Functional genomic analysis reveals overlapping and distinct features of chronologically long-lived yeast populations

Shared gene expression changes induced by CR and an ade4∆ mutation

Our lab previously identified components of the de novo purine biosynthesis pathway as genetic factors with direct functional impact on CLS. Specifically, deletion of any ADE gene upstream of AICAR production was sufficient to significantly extend lifespan [9]. This extension, particularly for ade4∆ (the first and rate-limiting step in the de novo pathway [19, 20]), was comparable to that observed for calorie restricted (0.5% glucose) cultures (Figure 1A). This finding, along with the observation that CR does not further extend the CLS of an ade4∆ mutant, led us to hypothesize there was overlap between the mechanisms required for CLS extension induced by either CR or the ade4∆ mutation [9].

To examine more specifically what molecular/cellular pathways could be involved in the overlap between CR and ade4∆, gene expression profiling analysis was performed on WT-NR, WT-CR, and ade4∆ NR cultures grown to log or stationary phase (day 8). Day 8 was chosen to ensure the cells were in stationary phase and to potentially enrich for changes in transcription that occur during the aging process. Affymetrix Yeast Genome 2.0 GeneChips were used for the array hybridizations with duplicate biological RNA samples. Genes differentially regulated in each of the long-lived conditions compared to the non-restricted WT culture were identified using a 20% False Discovery Rate (FDR) cutoff for each combination. There were surprisingly no significant expression differences in the log phase cultures that passed this liberal FDR cut-off.

At day 8, however, the WT CR and ade4∆ NR conditions were found to induce significant changes in the expression of 213 (p-value = 2.5e-90; 4-fold enrichment over random) common genes when compared to the WT NR cultures (Figure 1B, and Supplementary Table S1). Scatter plots (Figure 1C, R²=0.9002) and heat maps (Figure 1D) of the differential expression levels (log₂) revealed that the relative changes in expression for the shared genes were also highly concordant. Given the large number of changes at day 8 for this dataset, we focused on this time point throughout the rest of the study.

Effects of cell extrinsic factors on CLS and global gene expression

CR and ade4∆ conditions are known to have cell extrinsic effects on CLS, as demonstrated by previous media swapping experiments [9, 11]. In these studies, when the expired media from a non-restricted WT culture was replaced with the expired media from either a WT CR culture or an ade4∆ NR culture, CLS was dramatically increased. An example of this is shown for a CR media swap in Figure 2A. The reciprocal result was observed when cells grown in CR media were transferred into expired NR media (Figure 2A, right panel). Since expired media from CR or ade4∆ cultures contains less acetic acid, a simple interpretation was that swapping out acetic acid-laden media for acetic acid-depleted media was beneficial for CLS due to reduced toxicity [9, 11]. However, it was also possible that media from the CR or ade4∆ cultures contained one or more secreted factors that extend CLS. To test this idea, we collected 50 ml of expired media from 5-day old WT CR or NR cultures, and then concentrated the volumes to 1 ml. These concentrates were then added to fresh WT NR cultures (10 ml total volume) and CLS was tracked. As shown in Figure 2B, the CR concentrate dramatically extended CLS, strongly suggesting there was something present in the CR concentrate that extends CLS, and was absent from the NR concentrate. Similarly, concentrate from the non-restricted ade4∆ mutant also extended CLS when added to WT BY4741 growing in SC + 2% glucose (Figure 2C), further supporting the existence of extrinsic factors in the expired media.

We noticed there was residual acetic acid remaining in the NR concentrate (measured as 1.7 mM) that was absent in the CR concentrate and could potentially mask CLS extension by the NR concentrate. Along these same lines, it was also possible that the concentrated compounds in the CR concentrate could be buffering pH of the media and mitigating the negative lifespan effects of acid accumulation by WT yeast grown under NR conditions. To address the first possibility, we normalized the acetic acid to 1.7 mM for both the CR concentrate and NR concentrate and repeated the CLS assays. The CR concentrate again extended CLS and the NR concentrate did not (Figure 2C). To assess the buffering capacity of the NR and CR concentrates, the media pH of each culture was measured over the first 5 days of cell growth and stationary phase (Figure 2E). Although addition of the NR concentrate decreased the starting pH relative to the other cultures, by the first 24 hours there were no significant differences in media acidity between cultures treated with either the CR or NR concentrate, making pH buffering an unlikely cause for the effect on longevity. Experiments to identify positive CLS factors from these concentrates are ongoing, but for the purpose of the current study we have used the CR concentrate as a cell extrinsic means of extending CLS for microarray analysis and biomarker identification (see below).

