This paper characterizes transcriptional programs of fission yeast to a range of environmental stresses. We describe a common stress response, which is regulated primarily by the Sty1p MAPK pathway. In addition to the CESR, the cell initiates gene expression programs more specific to each stress or subsets of stresses. These specialized programs may involve Sty1p and/or as yet uncharacterized stress-specific regulatory factors. It should be noted that microarrays measure differences in mRNA levels, which may reflect regulatory changes in transcription and/or in mRNA turnover (e.g., Fan et al., 2002
The CESR contains genes whose expressions change stereotypically with stress. We have identified ~140 induced and ~100 repressed genes that provide a representative sample of the general response to stress. The predicted functions of CESR genes suggest that stressed cells selectively reprogram a wide range of activities, including carbohydrate metabolism, protein synthesis, and several other metabolic functions, possibly to save energy by limiting growth-related activities and to synthesize stress-protective molecules and cofactors. CESR genes may be controlled by known and predicted regulators that form part of the CESR, such as b-ZIP and Zn-finger transcription factors, phosphatases, a Sty1p-interacting protein kinase, and components of the PKA pathway. There was significant overlap between genes of the CESR and genes of the CER/ESR, recently described in S. cerevisiae
(see “Introduction”). This suggests that a general response to stress, involving similar gene sets, is evolutionarily conserved. This conservation is in contrast to genes induced during meiotic differentiation, where the overlap between S. pombe
and S. cerevisiae
is surprisingly small, given the large numbers of genes that are regulated (Mata et al., 2002
The CESR and ESR/CER responses discovered through DNA microarray analysis are manifestations of the general stress response described previously (reviewed by Siderius and Mager, 1997
). The general stress response was postulated to explain the phenomenon of cross-protection, wherein exposure to a nonlethal dose of one stress can protect against a potentially lethal dose of a seemingly unrelated stress. The degree of cross-protection varies depending on the stress and is not always reciprocal, indicating that stress-specific responses are required for full protection. General stress resistance is also associated with nutrient deprived cells, cells in stationary phase, and differentiated spores. Indeed, the CESR is activated during nitrogen starvation (J.M., unpublished data), and at least some of the CESR genes are also induced during sporulation (Mata et al., 2002
The CESR, irrespective of the type of stress, seems to be controlled predominantly by the Sty1p protein kinase and, to a lesser extent, by the Atf1p transcription factor. This is in contrast to S. cerevisiae,
where the ESR/CER is not governed by one “all-purpose” regulatory system. Instead, different signaling pathways and transcription factors, acting in response to specific stress conditions, control a common set of genes (Figure ). For example, in response to osmotic stress, the Hog1p MAPK pathway is critical for induction of ESR genes (O'Rourke et al., 2002
), whereas MMS induces similar genes through a pathway that requires the Mec1p kinase (Gasch et al., 2001
). Thus, S. pombe
and S. cerevisiae
appear to use different regulatory strategies to achieve similar outcomes.
Figure 7 Regulation of stress genes in budding and fission yeasts. Both yeasts control a similar core group of genes in response to all or most stresses. These genes are mainly regulated by stress-specific mechanisms in budding yeast, whereas in fission yeast, (more ...)
The sorbitol stress response in S. pombe
depended on Sty1p, both for CESR and stress-specific gene expression, possibly reflecting the evolutionary origin of the stress-activated MAPK pathway. The S. cerevisiae
Hog1p pathway, which is homologous to the Sty1p pathway, is required specifically for the response to osmotic stress (O'Rourke et al., 2002
). Thus, the stress-activated MAPK pathway may have evolved to control osmotic stress (as is still evident in budding yeast) and later acquired a more general role of stress regulation (as in fission yeast and Metazoa). Alternatively, the S. cerevisiae
Hog1p kinase evolved away from an ancestor with a more general function.
Regulation of the CESR by Sty1p appears to be complex, with some genes requiring the kinase for both basal and stress-induced expression and others requiring it only during stress. The only known transcription factor target for Sty1p is Atf1p, which binds the conserved ATF/CRE promoter motif (Hai and Hartman, 2001
). This motif was enriched in the CESR genes and other gene groups that showed Sty1p- and Atf1p-dependent transcription. The differences in expression profiles exhibited between sty1
mutants suggest that Sty1p may interact with additional downstream regulators to control various aspects of gene expression. We identified a potential regulatory motif for an unidentified transcription factor among the genes that depended on Sty1p, but not on Atf1p, for stress induction. Interestingly, the induction of most genes directly involved in stress defense depended on Atf1p, whereas genes with regulatory functions tended to be induced by Sty1p independently of Atf1p.
