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. 2008 Jan;19(1):308-17.
doi: 10.1091/mbc.e07-08-0735. Epub 2007 Nov 14.

Multiple pathways differentially regulate global oxidative stress responses in fission yeast

Dongrong Chen  1 , Caroline R M Wilkinson, Stephen Watt, Christopher J Penkett, W Mark Toone, Nic Jones, Jürg Bähler
Affiliations

Multiple pathways differentially regulate global oxidative stress responses in fission yeast

Dongrong Chen et al. Mol Biol Cell. 2008 Jan.

Abstract

Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.

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Figures

Figure 1.
Figure 1.
Changes in gene expression in response to different doses of HP. Hierarchical cluster analyses with columns representing experimental time points and rows representing genes. The mRNA levels at each time point relative to the levels in the same cells before HP treatments are color coded as indicated at the bottom, with missing data in gray. (A) Three independent biological repeats each of wild-type cells exposed to low, medium, and high doses of HP as indicated on top. Cells were collected before and at 5, 15, 30, and 60 min after exposure to HP. Data for 3466 genes with significant differential expression in at least one of the three doses are shown. Brackets on the left indicate clusters of genes described in the text. Annotated gene lists of these clusters are provided in Supplemental Table 3. (B) Expression profiles of CESR genes in wild-type cells exposed to low and medium doses of HP (with expression ratios from three repeats averaged). Data shown are for 888 genes that are included in the large lists of induced and repressed CESR genes from Chen et al. (2003), and they are also significantly regulated in the dose experiments. Brackets on the left indicate clusters of genes described in the text. Annotated gene lists of these clusters are provided in Supplemental Table 3. (C) Expression profiles of non-CESR genes in wild-type cells exposed to low and medium doses of HP as in B. Data shown are for 2578 genes that are not included in the CESR genes from Chen et al. (2003) but are significantly regulated in the dose experiments.
Figure 2.
Figure 2.
Changes in gene expression in response to four different oxidative stress conditions. Hierarchical cluster analysis with columns representing experimental time points, and rows representing the 1324 genes that show significant differential expression and at least twofold changes in one or more of the four stress conditions (with expression ratios from two or three repeats averaged). For each stress condition, data are shown from 0, 15, and 60 min after stress exposure as indicated on top. The mRNA levels at each time point relative to levels in the same cells before oxidative stress are color coded as indicated at the bottom, with missing data in gray. Brackets on the left indicate clusters of genes described in the text. Annotated gene lists of these clusters are provided in Supplemental Table 3.
Figure 3.
Figure 3.
Regulation of gene expression in response to low dose of HP and Md. Oxidative stress experiments performed in wild type (wt) and in different deletion mutant backgrounds as indicated on top; columns for each experiment represent data from 0, 15, and 60 min after stress exposure. The mRNA levels at each time point relative to levels in wild-type cells before oxidative stress are color-coded as indicated at the bottom with missing data in gray. The 69 genes that were significantly regulated in both stresses and showed >1.5-fold change in at least one stress in wild-type cells were used for clustering. The bracket indicates the 13 genes that require both Pap1p and Prr1p for induction in HP (Table 1).
Figure 4.
Figure 4.
Regulation of gene expression in response to TBH and medium dose of HP and differential requirements for the MAP kinases Sty1p and Pmk1p. Oxidative stress experiments performed in wt and in different mutant backgrounds as indicated on top; columns for each experiment represent data from 0, 15, and 60 min after stress exposure. The mRNA levels at each time point relative to levels in wild-type cells before oxidative stress are color coded as indicated at the bottom, with missing data in gray. The 2095 genes that were significantly regulated in either stress and showed >1.5-fold change in at least one stress in wild-type cells were used for clustering. These genes were first clustered into five main groups, and genes within each group were then clustered hierarchically. (A) Cluster of 620 genes that are induced in both stresses and in most regulatory mutants. (B) Cluster of 499 genes that are repressed in both stresses and in most regulatory mutants. (C) Cluster of 386 genes that are induced in both stresses and dependent on Atf1p and Sty1p. The bracket indicates genes that require Pmk1p for induction in TBH. (D) Cluster of 525 genes that are repressed in both stresses and dependent on Atf1p and Sty1p. (E) Serial dilutions of exponentially growing cultures of wt cells, sty1Δ and pmk1Δ single and double mutants, and atf1Δ cells were spotted on plates without oxidant (control) and with addition of TBH or HP. (F) Western analyses of protein extracts from TBH and HP stress-time course experiments. Top, antibody probing Sty1p phosphorylation; bottom: anti-HA antibody as input control of HA-tagged Sty1p.
Figure 5.
Figure 5.
Regulation of core genes induced in all four oxidative stress conditions. (A) Oxidative stress experiments performed in wt and in different deletion mutant backgrounds as indicated on top; columns for each experiment represent data before and at 15 and 60 min after stress exposure. The mRNA levels at each time point relative to levels in wild-type cells before oxidative stress are color coded as indicated at the bottom, with missing data in gray. The 41 genes that were significantly regulated and showed >1.5-fold change in all four stress experiments in wild-type cells were used for clustering. The gene names are indicated at left, and annotations are provided in Supplemental Table 2. (B) Average gene expression profiles for the 41 genes shown in A in the different stress conditions.
Figure 6.
Figure 6.
Regulation of CESR genes in oxidative stress. Expression profiles of CESR genes in the different stress conditions. Data shown are for 694 genes that are included in the large lists of induced and repressed CESR genes from Chen et al. (2003) and are also significantly regulated in the dose experiments. Oxidative stress experiments performed in wt and in different deletion mutant backgrounds as indicated on top; columns for each experiment represent data before, and at 15 and 60 min after stress exposure. The mRNA levels at each time point relative to levels in wild-type cells before oxidative stress are color coded as indicated in B, with missing data in gray. Brackets on the left indicate clusters enriched for genes encoding ribosomal biogenesis proteins (cluster 10) and ribosomal proteins (cluster 11). Annotated gene lists of these clusters are provided in Supplemental Table 3.
Figure 7.
Figure 7.
Hsr1p is involved in the response to HP stress. (A) Gene expression profiles of hsr1 in response to the medium dose of HP in wild type and different mutant strains and time points as indicated at the bottom. The mRNA levels at each time point relative to levels in the wild-type cells before HP treatments are indicated as expression ratios. (B) Average gene expression profiles of the 41 core oxidative stress genes (top; as in Figure 5) and 324 induced CESR genes (bottom; Chen et al., 2003) in response to the medium dose of HP in wild-type and different mutant strains and time points as indicated at the bottom. (C) Northern analyses of two genes whose induction in response to the medium dose of HP is much lower in hsr1Δ mutants compared with wt cells. Row 1, SPAC23D3.05c, an alcohol dehydrogenase pseudogene; row 2, SPBC1773.06c, predicted to encode an alcohol dehydrogenase. The rRNA amounts are shown as a loading control. (D) Serial dilutions of exponentially growing cultures of wt cells and hsr1Δ and sty1Δ mutants were spotted on plates without oxidant (control) or with addition of HP. (E) Scheme of the regulatory events in response to stress caused by HP or TBH. The gray arrows indicate weak links, and dashed arrows indicate a pathway that seems to be only activated in HP. See main text for details.
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