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Vol. 15, Issue 2, 851-860, February 2004
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* Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, United Kingdom;
The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom; and
MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxon OX11 ORD, United Kingdom
Submitted August 8, 2003;
Revised September 15, 2003;
Accepted October 6, 2003
Monitoring Editor: Pamela Silver
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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200 genes whose transcript levels were significantly altered at least twofold in response to 500 Gy of gamma IR in a temporally defined manner. The majority of induced genes were core environmental stress response genes, whereas the remaining genes define a transcriptional response to DNA damage in fission yeast. Surprisingly, few DNA repair and checkpoint genes were transcriptionally modulated in response to IR. We define a role for the stress-activated mitogen-activated protein kinase Sty1/Spc1 and the DNA damage checkpoint kinase Rad3 in regulating core environmental stress response genes and IR-specific response genes, both independently and in concert. These findings suggest a complex network of regulatory pathways coordinate gene expression responses to IR in eukaryotes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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It is well established that many physiological responses to DNA damage are regulated at the level of gene expression. In Escherichia coli, the SOS response induces the expression of a network of genes after DNA damage through the regulatory LexA and RecA proteins (Friedberg et al., 1995
). In lower eukaryotes, studies with the budding yeast Saccharomyces cerevisiae have identified a large number of damage-induced genes, including many involved in DNA metabolism and DNA repair (Aboussekhra et al., 1996; Gasch et al., 2001). In the distantly related fission yeast Schizosaccharomyces pombe, rhp51+, uvi15+, uvi31+, UVDE+, and rhp16+ have been shown to be DNA damage inducible (Bang et al., 1996; Davey et al., 1997; Shim et al., 2000).
DNA damage checkpoint pathways function to delay the eukaryotic cell cycle in response to DNA damage, thus providing an opportunity for DNA repair. Central to the DNA damage checkpoint pathway are two highly conserved phosphoinositol-related kinases, Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) and their yeast homologs SpTel1 and SpRad3 in S. pombe, and ScTel1 and ScMec1 in S. cerevisiae. Activation of ATM and ATR kinases by DNA damage leads to cell cycle arrest through a number of downstream effector molecules including the effector kinases CHK1/SpChk1/ScChk1 and CHK2/SpCds1/ScRad53 (for review, see Nyberg et al., 2002). In addition to regulating the cell cycle, DNA damage checkpoints also coordinate transcriptional responses that facilitate DNA synthesis and repair. In mammalian cells, genes encoding a number of key cell cycle regulators and proteins involved in DNA synthesis and DNA repair are regulated by the DNA damage checkpoint pathway. These include p21/Cip1, 14-3-3
, GADD45, and p53R2 and several genes encoding classical DNA repair factors (for review, see Khanna and Jackson, 2001).
In mammalian cells, the JNK and p38 stress-kinase pathways play a significant role in regulating the transcriptional stress responses to a variety of DNA damaging agents, including IR, UV light (UV), and oxidative stress. After their activation, JNK and p38 MAP kinases regulate the transcriptional responses to stress through phosphorylation and transactivation of transcription factors c-Jun (Hibi et al., 1993; Kyriakis et al., 1994), ATF2 (Gupta et al., 1995; Livingstone et al., 1995) and Elk-1 (Cavigelli et al., 1995; Whitmarsh et al., 1995). The activation of both the c-Jun NH2-terminal kinase (JNK) and p38 stress-kinases in response to IR is dependent on the ATM checkpoint kinase (Shafman et al., 1995), and a role for p38
in the DNA damage checkpoint has been identified (Wang et al., 2000). Thus, there seem to be significant links between the stress-kinase and the DNA damage checkpoint pathways in the response to ionizing radiation.
In S. cerevisiae, RNR2 and RNR3, which encode the small and large subunits of ribonucleotide reductase, respectively (Elledge and Davis, 1987; Hurd et al., 1987), are transcriptionally induced in a checkpoint-dependent manner: After activation of the Mec1-Rad53-Dun1 checkpoint pathway, Crt1 (a transcriptional repressor) is hyperphosphorylated. This leads to its promoter-dissociation and the subsequent derepession of the RNR2 gene in response to DNA damage (Zhou and Elledge, 1993; Allen et al., 1994; Huang et al., 1998). Microarray analysis has recently identified a Mec1-dependent gene expression signature in response to DNA-damaging agents that includes a large set of genes responsive to diverse environmental stresses (Gasch et al., 2001). A role for the DNA damage checkpoint pathway in regulating transcriptional responses to DNA-damaging agents has also been identified in fission yeast (Harris et al., 1996; Shim et al., 2000), although it is not known to what extent the checkpoint pathway coordinates damage-induced transcription in this microorganism.
