Genetic screen for mutants affecting spore lifespan and stress resilience in S. pombe.

(A) Experimental design for the parallel functional profiling of barcode-tagged deletion mutants using Bar-seq to identify genes required for spore lifespan or heat-stress resilience. Haploid deletion libraries were mated and sporulated; the resulting spores, each containing a specific gene deletion, were pooled, aged for 6 months in water at 25°C, and then an aliquot was heat-shocked at 55°C for 30 minutes. Aliquots were germinated, grown and sequenced at different times, as indicated, to quantify the relative abundance of the barcode-tagged mutant strains. At 6 months, unstressed spores were germinated in 2 technical replicates. The screen was performed in 2 independent biological repeats (Pools A and B). (B) Principal Component Analysis (PCA) of barcode counts (upper-quartile-normalised log2CPM [counts per million]) among the different repeated pooled spore samples. Dot colors represent the timepoints as indicated, and red triangles represent the heat-shocked samples. The variances represented in the principal components 1 and 2 are indicated on the axes. (C) Scatterplot comparing the log2 fold-changes (FC) in mutant barcodes in heat-stressed relative to unstressed spore pools and in old (6-month) relative to young (2-week) spore pools. Each point represents a deletion mutant. The Pearson correlation coefficient between spore stress resilience and longevity phenotypes is shown at top right, along with its significance. Green dots: mutants being significantly stress resistant and short-lived; blue dots: mutants being significantly stress sensitive and long-lived (FC >1.5; FDR < 0.05), with other mutants in grey. Enriched GO and KEGG terms are provided for stress-resistant but short-lived mutants (green) and stress-sensitive but long-lived mutants (blue), along with the gene counts for those terms present in the dataset. (D) Overlaps of lifespan mutants in stationary-phase cells (Romila et al. 2021) and in spores (this study). For overlaps that are higher than expected by chance, the p-values are indicated (Fisher’s exact test), along with enriched GO terms and gene counts for those terms present in the dataset. Only mutants that were detected in both screens are included in the comparison.

Genetic screen for mutants affecting spore lifespan and stress resilience.

(A) Viability of pooled spores as a function of time. Pools A and B are the two independent spore pools from the Bar-seq screen (Figure 1A). Mean spore viability was determined at each time by isolating 100 spores using a tetrad dissection microscope, plating them on YES, and counting the resulting cell colonies. (B) Left: Number of mutants detected at ≥3 reads/million in Pools A and B at the different time points indicated (of 3,206 mutants in the library). Right: Number of mutants detected at <3 reads/million in Pools A and B after 6 months in any of the 2 technical repeats; these mutants were discarded from the analysis. (C) Viability of 2-week-old wild-type spores maintained at 25° (Control) or subjected to a 30-minute heat stress at 55°C (Heat shocked). Each dot represents 1 of 4 independent repeats. (D) Volcano plot displaying changes in mutant (barcode) distributions, with normalisation done to the upper quartile. The mutants that significantly decreased or increased >3-fold at 6 months relative to 2 weeks are indicated in green and purple, respectively. Enriched GO terms and KEGG pathways for induced and repressed genes are shown, along with the gene counts for those terms present in the dataset. (E) Volcano plot displaying changes in mutant (barcode) distributions. The mutants that significantly decreased or increased >3-fold after heat stress relative to the unstressed condition are indicated in green and purple, respectively. Enriched GO terms and KEGG pathways for induced and repressed genes are indicated, along with the gene counts for those terms present in the dataset.

Transcriptome and proteome remodelling in yeast spores and killifish diapause embryos.