Notably, a number of genes were derepressed in sty1
mutants, both in stressed and unstressed cells. This may reflect indirect cellular responses to compensate for the problems caused by the absence of these regulators. For example, the basal gene expression levels of many CESR genes were reduced in both sty1
mutants, which may lead to stress. However, it is possible that Sty1p and/or Atf1p also play direct roles in repressing gene expression. In fact, a previous study has suggested that Atf1p can both induce and repress genes depending on the Sty1p phosphorylation state (Degols and Russell, 1997
). A role for Sty1p in down-regulating gene expression was also apparent from our finding that some CESR genes required Sty1p for repression. How Sty1p and/or Atf1p might control repression of gene expression is not known, but putative regulatory motifs were present in the promoters of Sty1p-repressed genes.
We also examined gene expression programs activated only under certain stress conditions. There were relatively few genes whose expression changes were absolutely specific to particular stresses. Analyses of SESR genes, either specific to a given stress or shared with other stresses, will aid in our understanding of the types of damage and alterations to metabolism caused by various stresses. Besides its role in the CESR, the Sty1p pathway also controls subsets of “stress-specific” genes, with the requirements varying depending on the stress. For example, heat-specific gene expression was independent of Sty1p, whereas H2
-specific gene expression depended to a large part on Sty1p. The characterization of the cation-specific induction of cta3
in S. pombe
illustrates how Sty1p may regulate stress-specific genes (Greenall et al., 2002
encodes a cation transporter, whose induction requires both activation of Sty1p and derepression of the Tup11/12p complex. In the absence of Tup11/12p, the cta3
promoter becomes responsive to other stresses regulated by Sty1p. Using similar mechanisms, Sty1p may be co-opted to regulate a range of specialized responses and thereby coordinate the CESR with the appropriate specific response.
Our results indicate that there are stress-specific regulators acting independently of Sty1p. Most of these factors, however, are as yet uncharacterized. Heat shock, for example, induced several genes functioning in protein folding or degradation in the absence of Sty1p. A subset of these genes was also induced with H2
and Cd. How only a portion of the heat shock response is activated by these other stresses is an interesting regulatory problem. A likely factor controlling the response to heat is Hsf1p. Intriguingly, S. pombe
Hsf1p contains distinct sequences in its C-terminal domain that are responsive to different stress stimuli and are required for the activation of different subsets of heat stress proteins (Saltsman et al., 1999
). This may explain how some genes of the heat shock response are also activated by other stresses.
Pap1p is a redox-sensitive transcription factor involved in regulating oxidative stress responses as well as drug and heavy metal resistance. Pap1p is particularly important for gene induction in response to low levels of H2
, whereas Atf1p becomes the predominant transcription factor with increasing levels of H2
, resulting in the induction of a different set of genes (Quinn et al., 2002
). We have characterized this dose-dependent switch in transcription factors and target gene expression at the whole genome level (D. Chen, W.M. Toone, J. Mata, G. Burns, N. Jones, and J. Bähler, manuscript in preparation). Similarly variegated responses are likely to be found by varying the intensity of other environmental stresses. In this study, 0.02% MMS induced only two stress-specific genes and failed to induce a DNA damage response. Using a similar MMS concentration with S. cerevisiae, Gasch et al. (2001)
observed induction of a surprisingly small cluster of DNA damage-specific genes. In contrast, Jelinsky et al. (1999)
used a higher dose (0.1% MMS) and observed a robust DNA damage response. These studies suggest that noxious compounds may cause different types of damage and induce different responses depending on the dose used.
Stress responses varied not only in the type of genes involved but also in the kinetics of gene expression. For example, heat and sorbitol stress showed a rapid and transient response to stress, whereas gene induction during H2O2 stress continued to increase over the course of the experiment. In all cases, stress was continually applied throughout the experiment. Thus, cells appear to adapt to some situations and return to homeostasis more readily than to others, although a delayed response may also reflect the time taken for a particular stress to take effect. It would be interesting to determine if the adaptation seen in heat and osmotic stress is due to active down-regulation of Sty1p and inactivation of the response or due to a more efficient adaptation in these conditions, eliminating the need for continued Sty1p activity.
Global transcriptional responses to stress have now been examined in some detail using gene expression profiling in both S. cerevisiae and S. pombe. The transcriptional programs consist of general and stress-specific responses. Although both yeasts induce a similar set of general stress genes, there are striking differences in the regulatory pathways used for stress responses. Stress adaptation and cross protection will likely require a combination of general and specific genes, depending on the type and intensity of the stress. In addition, some stresses cause similar types of damage and therefore induce similar sets of proteins. All eukaryotic cells have mechanisms for dealing with stress. Even within the protected confines of the human body, cells can experience fluctuations in osmolarity, exposure to noxious agents, and variations in the levels of oxygen and reactive oxygen species. Some responses, such as ischemic-reperfusion injury, are closely associated with pathology. Many of the factors important for regulating stress responses, including MAPK signaling pathways and AP-1 transcription factors, are conserved from yeast to human. It is therefore likely that lessons learned from stress responses in yeast will facilitate the understanding of how cells in general respond to a changing environment.