In fission yeast, the Sty1/Spc1 stress-kinase is closely related to the mammalian and Drosophila JNK and p38 stress-kinase pathways, and to the Hog1 pathway in S. cerevisiae (Toone and Jones, 1998). In contrast to the Hog1 pathway in budding yeast, Sty1/Spc1 is activated in response to a wide variety of environmental stresses, including UV, methyl-methane sulfonate (MMS), oxidative stress, osmotic stress, and heatshock (Millar et al., 1995; Shiozaki and Russell, 1995; Degols et al., 1996; Degols and Russell, 1997; Shieh et al., 1997). Sty1 coordinates the transcriptional response to stress, in part, through regulating the bZip transcription factors Atf1/Gad7 (Shiozaki and Russell, 1995; Takeda et al., 1995; Kanoh et al., 1996; Wilkinson et al., 1996; Gaits et al., 1998) and Pap1 (Toone et al., 1998). Deletion of these transcription factors results in altered sensitivity to a subset of stresses (Toda et al., 1991; Shiozaki and Russell, 1995; Degols and Russell, 1997; Nguyen et al., 2000; Quinn et al., 2002), indicating a role for these factors for modulating specific transcriptional stress responses. A central role for the Sty1 mitogen-activated protein kinase (MAPK) pathway has recently been identified in coordinating a core environmental stress response (CESR). The CESR defines a group of genes which are transcriptionally up-regulated in response to all, or most, environmental stresses, including oxidative stress, heat shock, osmotic shock, heavy metal stress, and the DNA-damaging agent MMS (Chen et al., 2003).
Fission yeast provide an excellent model system for studying the eukaryotic responses to DNA-damaging agents, because the cell cycle, DNA integrity checkpoint, stress-response, and DNA repair pathways are all highly conserved and well defined. The S. pombe genome has also been sequenced (Wood et al., 2002), thus facilitating a systematic analysis of global gene expression responses. We have therefore examined the global gene expression responses to IR in fission yeast, by using microarray technology. From these studies, we have defined both a general DNA damage response and a specific gamma radiation response signature for S. pombe, and have identified important roles for components of the DNA damage checkpoint and the stress-response pathways in regulating expression of a number of functional classes of genes in response to IR. Gene expression responses to IR in the budding yeast S. cerevisiae, an alternative but distantly related model eukaryote, have recently been reported. A comparison of IR-induced gene expression responses of fission yeast to those of budding yeast identifies commonalities likely to be shared among all eukaryotes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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4 x 106 cells/ml). Five OD units (25 ml) of cells were harvested by centrifugation and snap frozen in liquid nitrogen (unirradiated control) and immediately after, the remainder of the culture was exposed to 500 Gy of gamma irradiation at 12.5 Gy/min (a total of 40 min). Irradiated cells were allowed to recover for 20 or 120 min at 30°C, shaking at 200 rpm, before 5 OD units were harvested by centrifugation and snap frozen in liquid nitrogen. Asynchronously grown checkpoint mutant strains, including rad3
(ade6-704 leu1-32 rad3::ura4+ ura4-D18 h-), cds1 (leu1-32 cds::ura4+ ura4-D18 h-), and chk1 (chk1::ura4+ ura4-D18 h-); stress-activated MAP kinase mutant sty1 (sty1::ura4+ ura4-D18 h-); and a double delete rad3sty (rad3::ura4+ sty1::ura4+ h-) were cultured and irradiated under the same conditions. Mid-log phase wild-type (WT) cells were synchronized by centrifugal elutriation as described previously (Christensen et al., 2000), and G2 cells harvested before as well as after exposure to 500 Gy of gamma irradiation with further shaking 30°C at 200 rpm for 20, 50, 80, and 120 min. Each time course was repeated independently three times, and total RNA was extracted using a hot phenol method (Lyne et al., 2003).
