Cellular memory of acquired stress resistance in Saccharomyces cerevisiae

Abstract

Cellular memory of past experiences has been observed in several organisms and across a variety of experiences, including bacteria "remembering" prior nutritional status and amoeba "learning" to anticipate future environmental conditions. Here, we show that Saccharomyces cerevisiae maintains a multifaceted memory of prior stress exposure. We previously demonstrated that yeast cells exposed to a mild dose of salt acquire subsequent tolerance to severe doses of H(2)O(2). We set out to characterize the retention of acquired tolerance and in the process uncovered two distinct aspects of cellular memory. First, we found that H(2)O(2) resistance persisted for four to five generations after cells were removed from the prior salt treatment and was transmitted to daughter cells that never directly experienced the pretreatment. Maintenance of this memory did not require nascent protein synthesis after the initial salt pretreatment, but rather required long-lived cytosolic catalase Ctt1p that was synthesized during salt exposure and then distributed to daughter cells during subsequent cell divisions. In addition to and separable from the memory of H(2)O(2) resistance, these cells also displayed a faster gene-expression response to subsequent stress at >1000 genes, representing transcriptional memory. The faster gene-expression response requires the nuclear pore component Nup42p and serves an important function by facilitating faster reacquisition of H(2)O(2) tolerance after a second cycle of salt exposure. Memory of prior stress exposure likely provides a significant advantage to microbial populations living in ever-changing environments.

Figure 1

Cells retain a memory of acquired stress resistance. Cells were exposed to NaCl for 60 min and then returned to fresh stress-free YPD medium, and H₂O₂ tolerance was scored over time. (A) Cell viability is shown across 10 doses of H₂O₂ (ranging from 0.5 to 5 mM) at various times after removal from NaCl. (B) A single survival score was calculated from all H₂O₂ doses and at each time point (see Materials and Methods) and then normalized to the survival score of the mock-treated culture. Resistance relative to the maximum survival score at 60 min after NaCl treatment is shown (blue line). The percentage of original cells in the culture, as estimated by optical density, is shown by the gray line; the culture underwent between three to four doublings in the course of 360 min. Each plot shows the average and standard deviation of triplicate experiments.

Figure 2

Ctt1 protein persists over time. Levels of CTT1 mRNA (dark blue line) and FLAG-tagged Ctt1 protein (light blue line) were measured throughout the experiment by quantitative PCR and Western analysis, respectively. Fold-change in FLAG-tagged CTT1 mRNA was calculated relative to basal levels measured before stress. FLAG-tagged Ctt1p was normalized to an internal actin control before calculating fold-change relative to unstressed cells. Each plot represents the average and standard deviation of at least three biological replicates.

Figure 3

An exogenous pulse of Ctt1p produces a memory of H₂O₂ resistance. A strain in which the endogenous CTT1 was replaced with plasmid-borne FLAG-tagged CTT1 driven by the estradiol-regulated promoter was induced with 1 μM estradiol for 3 hr and then returned to stress-free medium. (A) H₂O₂ tolerance across 11 doses of H₂O₂ (ranging from 1 to 12 mM) was scored for 360 min after cells were removed from estradiol (red curve) and compared to cells harboring the native genomic, FLAG-tagged CTT1 induced with 0.15 M NaCl (blue curve). This dose of salt was chosen because it produces equivalent Ctt1p compared to estradiol induction. Estradiol-induced cells exposed to 0.7 M NaCl (gray curve) for the last 60 min showed no significant increase in H₂O₂ tolerance. Survival scores were adjusted to the maximum level survived after estradiol induction to represent the percentage maximal survival. (B) FLAG-tagged Ctt1p was measured by quantitative Western analysis, normalized to an internal actin control, under the conditions described in A. The fold-change in expression relative to unstressed cells is shown. Plots represent the average and standard deviation of at least three biological replicates.

Figure 4

Cells with prior NaCl exposure have a faster gene-expression response to H₂O₂. Cells were exposed to either 0.7 M NaCl or a mock treatment for 60 min and then grown in stress-free YPD medium for 240 min at which point 0.5 mM H₂O₂ was added to the culture. (A) The heatmap shows log₂ expression changes of 449 genes with significantly different H₂O₂-dependent expression in cells pretreated with NaCl (FDR < 0.01). Each row represents a gene and each column represents a microarray. Time points of the cellular response to 0.7 M NaCl or 0.5 mM H₂O₂ are labeled in minutes; the 0-min sample shows expression just before H₂O₂ treatment in mock- or NaCl-treated cells. Red represents gene induction and green represents gene repression relative to unstressed cells. Each data point is the average of biological replicates. Clusters (labeled 1–6) were manually identified in hierarchically clustered data. (B) The response to H₂O₂ was scored similarly in naive (N) and NaCl-pretreated (P) wild-type and nup42Δ cells at 10 and 20 min after treatment. Genes are organized as in A. (C) The average log₂ fold-change in expression of induced and repressed genes in B is shown for wild-type and nup42Δ cells. The average response of naive wild-type cells is shown in gray on the nup42Δ plot.

Figure 5

The effect of NPC subunits on the faster expression response of TSA2. The average log2 fold-change in TSA2 transcript was scored by quantitative PCR in wild-type, nup42Δ, nup59Δ, and nup100Δ cells as described in Figure 4 and in Materials and Methods. The response of the naive wild type is shown in gray on mutant plots.

Figure 6

Cells with a memory of stress exposure show faster reacquisition of H₂O₂ tolerance. (A) The experimental schema represents H₂O₂ tolerance in cells that were exposed to 0.7 M NaCl for 60 min (black trace), returned to stress-free YPD medium for 240 min, and then exposed to a second treatment of 0.7 M NaCl (blue trace). (B) The percentage maximal H₂O₂ tolerance is shown, as described in Figure 1B, during the first NaCl treatment (black, as depicted in A) and during the second NaCl treatment (blue) for wild-type (left) and nup42Δ (right) cells. To compare the fold-change in H₂O₂ tolerance provoked by each round of NaCl exposure, H₂O₂ tolerance was scaled to the tolerance measured immediately before each NaCl addition (dashed blue line in A). Data represent the average and standard deviation of biological triplicates. Points with statistically significant differences (P < 0.05) are indicated with an asterisk.

Figure 7

Sequences common to genes with faster expression response. (A) The characterized GRS1 zip code upstream of TSA2 is shown aligned to a similar sequence upstream of the CTT1 gene. Positions of identity are highlighted in gray. (B) MEME motif identified in the upstream regions of 77 genes induced with a faster response upon recurring stress (cluster 3, Figure 4). Information content (bits), where the height of each letter represents the frequency of the base at that position in the motif, is plotted.