(A) Experimental design for analyzing S. pombe spores and proliferating control cells. A homozygous strain was precultured in Yeast Extract Supplements (YES) liquid medium and then in Malt Extract Agar (MEA) plates as indicated. Three independent biological replicates were performed and processed for RNA-seq and mass spectrometry from the same samples. (B) Scatter plot comparing the log2 fold-changes (FC) in mRNA and protein levels in spores relative to those in proliferating cells. The Pearson correlation coefficient for mRNA and protein changes is shown at the top, along with its significance. The number of genes in each quadrant is indicated. Genes differentially expressed (FC >1.5×; FDR <0.05) for both mRNAs and proteins are highlighted in blue, with other genes in grey. (C) Enriched GO Biological Processes and KEGG pathways associated with repressed (green) and induced (red) mRNAs and proteins as indicated. The colour shade corresponds to the false discovery rate (FDR; top right). Enrichments were calculated with g:Profiler2 (Raudvere et al. 2019). (D) Principal Component Analysis (PCA) of transcriptomic signatures from spores (this study), quiescent cells (Marguerat et al. 2012), and their proliferating cell controls. Dot colours represent the conditions as indicated. The variances represented in the principal components 1 and 2 are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (E) Volcano plot showing mRNA expression in spores relative to quiescent cells. Blue dots: mRNAs that are significantly lower (497 genes) or higher (1189 genes) expressed in spores. Enriched GO and KEGG terms for induced and repressed genes are indicated, along with the proportions of genes with those terms. Red dots: 94 mRNAs encoding ribosomal proteins, 44 of which are significantly induced. Significance thresholds for differential expression: FC >1.5×; FDR <0.05. (F) Experimental design for analyzing N. fuzeri diapause embryos (GRZ strain). Embryos were incubated at 28°C for 4 days post-fertilisation, followed by incubation at 20°C for 14 days to obtain diapause embryos or for an extra 3 days at 28°C to obtain actively developing embryos at a similar stage to the diapause embryos. Three independent biological repeats, collected from different breeding times, were performed and processed for RNA-seq and mass spectrometry from the same samples. (G) Scatter plot comparing the log2 fold-changes (FC) in mRNA and protein levels in diapause relative to those in developing embryos. The Pearson correlation coefficient for mRNA and protein changes is shown at the top, along with its significance. The number of genes in each quadrant is indicated. Genes differentially expressed (FC >1.5×; FDR <0.05) for both mRNAs and proteins are highlighted in blue, with other genes in grey. All 64 genes encoding ribosomal proteins are highlighted in red. (H) Enriched GO Biological Processes and KEGG pathways associated with the human orthologs of the repressed (green) and induced (red) killifish mRNAs and proteins as indicated. The colour shade represents the FDR (top right). Enrichments were determined using g:Profiler2.

Transcriptome and proteome remodelling in yeast spores and killifish diapause embryos.

(A) Bar plots showing the numbers and proportions of mRNAs, proteins, and long non-coding RNAs (lncRNAs) being differentially expressed in spores relative to proliferating cells, colour-coded for genes that are induced (red), repressed (green), or non-differentially expressed (grey). Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (B) Overlap between mutants with altered spore heat resistance, spore longevity, and differentially expressed genes at RNA or protein levels in spores relative to exponentially growing cells. Only the 2396 genes detected in both Bar-seq and expression data (RNA or proteome) are included. The probability of enrichment in the overlap is included (hypergeometric distribution), with 615 genes expected to overlap by chance. (C) Principal Component Analysis (PCA) of proteomic signatures from spores, quiescent cells, and their proliferating control cells. Each dot colour shows the condition as indicated. The variances represented in the principal components are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (D) Volcano plot showing protein expression in spores relative to quiescent cells. Blue dots: proteins that are significantly lower or higher expressed in spores. Red dots: ribosomal proteins. Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (E) Overview of experimental design for analyzing S. pombe spores compared to early stationary-phase cells. A homozygous strain was cultured in Yeast Extract Supplements (YES) liquid medium, Malt Extract Agar plates (MEA) and YES Agar (YEA) plates as indicated. Three independent biological repeats were performed and processed for RNA-seq and mass spectrometry from the same samples. (F,G) Volcano plot showing mRNA (F) and protein (G) expression in spores relative to stationary-phase cells. Blue dots: genes that are significantly lower or higher expressed in spores. Enriched GO and KEGG terms for induced and repressed genes are indicated, along with the proportions of genes with those terms. Red dots: ribosomal protein genes. Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (H) Scatter plot of the log2 fold-change (FC) of mRNA levels in diapause embryos in our data compared to those of Reichwald et al. (2015). The Pearson correlation coefficient for the mRNA changes is shown at the top, along with its significance. (I) Bar graph representing the numbers and proportions of mRNAs and proteins being differentially expressed in diapause relative to developing embryos, colour-coded for genes that are induced (red), repressed (green), or non-differentially expressed (grey). Significance thresholds for differential expression: FC >1.5x; FDR <0.05.