Microarray Hybridization, Data Acquisition, and Visualization
Approximately 20 µg of total RNA was labeled by directly incorporating Cy-3- and Cy-5-dCTP by using Superscript (Invitrogen, Carlsbad, CA) reverse transcriptase, and the resulting cDNA was hybridized onto glass DNA microarrays containing PCR probes for 99.3% of all known and predicted fission yeast genes (for full details, see Lyne et al., 2003; http://www.sanger.ac.uk/PostGenomics/S_pombe/). Microarrays were scanned using a GenePix 4000B laser scanner and analyzed with GenePix Prosoftware (Axon Instruments, Foster City, CA). Unreliable signals were removed and data normalized using a Perl script (Lyne et al., 2003) and evaluated using Genespring software (Silicon Genetics, Redwood City, CA). Hierarchical clustering was performed with preselected log-transformed gene sets by using Cluster and Tree-View software (Eisen et al., 1998), with uncentered Pearson correlation and average linkage clustering.
Experimental Design
The IR time-course experiments with WT and mutant strains were performed as three independent biological repeats. For WT asynchronous, WT G2 synchronized and mutant strain time courses, labeled samples from each irradiated time point were hybridized with a labeled unirradiated sample. After data acquisition and normalization, the ratios represent the expression levels at each time point relative to the expression level of the untreated sample. Expression ratios for the biological repeats were averaged. Forty-eight microarrays in total were used in this study. The complete processed data are available from http://www.sanger.ac.uk/PostGenomics/S_pombe/
Identification of Checkpoint and Stress-dependent Genes
Gene expression was classified as dependent on checkpoint or stress-kinase genes when mean expression levels were statistically significantly different and changed by more than twofold between WT and mutant checkpoint or stress-kinase strains at the same time point. The Student's t-test was used to determine genes that were significantly different in mean expression level (p value cut-off of 0.05) and an F-test was run to determine whether the variances of the two populations were equal or unequal (p value cut-off of 0.05).
Northern Hybridization
Total RNA was extracted using the hot-phenol method described above. Northern blot analysis and hybridization of membranes to32P-labeled DNA probes were performed as described previously (Christensen et al., 2000). 32P-Labeled probes were synthesized using the Prime-It II Random Primer Labeling kit (Stratagene, La Jolla, CA) in accordance to the manufacturer's instructions.
Quantitative Polymerase Chain Reaction (PCR)
Approximately 20 µg of total RNA was treated with RQ1 RNAse free DNAse (Promega, Madison, WI) to remove contaminating genomic DNA, and reverse transcription was performed using Superscript (Invitrogen). Quantitative PCR (QPCR) was performed using Brilliant QPCR Core Reagent kit (Stratagene) and SYBR Green I (Molecular Probes, Eugene, OR) nucleic acid dye. Custom-made primers were designed using Primer 3 online software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi; Rozen and Skaletsky, 2000). Samples were run in duplicate on the MX4000 QPCR machine (Stratagene), and data were analyzed with the MX4000 software provided by the manufacturer. Only data from the log-linear portion of the amplification were chosen for analysis. Relative quantities of transcript were determined using the 2-Ct formula, where Ct is defined as the cycle at which fluorescence is determined to be statistically significantly above background; Ct is the difference in Ct of the gene of interest and Ct of the normalizer gene (cdc2); and Ct is the difference in Ct at time = t (irradiated) and Ct at time = 0 (unirradiated). Four fivefold serial dilutions of S. pombe genomic DNA were used to confirm that QPCR reaction efficiencies were equivalent.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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IR-induced Gene Expression in Wild-Type Fission Yeast
Genome-wide gene expression profiles of WT (501) S. pombe cultures grown asynchronously were examined after exposure to 500 Gy of gamma IR by using whole-genome microarrays. Genes (204 in total) were identified that exhibited a twofold or greater change in expression levels after IR at either time points. One hundred sixty genes were induced greater than twofold, and 45 genes were repressed greater than twofold (a list of all 204 IR response genes together with gene annotations is available from our Website http://www.sanger.ac.uk/PostGenomics/S_pombe/). The stress response gene rds1+ (Ludin et al., 1995) was induced greater than twofold after 60 min and repressed greater than twofold after 160 min. Only 25% of the 160 induced genes have been characterized. The number of IR-response genes listed here should be regarded as a conservative estimate because some genes failed to make the twofold cut-off in all experiments (see MATERIALS AND METHODS).