For the purpose of biomarker identification, we were interested in adding a 4^th long-lived growth condition for gene expression profiling that was unrelated to the CR concentrate. Isonicotinamide (INAM) is an isomer of nicotinamide that has been reported to enhance the NAD⁺-dependent histone deacetylase activity of sirtuins [21]. Although initially interested in its ability to promote Sir2 activity [22], we have also discovered the same concentration of INAM (25 mM) that enhances Sir2 activity in vivo also dramatically extends CLS, though independently of Sir2 or the other yeast sirtuins (Figure 3A and data not shown). Affymetrix expression profiles for the INAM and the CR concentrate were then generated from day 8 cultures for comparison with the CR and ade4∆ datasets.

Two distinct mechanisms of CLS extension revealed by expression profiles

Comparing expression profiles from the 4 long-lived populations (CR, ade4∆, INAM, and CR concentrate) relative to WT NR cultures revealed a wide diversity in the overlaps between each dataset, for both upregulated and downregulated genes (Figure 3B). This result was fully consistent with the disparate growth conditions used to extend CLS. Even with the 4-way comparison, the CR and ade4∆ datasets still showed the most overlap with each other (213 genes). The CR concentrate and INAM datasets were also highly overlapping with each other, showing 336 combined genes whose differential expression levels were highly correlated as shown in a heat map (Figure 3C) and scatter plot (Figure 3D, R²=0.9234). These overlapping genes are listed in Supplemental Table S2. Given the relatively fewer genes shared across the other combinations, we hypothesized that INAM and the CR concentrate may extend CLS through a primary mechanism distinct from the effects of CR or the ade4∆ mutation. In support of this idea, the top 10 GO terms for the upregulated and downregulated CR/ade4∆ genes (Supplemental Table S3) were different from GO terms for the upregulated and downregulated INAM/CR concentrate genes (Supplemental Table S4).

The most prevalent GO terms for the CR and ade4∆ samples indicated up-regulation of cellular processes related to the metabolism of organic acids such as acetic acid (Supplemental Table S3). By processing these metabolites, presumably in a manner that is energetically beneficial, CR and ade4∆ also prevent the accumulation of extracellular organic acids, as has been previously demonstrated [9, 11]. On the other hand, GO terms for the INAM and CR concentrate samples indicated an up-regulation of cell wall structural components (Supplemental Table S4), a general feature associated with exposure to acetic acid [23, 24]. The GO term emphasis on cell wall organization implies that yeast in these cultures are most likely producing the same organic acids as WT cultures but are also restructuring their cell walls to better cope with an increasingly toxic environment. Varied downregulation GO terms for CR and ade4∆ were not as informative, but included enrichment for transcription. For INAM and CR concentrate, the downregulation GO terms were enriched for vesicle and membrane fusion.

Based on the clear distinction in GO terms, we next measured acetic acid levels in the growth medium for each of the long-lived culture conditions across an 84-hour time course. The CR concentrate and INAM cultures accumulated significant concentrations of acetic acid, albeit to a lesser degree than WT (Figure 4A). In contrast, the CR and ade4∆ cultures accumulated very little acetic acid, even at the longer time points (Figure 4A), which was consistent with the up-regulation in organic acid metabolic processes and efficient consumption of acetic acid during the diauxic shift. The elevated acetic acid in INAM and CR concentrate cultures would theoretically require cells to become more resistant to the toxic environment in order to be long-lived, and thus the up-regulation of cell wall biosynthesis processes seems logical as functionally relevant GO terms. To model the extent that these differences in gene expression and resulting changes in metabolism could have on cell survival within each of the long-lived populations, we challenged each culture for 200 minutes with a toxic concentration of acetic acid (300 mM) at days 2 and 5 of the aging assay, and then measured the effects on cell viability (Figure 4B). As predicted from the array analysis and cell wall biosynthesis GO terms, INAM and CR-concentrate treated cells were best able to handle the acidic stress by day 5, maintaining significantly higher viability than WT, CR, and ade4∆ cultures.

Interestingly, all four long-lived populations exhibited slightly higher acetic acid tolerance at day 2 than WT before diverging into two distinct phenotypic subgroups, leaving open the possibility that there may still be some common cellular events occurring during early stationary phase responsible for all four increases in lifespan. To investigate this further, we evaluated the long-lived populations for other phenotypic features associated with increased longevity. Specifically, we checked ROS levels at early stationary phase (48 hours) as an indication of changes in mitochondrial function known to effect lifespan (Figure 4C). We could not, however, discern a common pattern among all of the conditions, with only CR and the CR concentrate exhibiting increased ROS levels. It is, therefore, unlikely that a common change in mitochondria function is responsible for the CLS increase observed in all four populations, though it is intriguing that both CR-associated conditions show the increase in ROS.

