GFP intensities were sorted and plated on the same plates with or without 4.4 mM H2O2. Survival rates were determined by counting colony-forming units after 5 d of growth. (B) Stationary-phase cells with high Tdh2-GFP tend to reenter the cell cycle faster than those with low Tdh2-GFP signal. Each dot of the scatterplot represents data from a single cell. Unsorted stationary-phase cells were placed on YPD agarose pads, and only unbudded cells were monitored using time-lapse microscopy. The x-axis indicates the initial Tdh2-GFP signal intensity for each cell, and the y-axis indicates the first rebudding time. The red dotted line represents a linear regression (Spearman’s rank correlation, n = 111, p = 8.131 × 10−12). (C) Stationary-phase cells with low Tdh2-GFP signal survive better than those with high signal when cells encounter heat stress after being re-fed with fresh nutrients (one-sided Wilcoxon rank-sum test, n = 4–5; p = 0.1995 for untreated samples, p = 0.2426 for heat-treated samples before nutrient refreshment, p = 0.0159 for heat-treated samples after nutrient refreshment). Survival rates were determined by counting colony-forming units after 3 d of growth. The median value for replicates is indicated by solid horizontal lines among groups of data points. *p < 0.05, ***p < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). GFP, green fluorescent protein; YPD, 1% yeast extract, 2% peptone, and 2% dextrose.
Fig 3.
Multiple genes in the evolved cells exhibit increased noise levels.
(A) Four reporter genes (TDH3-BFP, PGK1-BFP, ADK1-GFP, and GLY1-BFP) were engineered in modified ancestral (red circles) and evolved (blue squares) TDH2-GFP-carrying cells (see Materials and methods), and their expression noise was measured (one-sided Wilcoxon rank-sum test, n = 7–8; p = 4.534 × 10−4 for ADK1, p = 0.0027 for GLY1, p = 9.441 × 10−4 for PGK1, p = 7.158 × 10−4 for TDH3). TDH3 and PGK1 are involved in TDH2-related pathways, whereas ADK1 and GLY1 are not. (B) Transcriptional regulation is responsible for increased noise in the evolved cells (one-sided Wilcoxon rank-sum test, n = 7–8; p = 4.495 × 10−4). The TDH2 promoter was directly fused with the coding sequence of GFP. This construct was engineered in modified ancestral and evolved TDH2-GFP-carrying cells, and its expression noise was measured. The median value of replicates is indicated by horizontal solid lines among groups of data points. **p < 0.01, ***p < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). BFP, blue fluorescent protein; GFP, green fluorescent protein.
Fig 4.
The hmt1-G70D mutant recapitulates the increased noise phenotype observed in the evolved line.
(A) Reconstituting the hmt1-G70D mutation in the ancestral background increases Tdh2-GFP noise, whereas reversing that mutation in the evolved background decreases it (one-sided Wilcoxon rank-sum test, n = 6; p = 0.0011 for the ancestral background, p = 0.0025 for the evolved background). (B) Reconstituted hmt1-G70D mutant cells exhibit increased expression noise of the TDH2 promoter–GFP construct (one-sided Wilcoxon rank-sum test, n = 8; p = 0.0002). (C) Reconstituted hmt1-G70D mutant cells present increased noisy expression of Adk1-GFP, Gly1-BFP, and Tdh3-BFP (one-sided Wilcoxon rank-sum test, n = 7–8; p = 0.0030 for ADK1, p = 0.0011 for GLY1, p = 0.0030 for TDH3). All mutants were constructed using the CRISPR/Cas9 system. The median value of replicates is represented by horizontal solid lines among groups of data points. **p < 0.01, ***p < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). BFP, blue fluorescent protein; GFP, green fluorescent protein.
Fig 5.
Mutations of SWI/SNF components and the small ribosomal subunit Rps2 result in elevated noise.