Comparison of the IR-response genes with the S. pombe core environmental stress response (Chen et al., 2003) reveals that 108 of the 160 IR-induced genes are present in the CESR. Therefore, the majority of IR-induced genes encode proteins known or predicted to be involved in the environmental stress response, thus indicating an underlying stress response to IR (Figure 1A). IR-response genes common to the CESR include antioxidants such as cta1+, grx1+, trx2+, and gst2+ and genes involved in carbohydrate metabolism such as tps1+, zwf1+, and ntp1+. These stress response genes largely exhibited similar expression profiles in response to IR: a severalfold induction was observed 60 min after initiating exposure to IR, which had returned to approximately unirradiated levels by 160 min (Figure 2A).
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The remaining 52 induced IR-response genes not present in the CESR are involved in a variety of functions, including cell cycle control, signal transduction, transcriptional regulation, and cell metabolism (Table 1). Of the 52 non-CESR-induced IR genes, surprisingly few are known to be involved in DNA repair. These include genes encoding the homologous recombination proteins rhp51+ and rhp54+, the putative translesion DNA synthesis repair enzyme SPCC553.07c (dinB+), and the meiotic recombination repair protein rad50+. The transcriptional induction of rhp51+ by a variety of DNA-damaging agents, including UV and MMS has been reported previously (Park et al., 1998). Notably, the mRNA levels of checkpoint genes rad3+, chk1+, and cds1+ were found not to be significantly modulated in response to ionizing radiation, consistent with the finding that most checkpoint proteins are modulated posttranslationally in response to DNA damage in wild-type S. pombe (Humphrey, 2000).
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Further analysis of the 52 IR non-CESR genes allows the characterization of a DNA damage-specific transcriptional response. The non-CESR transcriptional responses of S. pombe to DNA-damaging agents H2O2 and MMS (Chen et al., 2003) and IR (this study) were compared and 12 genes identified that were common to all three DNA-damaging agents (Figure 1B and Table 1). This list includes genes involved in the initiation of DNA replication (cdc18+ and cdt2+) and stress response (trr1+).
The 17 induced IR-response genes whose expression was not modulated by MMS or H2O2 may represent an IR-specific response. However, these gene numbers can only be approximate and will change depending on the chosen cutoff. Moreover, both lists may be narrowed as more DNA-damaging agents and types of cellular stress are studied using microarray technology. For example, the DNA repair genes rhp51+ and dinB+, both present in the IR-specific gene list, are also induced by UV (Park et al., 1998) and MMS, respectively (Kai and Wang, 2003).
Of the 45 genes whose expression was repressed in response to IR, only five genes are involved the CESR. However, more than one-half show periodic cell cycle expression in G1/S (Rustici and Bähler, personal communication): Expression of the histone H4 gene (hhf2+), found to be repressed after exposure to IR, is S-phase specific (Aves et al., 1985). Similarly cdc15+, which is transcribed at the M-G1 transition (Anderson et al., 2002), was found to be repressed after exposure to IR, as was eng1+, which is transcribed after septation (Martin-Cuadrado et al., 2003). It is likely that the repression is an indirect consequence of the IR-induced checkpoint-dependent G2-arrest in WT cells, which is expected to lead to reduced expression levels relative to unirradiated controls after DNA damage.
Confirmation and Extension of Array Data by Using QPCR
To verify the array data by a second method, and to extend the analysis in certain cases, QPCR was performed.
trr1+ encodes thioredoxin reductase, which responds to redox changes, and is induced by H2O2 in S. pombe (Toone et al., 1998; Chen et al., 2003). QPCR confirmed the microarray observation that trr1+ was induced severalfold after IR (see our Website).
dinB+ is the S. pombe homolog of the E. coli DinB gene whose product is involved in transletion synthesis repair (Kai and Wang, 2003). The microarray data indicate that dinB+ transcript levels were increased in asynchronous WT cells after exposure to IR and this up-regulation of dinB+ in WT cells was confirmed using QPCR (see our Website).
rev1+, like dinB+, is a member of the Y-family of polymerases that are involved in error-prone translesion synthesis repair (Ohmori et al., 2001). Microarray data was not obtained for rev1+ expression. QPCR, however, revealed a transcription profile for rev1+ in which an approximately threefold increase was observed in WT cells after exposure to IR (see our Website).
