BACKGROUND
cell cycle progression is fundamental for all proliferation.
transition from one cell-cycle phase to the next is often brought about by a changed transcriptional pattern: repression of specific genes and/or expression of new genes promote progression from one phase into the next.
the regulation of transcriptional patterns during the cell cycle is conserved from yeast to humans  <cit> , although the actual genes and transcriptional factors involved are not necessarily conserved.
in addition to transcriptional regulation the key components of the cell cycle are also frequently regulated at the translational and the post-translational levels.
regulation of transcription is important in the cellular response to environmental stress.
exposure to radiation, toxic chemicals, fluctuations in temperature, osmolarity or nutrient availability profoundly affect cell growth and the genomic expression programme is adjusted to adapt to the different challenges.
microarray technology has been used to characterise global gene expression profiles for several different stress conditions in the model organism schizosaccharomyces pombe  <cit> .
a common set of genes responding to many different forms of stress has been identified in both fission and budding yeast.
these genes are known as core environmental stress response genes, cesr  <cit>  in s. pombe and environmental stress response genes  <cit>  or the common environmental response genes  <cit>  in saccharomyces cerevisiae.
in addition to this common pattern there are genes that are specifically expressed in response to each individual stress treatment.
in general, stress represents a threat to genome stability.
depending on the type of damage inflicted and the position in the cell cycle different strategies are used for handling a stress situation.
checkpoint mechanisms delay the cell cycle to allow the cells to repair dna damage and to ensure stable inheritance of the genome.
several checkpoint pathways target the transcription machinery to ensure the appropriate expression levels of genes involved in the response to the insult.
in s. pombe there are separate checkpoints that inhibit mitosis when the dna is damaged  or when s phase has not been completed  and that inhibits dna replication when the dna is damaged   <cit> .
these checkpoints have at least two features in common: they all operate through the five so-called checkpoint rad proteins and they all bring about the cell-cycle delay via inhibition of the cdc <dig> protein kinase, the key regulator of cell-cycle progression  <cit> .
in addition, they also include rad3-dependent transcriptional responses  <cit> .
in g <dig> phase the cell decides whether to commit to a new round of the cell cycle or to enter stationary phase or meiosis.
the g1/s dna damage checkpoint regulates the transition into s phase  <cit> , and insensitivity to growth-inhibitory signals, especially in the g <dig> phase, is one of the hallmarks of cancer  <cit> .
we have recently discovered and partly characterised a novel checkpoint mechanism in s. pombe which delays s-phase entry after uvc irradiation in a gcn2-dependent manner  <cit> .
in the present work we have investigated whether the response to uvc in g <dig> phase involves a specific transcriptional response and searched for possible genes to be involved in the g1/s checkpoint.
furthermore, we compare the genes differentially expressed after uvc irradiation with the transcriptional response to oxidative stress  and ionising radiation .
RESULTS
we have performed genome-wide expression analyses of uvc-irradiated g1-phase fission yeast cells to further characterise the g1/s checkpoint  <cit> .
we have investigated what kind of transcriptional response uvc irradiation imposes on the cells and searched for potential candidate genes involved in the regulatory process of the g1/s checkpoint.
the cells were synchronised by employing a temperature-sensitive version of cdc <dig>  a transcription factor required for progression from g <dig> into s phase.
this method gives good synchrony and allows convenient detection of the g1/s checkpoint.
during a four-hour shift to 36°c the cells were arrested in g <dig> phase and could be released synchronously into the cell cycle or kept in g <dig> phase.
total rna was isolated from both uvc-irradiated cells and unirradiated control cells.
the rna was subjected to total genomic microarray analysis.
the two experiments
two distinct experiments were performed : first, g1-phase cells were irradiated and kept at the restrictive temperature .
thirty minutes after the time of irradiation the uvc-irradiated  and unirradiated control  cells were collected.