Genome regulation in yeast spores, killifish diapause embryos and human dormant cancer cells.

(A) Scatter plots comparing log2 fold-changes (FC) in mRNA levels of orthologous genes in dormant cells relative to active control cells. Top left: yeast spores vs killifish diapause embryos; top right: human dormant cancer cells vs killifish diapause embryos; bottom left: human dormant cancer cells vs yeast spores. Each point represents either an individual mRNA (blue) or the mean log2FC of all genes within a GO Biological Process (red). The Pearson correlation coefficients for individual mRNAs (blue) or Biological Processeses (BP, red) are indicated at the top right of each plot, with asterisks indicating significant correlations (p <0.05). (B) Scatter plot comparing the log2 fold-changes (FC) in protein levels of orthologous genes in yeast spores relative to proliferating cells and in killifish diapause embryos relative to developing embryos. Each point represents either an individual protein (blue) or the mean log2FC of all genes within a GO Biological Process (red). The Pearson correlation coefficients for individual mRNAs (blue) or Biological Processeses (BP, red) are indicated at top right, with asterisks indicating significant correlations (p <0.05). (C) Overlaps among mRNAs induced (fold-change >1.5x; FDR <0.05) in yeast spores, killifish diapause embryos, and dormant cancer cells. In total, 2239 human genes with orthologs in killifish and yeast were detected in all 3 datasets. The p-values are indicated (Fisher’s exact test) for the overlaps that are higher than expected by chance. (D) Functional enrichments for mRNAs induced in at least 2 of the 3 dormant states. Green: GO Biological Processes; blue: KEGG pathways. The x-axis displays the –log10 of the false discovery rate (FDR), indicating the significance of the enrichment, determined using g:Profiler2. (E) Expression changes of conserved genes (columns) encoding cytoplasmic ribosomal proteins and autophagy proteins across the different datasets (rows) as labelled at right, including data for mRNAs (blue), proteins (red), and mutants (brown). Data for 58 ribosomal genes and 21 autophagy genes conserved in yeast, killifish, and human are shown, with log2 FC color-coded as indicated at left. (F) Overlap among proteins induced (fold-change >1.5x; FDR <0.05) in yeast spores and killifish diapause. In total, 1103 orthologous proteins were detected in both species. The p-value is indicated for the overlap (Fisher’s exact test). (G) Overlaps among mRNAs repressed (fold-change >1.5x; FDR <0.05) in yeast spores, killifish diapause, and dormant cancer cells. The p-value is indicated for the overlap (Fisher’s exact test). (H) Functional enrichments for mRNAs that are repressed in at least 2 of the 3 dormant states, as in (D). (I) Overlap among proteins repressed (fold-change >1.5x; FDR <0.05) in yeast spores and killifish diapause. The p-value is indicated for the overlap (Fisher’s exact test). (J) Functional enrichments for proteins that are repressed in both spores and killifish diapause, as in (D).