Ribonuclueotide reductase (RNR) is the rate-limiting enzyme for DNA synthesis and catalyzes the conversion of ribonucleoside diphosphates to their corresponding deoxyribonucleotides. The small subunit of RNR in S. pombe is encoded by the constitutively expressed suc22+ through a 1.5-kb transcript (suc22S). It has been demonstrated that a second larger transcript of 1.9 kb (suc22L) is induced after DNA damage and heat shock (Harris et al., 1996). The microarray analyses used in this study cannot distinguish between the smaller and larger suc22 transcript. By using QPCR, an 11-fold induction was observed after 160 min in asynchronous cells for the larger transcript (see our Website). The more abundant suc22S transcript remains relatively unchanged after IR as determined from the array, confirming that it is the larger suc22L transcript that becomes more abundant in response to ionizing radiation.
Regulation of IR-induced Gene Expression Responses
We wished to examine the potential roles of the DNA checkpoint and stress response pathways in regulating IR-induced gene expression responses in fission yeast. To this end, microarray analysis was performed to determine transcript levels from cultures of three DNA checkpoint deletion mutants (rad3, chk1, and cds1), an MAP kinase (sty1) deletion mutant, and a strain in which both pathways were disrupted (rad3 sty1) after exposure to IR. Using hierarchical clustering, the expression profiles of the 200 genes whose expression was modulated greater than twofold in asynchronous WT cells were viewed in all genetic backgrounds at both time points. The gene list was subdivided into induced non-CESR, induced CESR, and repressed IR genes, and cluster analysis was performed (Figure 2A).
To determine whether expression of a particular gene was dependent on any of the above-mentioned kinases, mean expression levels in both WT and mutant cells after exposure to IR were compared. Genes whose expression levels exhibited a 
2-fold change in expression in the mutant relative to WT after exposure to IR were classed as "dependent" on that gene (see MATERIALS AND METHODS). The analysis was again subdivided into induced IR genes (divided into non-CESR and CESR genes) and repressed IR genes, and a complete list of all checkpoint and MAP kinase-dependent IR genes is available from our Website. The percentage of genes whose expression was found to be dependent on each particular kinase after exposure to IR was determined (Figure 2B).
Regulation of non-CESR Genes
Analysis of gene expression in strains in which the DNA checkpoint or stress-response pathways were disrupted indicated that deletion of rad3+ or sty1+ had only a minor effect on the induction of non-CESR genes, where 12% of induced fast response non-CESR genes were found to be modulated in a Rad3-dependent manner, and only 6% to be modulated in a Sty1-dependent manner. Similar percentages of Rad3- and Sty1-dependent non-CESR genes (17 and 10, respectively) were observed for the slow response (Figure 2A, 1; and B). From the cluster analysis, it is clear that the effect on gene expression in the rad3 sty1 double deletion strain was greater than the one seen for the single mutants (Figure 2A). The percentage of IR-induced fast-response genes whose expression was significantly reduced in the double mutant (21%) was greater than the sum of single deletion strains (Figure 2B, top), suggesting that although the DNA checkpoint and stress-response pathways seem to function largely independently, they may both contribute to the expression of particular genes in response to IR.
Non-CESR genes whose expression in response to IR was determined to be Rad3-dependent included homologous recombination genes rhp51+ and rhp54+ and the translesion DNA synthesis repair gene dinB+ (Table 2). These same genes show down-regulation in rad3 sty1 double mutants but were unchanged in sty1 cells compared with WT cells. These genes were however not Cds1 nor Chk1 dependent. The expression of these genes in response to IR is thus likely to be specifically regulated by a subbranch of the DNA checkpoint pathway.
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A number of repressed IR genes showed significantly reduced levels of repression in response to IR in either rad3 or chk1 strains, especially at the later time point. This group again contains a number of cell cycle genes, including histone H4 gene (hhf2+) and eng1+, suggesting that these genes are inappropriately expressed as a result of disruption of the DNA checkpoint in these mutant strains (Figure 2A, 3; and B, bottom). Deletion of cds1+, in contrast to deletion of rad3+ and chk1+, had little effect on the number of genes induced or repressed in response to IR, consistent with checkpoint dependent G2 arrest in this mutant (Murakami and Okayama, 1995).