these cells were still in early g <dig> phase due to the continued inactivation of cdc <dig>  second, g1-cells were released into the cell cycle after synchronisation by reducing the temperature to 25°c, thus reactivating cdc <dig>  allowing the cells to continue in the cell cycle.
cell samples were collected at  <dig>   <dig> and  <dig> min after irradiation .
samples of irradiated and unirradiated cells were analyzed on microarrays and compared to a common reference pool .
flow cytometry  demonstrated that the control cells had entered s phase by  <dig> minutes after release, whereas the uvc-irradiated cells delayed in g <dig> phase and moved from g <dig> to s phase around  <dig> minutes after release, in agreement with previous data  <cit> .
we have previously shown the existence of the g1/s checkpoint using several synchronisation methods  <cit> , but none of the other methods provided good enough synchrony to perform similar analyses on global transcription.
we have looked at our data for all known cdc <dig> targets in the  <dig> arrays from the time-course experiment and observed no trend showing that uvc delays the occurrence of these transcripts.
this is consistent with our rna blots of the two selected transcripts cdc <dig> and cig <dig>  which are not delayed by uvc in block-and-release experiments .
the restrictive-temperature experiment
in this experiment we searched for genes that altered their expression more than twofold as a consequence of uvc irradiation in g <dig> phase.
of almost  <dig> genes represented on the microarrays as few as  <dig> genes were induced twofold or more and  <dig> of these were non-cesr genes .
most of the  <dig> genes were induced two- to threefold, and only three genes were induced more than fivefold.
most of the induced non-cesr genes are likely to be uvc-specific and not cell-cycle related, since the cells did not move into s phase during the time of the experiment.
no non-cesr genes were found to be induced by both uvc and h2o <dig>  <cit> , but two genes, spcc <dig> c and spbc16a <dig> c were induced after both uvc  and ir treatment  <cit>  .
we categorised the  <dig> non-cesr genes into eight different groups according to the functions of their products .
these genes are involved in a variety of functions such as signalling and stress response, ribosome biogenesis and translation, dna/rna binding, and as many as  <dig> of the genes are involved in transport mechanisms.
there were no obvious candidates for genes involved in the g1/s checkpoint amongst the  <dig> upregulated genes.
surprisingly, only one of the induced genes, rhp4b, encoding a nucleotide excision repair factor, is involved in dna repair of uvc-induced lesions, strongly suggesting that the capacity to perform dna repair is not regulated at the transcriptional level.
another possibly interesting induced gene is pyp <dig>  a protein-tyrosine phosphatase that acts on sty <dig>  the mapk  that regulates various stress responses in s. pombe  <cit> .
we have shown that the g1/s checkpoint does not require sty <dig>  <cit> , but it is possible that pyp <dig> has additional targets in the cell besides sty <dig>
gene annotations are from genedb http://www.genedb.org/genedb/pombe/index.jsp.
we also identified  <dig> genes that were repressed more than twofold and almost all of them  were non-cesr genes .
these  <dig> genes were categorised according to the function of their products , and, like for the proteins encoded by the induced genes, were known to be involved in a wide variety of activities including transport, metabolism, mating and mitochondrial activities.
amongst the repressed genes no obvious candidate genes for checkpoint regulators were found.
the time-course experiment
in the unirradiated control cells only  <dig> genes from the whole dataset were upregulated  and  <dig> were downregulated in either the c <dig> and/or the c <dig> sample relative to the situation in cells at the start of the experiment  .
in comparison, altogether  <dig> genes were upregulated and  <dig> downregulated in either the uv <dig>  uv <dig> or the uv <dig> sample relative to c <dig> during the time-course.
like in the restrictive-temperature experiment, most of the induced genes were only upregulated two- or threefold.
only four genes in either c or uv were induced more than fivefold in both repeats of the experiment and this was only found for the c <dig> sample.
it is striking that only about 1% of around  <dig> genes has a changed expression after cell cycle progression and uvc irradiation.
furthermore, we searched for genes differentially expressed between 0/ <dig>  30/ <dig> and 0/ <dig> minutes in both the c and uv samples.