Transcriptome and proteome changes in older and stressed spores and their offspring

(A) Scheme of the experimental design. Spores were aged and heat-shocked as indicated, followed by transcriptome and proteome analyses of the different spore samples (top) and of the offspring cells derived from germinating the different spore samples and growing for ∼18 hours to exponential phase (bottom). For the 2-week-old spores, with and without heat shock, 3 biological replicates were analysed for each condition, whereas for the 3– and 5-month-old spores, 2 biological replicates were analysed. For all offspring cells, 3 biological replicates were analysed for each condition. (B) Principal Component Analysis (PCA) of transcriptomic signatures from spore samples after different times or heat shock. Each dot represents a biological replicate, and the colors correspond to the conditions as indicated. The variances represented in the principal components 1 and 2 are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (C) Scatterplot comparing the log2 fold-changes (FC) in mRNA and protein levels in 5-month-old relative to 2-week-old spores. The Pearson correlation coefficient (R) and its significance are displayed on top. Genes differentially expressed (FC >1.5x; FDR <0.05) at both the mRNA and protein level are highlighted in blue, and their numbers are indicated for each quadrant. (D) Enriched GO Biological Processes and KEGG pathways associated with repressed (green) and induced (red) mRNAs and proteins as indicated in 5-month-old compared to 2-week-old spores. The colour shade corresponds to the false discovery rate (FDR; top right). Enrichments were calculated with g:Profiler2. (E) Scatterplot showing the log2 FC in mRNA levels in 5-month-old vs 2-week-old spores compared to old vs young stationary-phase cells at 100% and 50% viability (Atkinson et al. 2018). The Pearson correlation coefficient and its significance are shown on top. Genes differentially expressed (FC >1.5x; FDR <0.05) in both spores and cells are highlighted in blue, with other genes in grey. The number of significant genes along with enriched GO and KEGG terms is indicated for each quadrant (g:Profiler2). (F) Scatterplot showing the log2 FC in protein levels in 5-month-old vs 2-week-old spores compared to heat-shocked vs control spores. The Pearson correlation coefficient and its significance are shown on top. Proteins differentially expressed (FC >1.5x; FDR <0.05 in at least one condition) in both old spores and heat-shocked spores are highlighted in blue, with other proteins in grey. The number of significant proteins, along with enriched GO terms (g:Profiler2), is indicated for each quadrant. (G) PCA of transcriptomic signatures from offspring cells derived from spores after different times or heat shock. Each dot represents a biological replicate, and the colors correspond to the spore conditions as indicated. The variances represented in the principal components 1 and 2 are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (H) Volcano plot showing mRNA expression in offspring cells from 5-month-old spores relative to offspring from 2-week-old spores. Blue dots: genes that are significantly lower (72 genes) or higher (160 genes) expressed in the offspring from old spores, with other genes in grey. Enriched GO and KEGG terms for induced and repressed genes are indicated. Significance thresholds for differential expression: FC >1.32x; FDR <0.05. (I) Volcano plot showing protein expression in offspring cells from 5-month-old spores relative to offspring from 2-week-old spores. Blue dots: proteins that are significantly lower (65 proteins) or higher (158 proteins) expressed in the offspring from old spores, with other proteins in grey. Enriched GO and KEGG terms for induced and repressed proteins are indicated. Significance thresholds for differential expression: FC >1.32x; FDR <0.05. (K) Top: Normalized chronological lifespan proxies for cells derived from young (grey) and old (green) spores, with the p-value for the difference in lifespan indicated (T-test). Cells germinated from spores in YES medium were grown to stationary phase, diluted and grown again to stationary phase, with Day 1 being the time when cells reached maximum density. The lifespan proxies represent the time required for cells derived from old spores to reach 1% viability, relative to those from young spores. The lifespans were measured for 6 (young spores) and 8 (old spores) independent repeats across 3 experiments, using a robotics-based colony-forming unit assay (Romila et al. 2021), with values normalized to Day 1 (100% viability). Bottom: Chronological lifespan curves for Experiment 1, including 2 independent repeats for offspring from young spores (grey) and 3 repeats for offspring from old spores (green). The lifespan curves of Experiments 2 and 3 are provided in Figure S3E. (L) Top: Normalized chronological lifespan proxies for cells derived from control spores (grey) and spores heat-shocked for 30 minutes at 50°C (red), determined as in (K), with the p-value for the difference in lifespan indicated (T-test). Bottom: Chronological lifespan curves for this experiment, including 3 independent repeats for offspring from control spores (grey) and 3 repeats for offspring from heat-stressed spores (red). (M) Spot test of cells derived from young, old, and heat-shocked spores. Cells germinated from spores in YES medium were grown to stationary phase, diluted and grown again to stationary phase. Cells were 3-fold serially diluted and spotted onto YES plates. To test for stress resistance, cells were incubated at 43°C or 45°C for 30 min before plating.