Regulation of CESR Genes
Deletion of the stress-activated MAP kinase gene sty1+ has a pronounced effect on the expression of IR-induced CESR genes, where the expression of 39% of the IR-induced fast response CESR genes was reduced by more than twofold in a sty1 strain compared with wild-type (Figure 2A, 2; and B, middle). The Sty1 dependency of these CESR genes is mostly restricted to the 60-min time point after exposure to IR, consistent with a role for Sty1 in the transient expression of these genes in response to IR (Figure 2A, 2; and B, middle). The expression of a number of Sty1-dependent IR-response genes identified here had previously been shown to be Sty1-MAP kinase dependent after exposure to environmental stress, including rds1+, cgs1+, tps1+, and gpd1+(Chen et al., 2003). These same genes show down-regulation in rad3 sty1 double mutants but were unchanged in rad3 cells compared with WT cells.
Surprisingly, deletion of rad3+ was also shown to significantly reduce expression of 8% of IR-induced fast response CESR genes (Figure 2A, 2; and B, middle), and many CESR genes showed a less than twofold reduction in expression in a rad3 strain. As observed with non-CESR genes, the percentage of CESR fast-response genes whose expression was modulated in response to IR in the double rad3 sty1 mutant (56%) was greater than the sum of the individual sty1 (39%) and rad3 (8%) mutants, again suggesting the DNA checkpoint and stress-response pathways functioned in concert to modulate particular genes in response to IR. Several uncharacterized induced CESR genes, including SPAC27D7.09c, SPAC4H3.08, and SPBC1271.08c show both Rad3 and Sty1 dependency (list available from our Website).
Surprisingly, some genes that are regulated in a Sty1-dependent manner under environmental stress conditions were found to be modulated in a Rad3-dependent manner in response to IR: Catalase, which is encoded by the cta1+ gene in S. pombe and converts H2O2 to H2O + O2, has been previously shown to be transcriptionally regulated by the Sty1-MAP kinase signal pathway after oxidative stress (Toone et al., 1998; Nguyen et al., 2000). Studies here indicated cta1+ to be rapidly and significantly (80-fold) induced in response to IR. Surprisingly, IR induction of cta1+ was Rad3 dependent, whereas cta1+ levels in sty1 cells were not significantly different from those of WT cells after IR.
The hsp16+ gene encodes a polypeptide of predicted molecular mass 16 kDa that belongs to the HSP20/alpha-crystallin family. Expression of hsp16+ in response to heat shock and nucleotide depletion is regulated via the Sty1-MAPK pathway (Taricani et al., 2001). After exposure to IR, hsp16+ transcript levels were found to be increased approximately fourfold in WT cells, and although our data suggest that the increase is Sty1-dependent, consistent with what has been described previously, transcriptional induction was also Rad3 dependent.
Deletion of chk1+ was also observed to significantly increase the IR-induced expression of several CESR genes at the later time point relative to WT cells (Figure 2A, 2; and B, middle). These genes include stress response genes cgs1+, cta1+, hsp16+, rds1+, slt1+, and tps1+ (Table 2). The IR-induced expression of these genes seems to be extended in chk1 cells compared with wild type. For example, cgs1+ is induced approximately threefold in both WT and chk1 cells after 60 min, and in WT cells the expression level has returned to unirradiated levels after 160 min. In contrast, expression of cgs1+ in chk1 cells is still elevated more than twofold after 160 min after exposure to IR. Unlike rad3 and cds1 mutants, chk1 cells can accumulate in S phase after IR through activation of the intra-S checkpoint. One possible explanation for these findings is that genes whose expression is elevated in chk1 at the later time point may be S-phase specific. Alternatively, these findings may point to an independent role for Chk1 in attenuating expression of these genes in response to IR.
IR-induced Gene Expression in G2 Synchronized Cells
To eliminate genes whose transcriptional "induction" after IR was an indirect result of cell cycle accumulation, we next examined the effects of irradiating cells that had been synchronized in G2. G2-synchronized cells were irradiated with 500 Gy of gamma IR and sampled at times 60, 90, 120, and 160 min and analyzed using microarrays (see MATERIALS AND METHODS). Genes (118 in total) were identified that exhibited a twofold or greater change in expression levels in G2-synchronized cells after IR. One hundred fourteen genes were induced greater than twofold, and only four genes were repressed greater than twofold in G2-synchronized cells after IR. However, there is relatively little overlap with those genes modulated in asynchronous cells after IR (Figure 3A). This was surprising because the majority (70%) of asynchronous S. pombe cells are within the G2 period of the cell cycle. Fewer CESR genes are modulated in response to IR in G2 cells compared with those of asynchronous cells (Figure 3B). A complete list of genes whose expression was modulated more than twofold after irradiation of G2-synchronized cells is available from our Website.