Comparative gene expression profiling of all 4 long-lived populations yields potential biomarkers of extended CLS

Hidden among the diversity of gene expression changes between the 4 different lifespan extending conditions, were 18 commonly upregulated and 5 downregulated genes (Figure 3B and 5A). By qRT-PCR with a p-value cut-off of 0.05, we could confirm 12 of the 18 upregulated genes in all four conditions (Figure 5B and C), and 3 of the 5 downregulated genes (Figure 6C). For one of the downregulated genes, YAR029W, its mRNA was below the level of detection for RT-PCR confirmation (data not shown). To test the predictive value of these 15 confirmed genes as biomarkers for CLS extension, we used qRT-PCR to measure their expression from two additional long-lived deletion mutants, mba1∆ and bna2∆, that were randomly selected from a previous genome-wide screen for longevity genes [9]. Although both mutants consistently showed extended CLS (Figure 6A), we could only show significant expression changes for 1 of the original 12 upregulated genes, CWP2, in both strains at day 8 (Figure 6B). Most of the upregulated genes other than CWP2 are therefore not effective biomarkers for CLS extension beyond the 4 original long-lived conditions used for the expression profiling. However, CLS extension for the mba1∆ and bna2∆ mutants was not nearly as extensive compared to CR, ade4∆, INAM, or the CR-concentrate, making it possible that most of these commonly upregulated genes only hold predictive value in cases of more extreme CLS extension.

Success at validating biomarkers improved with the 3 confirmed downregulated genes - LEU3, ADD66, and FYV6. Similar to the initial 4 long-lived conditions, expression of all three genes was downregulated in the mba1∆ and bna2∆ mutants on day 8 (Figure 6C). For additional validation we measured expression of the downregulated genes in a tor1∆ mutant that was well known to extend CLS [10]. Expression of LEU3, ADD66, and FYV6 was also reduced in the tor1∆ mutant (Figure 6C), making it possible that down-regulation of these 3 genes is a common feature of chronologically long-lived yeast strains, and suggesting they could be used as biomarkers for future screening purposes.

To this end, we designed a small blinded experiment to test the predictive power of the four CLS biomarkers (LEU3, ADD66, FYV6, and CWP2). Two known long-lived conditions, 0.5M sorbitol treatment and a gln3∆ mutant, were chosen as test cases [10, 25], along with WT BY4741 and 4 randomly selected deletion mutants from the yeast KO collection. Each sample was blindly assigned a random numerical value by another lab member, and then CLS and relative mRNA biomarker expression were tested in triplicate (Figure 7). The average expression level of each biomarker was normalized to the corresponding average of the WT. Those samples with normalized expression values <1 for the three downregulated biomarkers and a value >1 for the upregulated biomarker CWP2 were predicted to be long-lived (sorbitol, gln3∆, and eaf3∆). When compared to the CLS data, we had successfully predicted the lifespan for 5 out of 6 strains, with the eaf3∆ mutant called as a false positive for extended CLS.

Comparative gene expression profiling of long-lived populations yields genes with a functional impact on CLS

While only 4 of the commonly regulated genes were found to be effective biomarkers of CLS extension, we hypothesized that the overall list was enriched for genes that functionally impact aging in the BY4741 background. To test this idea, we obtained deletion mutants from the yeast knockout collection for 21 of the 23 original shared genes and determined their relative CLS (Figure 8). Of the mutants not shown, fas2∆, qns1∆, and erg1∆ could not be tested because the genes are essential, and the others had no effect on CLS d to WT. The rationale to also test deletion mutants for the unconfirmed genes was because YAR029W expression was below detection limits, and ASH1, GAL80, QNS1 were either trending or significantly upregulated in the CR, ade4∆, and INAM cultures (Figure 5B), suggesting they could still be functionally relevant. We initially predicted that deleting a gene that is downregulated during aging would be beneficial to stationary phase survival and result in CLS extension. However, while all four of the downregulated deletion mutants functionally impacted CLS, only leu3∆ and add66∆ were long-lived compared to WT (Figure 8A). Extended CLS of the leu3∆ mutant was reported previously [26, 27]. Add66 is involved in proteasome assembly and the function of YAR029W remains unknown. Fyv6 has a stationary phase specific role in double stranded break repair [28], which may explain poor survival of the fyv6∆ mutant (Figure 8A).