Transcriptome and proteome changes in older and stressed spores and their offspring

(A) Heatmaps of transcriptome (left) and proteome (right) changes in spores after 2 weeks, 3 months, and 5 months. Rows represent genes, and the colors show the fold-changes (FC) in expression relative to proliferating cells. Columns represent the different conditions, with data for the 2 to 3 biological repeats provided separately. Only genes that are differentially expressed (FC >1.5x; FDR <0.05) in at least 1 condition are shown. Gene expression patterns are grouped into 6 clusters using k-means. (B) Boxplot showing the percentage viability of 2-week-old spores following a 30-minute heat-shock treatment at the indicated stress temperatures. (C) Volcano plot showing protein expression in heat-shocked relative to control spores. Blue dots: proteins that are significantly lower or higher expressed in heat-shocked spores. Enriched GO terms for induced proteins are indicated. Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (D) Scatterplot comparing the log2 fold-changes (FC) in mRNA levels in 5-month-old vs 2-week-old spores compared to mRNA levels in heat-shocked vs control spores. The Pearson correlation coefficient (R) and its significance are displayed on top. Genes differentially expressed (FC >1.5x; FDR <0.05) in both old and heat-shocked spores are highlighted in blue, and the numbers of these genes are indicated for each quadrant. (E) Chronological lifespan curves of Experiments 2 and 3 for offspring from young (grey) and old (green) spores for the results shown in Figure 3K.

Transcriptome and proteome changes in late and stressed diapause and post-diapause embryos.