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A striking difference in the G2 and asynchronous expression profiles after IR exposure was observed for several Cdc10 targets, including cdc22+, cdt1+, cdt2+, and cdc18+ (Figure 3, C and D). These Cdc10 targets are transiently expressed during S phase (Lowndes et al., 1992; Kelly et al., 1993; Hofmann and Beach, 1994) and are under control of the DSC1 (DNA synthesis control) transcription factor (Lowndes et al., 1992; Hofmann and Beach, 1994; Baum et al., 1997). In asynchronous cells, IR induction levels of between approximately twofold (cdc22+) and approximately fourfold (cdt2+) after 160 min were observed (Figure 3C) and contrasted markedly with IR-induction levels of between approximately sixfold (cdt2+) and 30-fold (cdc22+) in irradiated G2-synchronized cells (Figure 3D). Further analysis of the expression levels of these genes by QPCR revealed their expression levels to be significantly reduced, by between fivefold (cdt1+ and cdt2+) and 16-fold (cdc22+ and cdc18+) in the unirradiated G2 synchronized cells compared with those of unirradiated asynchronous cells (our unpublished data). Such differences in expression profiles are likely for genes that are expressed at lower levels in G2 compared with other phases of the cell cycle and are likely to account for the differences in IR expression profiles recorded between asynchronous and G2-synchronized cultures. These data raise the possibility that, in a G2 cell subjected to IR-induced damage, the checkpoint pathway activates the DSC1/Cdc10-dependent transcriptional machinery, possibly to ensure an adequate supply of factors for the DNA synthesis associated with repair.
Further examination of genes induced in G2 cells after IR revealed several genes involved in cell cycle control not identified after the IR treatment of asynchronous cells, including smc3+ (2.5-fold induced), pol1+ (twofold induced), rep2+ (threefold induced), and cig2+ (fivefold induced). The cohesin and DNA repair gene rad21+ was also found to be induced threefold after radiation of G2-synchroninzed cultures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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200 genes was identified that exhibited a greater than twofold change in expression levels in response to IR in asynchronous cells. Independent analysis of specific genes by using QPCR was used to confirm a subset of these results. Genes whose expression was found to be induced in response to 500 Gy of gamma IR fall into a variety of predicted functional categories, including DNA damage response, cell cycle control, signal transducers, stress-response genes, and genes involved in carbohydrate, lipid, and protein metabolism. The largest category was a subset of >100 CESR genes, indicating that exposure to ionizing radiation induces a general stress response in S. pombe.
Comparative analyses of the 50 induced IR genes not present in the CESR with non-CESR genes whose expression was modulated in response to H2O2 and MMS revealed potentially both IR-specific and DNA damage response gene expression profiles. Although the majority of these genes have yet to be characterized in fission yeast, comparative sequence analysis indicates that these IR-specific and DNA damage-specific genes are likely to function in a variety of cellular processes. An overview of IR-specific and DNA damage-specific expression profiles revealed that several genes increase induction levels at the later time point. Such transcriptional up-regulation could function to confer an adaptive response to IR and DNA damage.
IR-Response Pathways
Our data indicate an important role for the Sty1-MAP kinase in coordinating gene-expression responses to IR. A primary role would seem to be the regulation of CESR responses, where 39% of CESR genes induced at an early time point were found to be modulated in response to IR in a Sty1-dependent manner. However, it seems that Sty1 has less of a role in the transcriptional response to IR than with other stresses, where 70-90% of CESR genes were found to be Sty1-dependent (Chen et al., 2003). As expected, genes were identified in this study whose expression was found to be Sty1-dependent specifically in response to IR, and not other stresses, as determined by Chen et al. (2003), and are likely to be involved in responding to IR-specific lesions and/or stress.
Our data further indicate a role for the DNA integrity checkpoint pathway in coordinating gene expression responses to IR. Approximately 9% of all fast response-induced genes and 8% of all slow response-induced genes were identified whose expression levels were significantly modulated in a Rad3-dependent manner in response to IR. Such genes included DNA repair rhp51+, rhp54+, and dinB+. This cluster of genes was regulated by Rad3, independently of Chk1 and Cds1 checkpoint kinases in response to IR, suggesting that these genes are controlled by Rad3 through distinct downstream effectors. The expression of many genes was also found to be elevated in rad3 and chk1 strains, suggesting a role for these genes in transcriptional repression of these transcripts in response to IR. However, more than one-half of these genes are transcriptionally regulated through the cell cycle, suggesting that the elevated levels of many of these transcripts may have resulted indirectly through inappropriate cell cycle advance in these checkpoint mutants after exposure to IR.