(A) Attenuating RPS2, SNF2, or SNF5 gene expression in the ancestral background results in increased noise (one-sided Wilcoxon rank-sum test, n = 6–19; p = 0.0039 for rps2-DAmP/+, p = 0.0025 for snf2Δ/+, p = 0.0008 for snf5Δ/+). Rps2 and Snf2 are methylation substrates of Hmt1. Snf2, Snf5, and Snf6 are essential components of the SWI/SNF chromatin remodeler. Since haploid mutants of rps2-DAmP, snf2Δ, and snf5Δ exhibit severe growth defects and because slow growth has been shown to increase expression noise [91], we constructed heterozygous mutant diploids to measure noise. (B) Deletions of NPL3 or SBP1 in haploid cells do not result in increased noise (one-sided Wilcoxon rank-sum test, n = 5; p = 0.0079 for npl3Δ, p = 0.206 for sbp1Δ). Npl3 and Sbp1 are methylation substrates of Hmt1. (C) Deleting SNF6 in the ancestral background results in increased noise (one-sided Wilcoxon rank-sum test, n = 5; p = 0.0087). Haploid snf6Δ mutants exhibit defective respiration, so we used a haploid rho- ancestral strain in this experiment, and cells were grown in YPD medium. The median value of replicates is represented by horizontal solid lines among groups of data points. (D) The activity of SWI/SNF chromatin-remodeling complexes is compromised in the hmt1-G70D mutant. It has been shown previously that compromising SWI/SNF complexes results in attenuated transcriptional activation of CHA1 [52]. Total RNA was isolated from ancestral, hmt1-G70D, and snf5Δ haploid cells 0, 5, and 15 min after adding 0.1% L-Serine (an inducer of CHA1 expression). Specific mRNA levels were assessed by Q-PCR. In the figure, CHA1 mRNA levels were normalized to those of PYK1, with this latter acting as an internal control. For all time points, CHA1 mRNA levels in ancestral cells (blue) were significantly higher than those in hmt1-G70D (red) or snf5Δ (green) mutant cells (one-sided Student’s t test, n = 9; p < 0.01). (E) hmt1-G70D mutant cells display higher nucleosome occupancy in the promoter and partial coding regions of TDH2 and GLY1. The chromatin status of the TDH2 promoter was established by ChIP against H3 coupled with Q-PCR. The numbers in the x-axis indicate the distance (in bp) from the transcription start site. The y-axis represents relative enrichment of H3 signals for the amplicons at the indicated regions (one-sided Student’s t test, n = 10–12). Black, gray, and dashed circles indicate confirmed, fuzzy, and condition-specific nucleosome-occupied regions, respectively. Error bars represent standards errors. *p < 0.05, **p < 0.01, ***p < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). ChIP, chromatin immunoprecipitation; DAmP, decreased abundance by mRNA perturbation; GFP, green fluorescent protein; H3, histone 3; Q-PCR, quantitative PCR; YPD, 1% yeast extract, 2% peptone, and 2% dextrose.
Fig 6.
Hmt1-mediated noise suppression can be released under nonoptimal growth conditions as a conserved cell survival strategy.
(A) HMT1 transcripts are down-regulated under nonoptimal growth conditions (one-sided paired Student’s t test, n = 3–4; p = 0.0008 for heat stress, p = 0.003 for oxidative stress, p = 0.002 for osmotic stress, p = 0.007 for diauxic shift). The level of mRNA was measured using Q-PCR, and DET1 mRNA was used as the internal control. Error bars indicate standard errors. (B) Nonoptimal growth conditions result in increased Tdh2-GFP noise (one-sided Wilcoxon rank-sum test, n = 4; p = 0.015 for heat and oxidative and osmotic stress, p = 0.014 for diauxic shift). Noise was measured 2.5 h after cells had been shifted to the indicated conditions or was measured at different growth stages. (C) hmt1-G70D mutant populations survive better than the ancestral line in medium containing H2O2 (Fisher’s exact test; p < 2.2 × 10−16). Stationary-phase cells were spread on plates with 5.3 mM H2O2, and survival rates were determined by counting colony-forming units after 5 d of growth. (D) Mutation of the HMT1 ortholog in the fission yeast Schizosaccharomyces pombe also results in increased expression noise. rmt1 is the ortholog of HMT1, whereas tdh1 and gpd3 are TDH2 orthologs. Deletion of rmt1 increased protein noise of both tdh1-YFP and gpd3-YFP (one-sided Wilcoxon rank-sum test, n = 10–12; p = 0.0005 for tdh1-YFP, p = 0.037 for gpd3-YFP). The median value of replicates is indicated with horizontal solid lines among groups of data points. *p < 0.05, **p < 0.01, ***p < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). GFP, green fluorescent protein; Q-PCR, quantitative PCR; YFP, yellow fluorescent protein.
Fig 7.
A model showing how Hmt1 modulates cell-to-cell heterogeneity in response to environmental stress.
Hmt1 methylates and enhances the function of the SWI/SNF chromatin remodeler and small ribosomal subunits to reduce stochastic noise in gene expression. Under normal conditions, cells maintain a high level of Hmt1 and exhibit homogeneous gene expression in most cells. However, when the population encounters environmental stress, HMT1 expression is down-regulated, inhibiting the functions of Hmt1 targets. Accordingly, expression of Hmt1 gene targets becomes noisier, so individual cells exhibit heterogeneous cell physiologies. The likelihood of population survival is enhanced because of this heterogeneity. Me, methylation.