(A) Scheme of the experimental design. N. furzeri diapause embryos (MZCS strain) were maintained for 2 weeks (early, with or without heat shock) and 3 months (late) as indicated, followed by transcriptome and proteome analyses of the different diapause samples (top) and the post-diapause embryos derived from the diapause samples by triggering diapause exit at increased temperature and development for one week to the prehatching stage (bottom). For the early diapause samples, 3 biological replicates were analysed; for late diapause, 2 and 3 biological replicates were analysed for transcriptomics and proteomics, respectively; for all post-diapause samples, 3 biological replicates were analysed. For diapause samples, batches of ∼50 and ∼15 embryos were used for transcriptomics and proteomics, respectively; for post-diapause samples, batches of ∼10 and ∼ 1 embryos were used for transcriptomics and proteomics, respectively. (B) Principal Component Analysis (PCA) of transcriptomic signatures from early, late, and heat-shocked diapause samples. Each dot represents a biological replicate, and the colors correspond to the conditions as indicated. The variances represented in the principal components 1 and 2 are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (C) Scatterplot comparing the log2 fold-changes (FC) in mRNA and protein levels in 3-month-old relative to 2-week-old diapause embryos. The Pearson correlation coefficient (R) and its significance are displayed on top. Genes differentially expressed (FC >1.5x; FDR <0.05) at both the mRNA and protein level are highlighted in blue, and their numbers are indicated for each quadrant. (D) Enriched GO Biological Processes and KEGG pathways associated with repressed (green) and induced (red) mRNAs and proteins as indicated in 3-month compared to 2-week diapause embryos. The colour shade corresponds to the false discovery rate (FDR; top right). Enrichments were calculated with g:Profiler2. (E) Scatterplot showing the log2 FC in mRNA levels in 3-month vs 2-week diapause embryos compared to old vs young brain cells (Baumgart et al. 2014). The Pearson correlation coefficient and its significance are shown on top. Genes differentially expressed (FC >1.5x; FDR <0.05) in both diapause and brain cells are highlighted in blue, with other genes in grey. The number of significant genes and enriched GO/KEGG terms are indicated for each quadrant (g:Profiler2). (F) Scatterplot showing the log2 FC in protein levels in 3-month vs 2-week diapause embryos compared to heat-shocked vs control embryos. The Pearson correlation coefficient and its significance are shown on top. Proteins differentially expressed (FC >1.32; FDR <0.05 in ≥1 condition) in both late and heat-shocked diapause embryos are highlighted in blue, with other proteins in grey. The number of significant proteins, along with enriched GO terms (g:Profiler2), is indicated for each quadrant. (G) PCA of transcriptomic signatures from post-diapause embryos derived from early, late, or heat-shocked diapause. Each dot represents a biological replicate, and the colors correspond to the spore conditions as indicated. The variances represented in the principal components 1 and 2 are indicated on the axes. The gene expression values used for the PCA are normalized read counts derived from DESeq2. (H) Volcano plot showing mRNA (left graph) and protein (right graph) expression in post-diapause embryos derived from 3-month vs 2-week diapause. Blue dots: genes that are significantly lower or higher expressed in the post-diapause embryos from 3-month diapause, with other genes in grey. Enriched GO and KEGG terms for induced and repressed genes are indicated. Significance thresholds for differential expression: FC >1.32x; FDR <0.05.

Transcriptome and proteome changes in late and stressed diapause and post-diapause embryos.

(A) Viability of diapause embryos (blue) and developing control embryos (green) after a 1-hour heat-shock at the indicated temperatures. Survival was assessed visually 24 hours after the heat shock. Bars indicate the mean survival for each condition, and dots represent independent repeats. For developing samples, batches of 25 embryos were analysed, whereas for diapause samples, batches ranged from 15 to 23 embryos. (B) Scatterplot comparing the log2 fold-changes (FC) in mRNA and protein levels in heat-shocked relative to control diapause embryos. The Pearson correlation coefficient (R) and its significance are displayed on top. Genes differentially expressed (FC >1.5x; FDR <0.05) at both the mRNA and protein level are highlighted in blue, with other genes in grey. The number of significant genes and the enriched GO terms are indicated for each quadrant. (C) Volcano plot showing mRNA expression in heat-shocked relative to control diapause embryos. Blue dots: genes that are significantly lower or higher expressed after the heat shock. Enriched GO terms for repressed and induced mRNAs are indicated. Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (D) Volcano plot showing protein expression in heat-shocked relative to control diapause embryos. Blue dots: proteins that are significantly lower or higher expressed after the heat shock. Enriched GO terms for induced proteins are indicated. Significance thresholds for differential expression: FC >1.5x; FDR <0.05. (E) Scatterplot showing the log2FC in mRNA levels in 3-month vs 2-week diapause embryos compared to heat-shocked vs control diapause embryos. The Pearson correlation coefficient and its significance are shown on top. Genes differentially expressed (FC >1.32x; FDR <0.05) in both conditions are highlighted in blue, with other genes in grey. The number of significant genes and the enriched GO terms are indicated in the quadrants. (F) Scatter plots comparing log2FC in protein levels in post-diapause embryos from heat-shocked vs control diapause compared to post-diapause embryos from 3-month vs 2-week diapause. The Pearson correlation coefficient (R) and its significance are displayed. Genes differentially expressed (FC >1.32x; FDR <0.05) in both conditions are highlighted in blue, with other genes in grey. The number of significant genes and the enriched GO terms are indicated for each quadrant.