The Rad3-checkpoint and Sty1-stress kinase pathways seem to function largely independently to modulate transcript levels in response to IR. However, a number of unexpected regulatory interactions were identified between the Sty1-MAP kinase pathway and the Rad3-dependent checkpoint pathway. After exposure to IR, a role for the Rad3-dependent checkpoint pathway was identified in coordinating expression of a number of CESR or specific stress-response genes that had previously been demonstrated to be regulated by the Sty1-MAPK pathway. The most striking example was that of cta1+, which is transcriptionally induced in response to oxidative stress in a Sty1-dependent manner (Wilkinson et al., 1996). Surprisingly, we found cta1+ transcript levels to be significantly increased after exposure to IR in a Rad3-dependent manner, and this transcriptional response to IR was largely unaffected in sty1 cells. However, Rad3 may not be the only regulator as cta1+ is still induced 35-fold in rad3 cells. These results indicate that cta1+ expression levels can be modulated by either Rad3-checkpoint or Sty1-MAPK pathways in response to different stresses.
Although important roles for the Rad3 checkpoint and Sty1-MAP kinases have been identified in modulating transcript levels in response to IR, whether these pathways function to modulate transcription or mRNA turnover is currently unknown. Moreover, the majority of CESR and non-CESR genes responding to IR did so independently of these pathways (Figure 4). Thus, understanding how the expression of these genes is regulated in response to IR remains an important goal.
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Eukaryotic IR-Response Genes
An aim of this study was to define a eukaryotic IR-response signature through comparative analysis of the IR responses of fission and budding yeasts. However, the number of genes whose expression levels are altered in response to IR in budding is considerably greater than the numbers recorded in this study for fission yeast (Gasch et al., 2001). Thus, the functional significance of the identification of homologous genes in both yeasts whose expression is modulated by IR is unclear. Indeed, both numbers of genes and gene expression profiles may alter in response to exposure to different radiation doses in fission yeast. However, a comparison of the transcriptional responses to a number of DNA-damaging agents has identified a small group of nine genes comprising the DNA damage response signature in S. cerevisiae (Gasch et al., 2001). Functional homologs of RAD51, RAD54, RNR2 and RNR4, namely, rhp51+, rhp54+, and suc22+ respectively, were found to be transcriptionally induced in response to IR in fission yeast. Moreover, the IR-induced expression of these genes is regulated by the highly conserved DNA damage checkpoint pathway in both yeasts, suggesting that such conserved responses to IR represent fundamental survival pathways common to all eukaryotes. These studies further identified a role for Rad3 in modulating the expression of CESR genes in response to IR in S. pombe. In S. cerevisiae, the Mec1 pathway was similarly found to coordinate the induction of a large number of environmental stress response genes after exposure to IR (De Sanctis et al., 2001; Gasch et al., 2001), suggesting a conserved role for the DNA integrity checkpoint pathway in coordinating environmental stress response genes after exposure to IR. These CESR genes function to mount a protective response to a variety of environmental stresses, including oxidative stress. Both Rad3 and Mec1 are structurally related to the checkpoint kinase ATM, which when absent results in the disease Ataxia telangiectasia (A-T). A-T patients suffer from significant neurodegeneration associated with cerebellar ataxia in addition to immunodeficiency, premature ageing, acute radiosensitivity, and cancer predisposition (for review, see Shiloh, 2001). It has been suggested that many of the A-T features might result from elevated levels of oxidative stress in A-T cells or tissues (for review, see Barzilai et al., 2002). Our data suggest that one possible mechanism by which the ATM checkpoint kinase may function to coordinate the cellular response to oxidative stress in mammalian cells is through modulating the transcription of CESR genes. Understanding the underlying mechanisms by which the DNA checkpoints, stress response and other pathways function to maintain homoeostasis in response to ionizing radiation and other stresses will therefore be of significant biomedical interest.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Online version of this article contains supplemental tables. Online version is available at www.molbiolcell.org. 
Corresponding author. E-mail address: t.humphrey{at}har.mrc.ac.uk.
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REFERENCES |
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