The upregulated genes we tested for functional relevance had less variation in their CLS effect. Of the 12 tested deletion mutants, 8 had a measurable impact on CLS (Figure 8B). Of these 8 mutants, 7 had reduced CLS relative to WT, which was anticipated if up-regulation of these genes was beneficial to stationary phase survival. However, none of the CLS reductions were severe compared to WT (all mutants persisted until day 18 at least), making it unlikely that any of these factors are individually major contributors to stationary phase survival. Instead, the data are consistent with the idea that yeast chronological lifespan is defined by a complex combination of numerous factors, and is a highly multigenic process regulated at the transcriptional level.

Mutations in the adenosine salvage pathway extend CLS similarly to those in the adenine salvage pathway

The only shared upregulated gene to cause CLS extension when deleted was ADO1 (Figure 9A), which encodes an adenosine kinase that salvages adenosine derived from the methyl cycle by converting it to AMP [29]. Ado1 also converts 5-amino-4-imidazole-carboxamide ribonucleoside (AICAR) into the active phosphorylated form [30]. While the extended CLS for this mutant was weaker than deletions of de novo purine biosynthesis genes such as ade4∆ (Figure 9A), it was similar to the effects of deleting APT1 or AAH1 [9], both of which are involved in salvaging adenine to produce AMP (see adenine salvage pathway in Figure 9C). We had earlier proposed that the modest lifespan extension caused by mutants such as apt1∆ or aah1∆ was possibly due to secondary effects on expression of the de novo purine synthesis pathway [9]. To test this idea for the ado1∆ mutant, we analyzed the effect of this mutation on ADE4 expression and found it to be reduced at day 8 (Figure 9B), consistent with such a model. On the other hand, up-regulation of ADO1 in the ade4∆ mutant (Figure 5C and 9B) suggested that adenosine salvage from the methyl cycle was important in compensating for loss of de novo purine biosynthesis. Indeed, deleting ADO1 completely blocked the ade4∆-induced CLS extension, with the double mutant having a CLS even shorter than WT (Figure 9A). Eliminating two pathways to AMP synthesis was apparently too detrimental for proper stationary phase survival.

Yeast strains and media

The wild type strain used in this study was BY4741 (MATa his3∆1 leu2∆0 met15∆0 ura3∆0). Strains containing gene deletions were obtained yeast knockout collection [53], or generated by PCR-mediated gene replacement using the G418 resistance marker KANMX4 from pRS400 [54]. Synthetic complete (SC) media was used for all experiments, with glucose added to a final concentration of 2.0% (NR; non restricted) or 0.5% (CR; calorie restricted) [9, 55]. For isonicotinamide (INAM) cultures, a 1 M INAM stock was used to bring the final concentration to 25 mM INAM in the SC media. All cultures were grown at 30°C.

Chronological lifespan assays

Semi-quantitative CLS assays were performed as described previously [25]. Briefly, overnight 10 ml cultures were started from single colonies and grown at 30°C in 15 ml glass culture tubes with loose fitting metal caps on a New Brunswick Scientific roller drum. 100 μl of the overnight cultures was used to inoculate fresh 10 ml cultures in the indicated SC medium. For each time point, 20 μl aliquots were removed and serially diluted 1:10 with sterile H₂O in a 96-well plate. Then 2.5 μl of each dilution was spotted onto YPD plates and colonies allowed to grow for 2 days to measure cell viability. Digital photos were then taken of each plate. At the end of each experiment, images of the serial dilutions on YPD plates for each sample and time point were cropped and compiled into a single image of the time course.

Media swap

BY4741 was grown in 10 mL SC cultures with either 2.0% (NR) or 0.5% (CR) glucose at 30°C for 5 days. On day 5, cultures were centrifuged at 2,500 RPM in an Eppendorf 5810-R table-top centrifuge with a swinging buck rotor. The supernatants were collected and passed through a 0.2 micron filter to remove residual cells. The cell pellets were then resuspended in the opposite sterilized culture supernatant and the swapped cultures allowed to age at 30°C in the roller drum. Semi-quantitative CLS assays were then carried out at the indicated time points.

Expired media concentrate

BY4741 was grown in 50 mL SC cultures (with either NR or CR levels of glucose) at 30°C for 5 days. The cultures were centrifuged at 2,500 RPM in an Eppendorf 5810-R table-top centrifuge with a swinging buck rotor. The supernatants were collected and condensed from 50 mL down to 1 mL using a Büchi Rotavapor-R apparatus, then sterilized by passing through a 0.2 micron filter. For each experimental culture, 1 mL of media concentrate (derived from either CR or NR cultures) was added to 9 mL of non-restricted SC media. As a control, 1 ml of sterile H₂O was added instead. The cultures were then inoculated and CLS measured. For the ade4∆ media concentrate, the ade4∆ mutant (SY403) from the yeast knockout collection was grown in 50 mL of SC (NR), and then the expired media concentrated in the same manner.

Microarray analysis

Total RNA was harvested from duplicate 10 mL BY4741 NR, CR, CR conc., INAM, and ade4∆ (SC, NR) cultures on day 8 using the acid phenol method [56]. The gene expression profile of each sample (4 μg RNA) was then determined by using Affymetrix Yeast Genome 2.0 GeneChip arrays as previously described [57]. Raw data from the CEL files were quantile normalized and expression values estimated using GCRMA in Bioconductor [58]. Lists of differentially expressed genes for all pair-wise comparisons (i.e. long-lived condition versus normal control) were derived using Limma [59], which applies a modified t-test, correcting for multiple hypothesis testing by applying False Discovery Rate (FDR) correction to the p-values [58] and applying a 20% FDR cutoff. Hierarchical cluster heat maps of differential expression levels (log₂) between the long-lived conditions versus normal control samples of genes that passed the 20% FDR cutoff (y-axis) vs. long-lived condition (x-axis) were generated using TM4 MultiExperiment Viewer. The Affymetrix array data sets have been deposited in the NCBI Gene Expression Omnibus, and are accessible through GEO Series accession number GSE61341.

qRT-PCR confirmation of microarray data

cDNA was synthesized from 1 μg total RNA using a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems) per manufacturer's instructions, then quantitated by PCR reactions using SYBR Hi-ROX Mastermix (Bioline) and a StepOnePlus Real Time PCR System (Applied Biosystems). Samples were isolated from day 8 cultures. Transcript levels were normalized to levels of ACT1 mRNA. Standard deviation for expression level of each transcript of interest was calculated for each condition.

Randomized, blinded test of biomarker utility

We chose 2 independent means of extending CLS for this experiment. A long-lived gln3∆ mutant was used and the WT BY4741 strain was also treated with 0.5M sorbitol to extend CLS by osmotic stress [25]. Four single gene deletion strains of unknown chronological lifespan were also randomly selected from the BY4741 knockout collection (snx41∆, eaf3∆, ydl119c∆, and sds23∆). Each test strain and the BY4741 control were grown in 2 sets of biological triplicates (10 mL SC 2% glucose media). Each triplicate set was assigned an independent, random numerical value by a third party. The CLS of one set of cultures was assessed using the semi-quantitative CLS assay, and the other was used for measuring the relative mRNA expression of each of the putative biomarkers LEU3, ADD66, FYV6, and CWP2 using qRT-PCR (described above) on day 8. The average expression values for each set of unknown triplicate was then normalized to the known WT expression values. Those samples with normalized values <1 for the downregulated biomarkers (LEU3, ADD66, and FYV6) and >1 for the upregulated biomarker (CWP2) were predicted to correspond to the long-lived samples of the first set.

Acetic acid measurements and treatments

To measure acetic acid in the growth media, 100 μL aliquots were taken at designated time points from each aging 10 mL culture. Cells were pelleted by centrifugation (2,500 rpm at 4°), and 50 μL of supernatant was removed and stored at −80°C. Acetic acid concentration for each sample was then later determined using the Acetic Acid Kit (Biopharm AG) per manufacturer's instructions. The acetic acid concentrations and standard deviations provided are an average of three biological replicates for each condition. To assess sensitivity to extracellular acetic acid, the cell viability of each aging culture was quantitated before and after treatment with 300 mM acetic acid for 200 minutes at 30°C. To quantitate viability, 20 μl aliquots were taken from each culture, diluted in water, and spread onto YPD (2% glucose) plates. The total number of colonies for each group was counted and the viability ratio of pre-acetic acid treatment to post-treatment was calculated. Acetic acid tolerance was assessed on both day 2 and day 5 of aging cultures for each condition. For each condition and time point, three biological replicates were tested.

ROS measurements

Cell cultures were started at OD₆₀₀ = 0.015 in SC medium. After 48 hours, cells were harvested and washed with incubation buffer (10 mM MES, 0.1 M KCl, 0.14 M NaCl, pH 6.5). Equal numbers of cells (1×10⁷) for each condition were resuspended in 1 mL of incubation buffer. Dihydrorhodamine 123 (1 mg/ml) was added to each reaction (1×10⁷cells/ml) to obtain a final concentration of 5 μM. Cells were incubated for 10 min at room temperature, and the fluorescence intensity was then measured using a Molecular Devices Spectramax M2 plate reader at excitation 504/emission 534 nm. For each condition, three biological replicates were tested and normalized to WT NR samples.