to this end we used moderated t-statistics with a p-value cut-off of  <dig>  .
in these experiments each gene could potentially be assigned  <dig> expression values .
about 35% of the values were missing in the entire dataset.
the reasons for the missing values will be discussed below.
we decided to remove the data for a gene if four or more of the  <dig> possible data values were missing.
this action reduced the dataset from  <dig> to  <dig> genes, and data for the resulting 54% of the genes was considered more reliable and was used for further analysis.
cell-cycle-regulated genes
the rna samples from irradiated cells reflect gene expression changes that occur for two separate reasons: first, the cells are progressing through the cell cycle and will necessarily change gene expression  <cit>  and, second, the cells have been exposed to uvc light and will display stress-related and uvc-specific changes.
in contrast, differences between the samples in the restrictive-temperature experiment  should reflect only the stress-related and uvc-specific changes.
in the present analysis, we attempt to identify the genes specifically affected by uvc exposure.
for this reason we identified the genes in unirradiated control cells whose expression varied after release into the cell cycle and these genes were classified as not specifically responding to uvc.
in the unirradiated cells altogether  <dig> genes were found to be differentially expressed between g <dig> phase  and s/g <dig> phase  .
only  <dig> genes  were differentially expressed at  <dig> minutes after release into the cell cycle .
in comparing c <dig>  with c <dig>   <dig> genes  were found to be differentially expressed.
finally,  <dig> genes  had a changed transcriptional level when comparing c <dig>  to c <dig> .
this means that, not surprisingly, most of the induced genes were found in cells entering s phase .
well-known g1-specific genes, like cdc <dig>  cig <dig>  cdc <dig> and cdt <dig>  were induced normally during g <dig> phase  in our study , serving as convenient controls.
uvc-regulated genes
using the p-value cut-off of  <dig> , we identified as many as  <dig> genes in total that were differentially expressed in the uvc-irradiated cells after release into the cell cycle .
of all the  <dig> genes only  <dig> were different between uv <dig> and uv <dig>  so little was happening at the transcriptional level during the first  <dig> minutes after irradiation.
no genes were determined to be differentially expressed when comparing uv <dig>  and uv <dig> .
however, as many as  <dig> genes were defined to be different between uv <dig>  and uv <dig> .
the apparent discrepancy between the numbers of regulated genes between uv0/uv <dig> and uv30/uv <dig> on the one hand and between uv0/uv <dig> on the other can be explained as follows: expression of a number of genes is different between uv <dig> and uv <dig>  but only  <dig> genes were significantly different .
similarly, a number of genes changed their expression between uv <dig> and uv <dig>  but no difference passed the threshold we had set.
however, when comparing uv <dig> and uv <dig>  a number of genes  had altered their expression sufficiently during the total interval.
it follows that the level of change for all of these genes was low.
some of the  <dig> genes that were up- or downregulated after uvc in this experiment are regulated as a consequence of the cell-cycle progression and not of the uvc irradiation.
to identify the uvc-specific transcripts, we excluded the  <dig> cell-cycle-regulated genes identified above , resulting in  <dig> uvc-regulated genes , of which  <dig> are non-cesr genes.
of these  <dig> genes as many as  <dig> genes are dedicated to the translational machinery.
nine of the  <dig> genes were specifically upregulated in uv <dig> , which is at a time when cells are arrested in g <dig> phase by the g1/s checkpoint.
these  <dig> genes were compared to the set of non-cesr genes induced by h2o <dig> and ir  <cit> .
no genes were found to be induced by both uvc and h2o <dig> treatment.
only two genes, gst <dig> and fip <dig>  were induced after both uvc  and ir treatment.
none of the  <dig> uvc-specific genes are likely to be regulators of the g1/s checkpoint, judging from their annotations.
spac23h <dig> c
gene annotations are from genedb http://www.genedb.org/genedb/pombe/index.jsp.
profile analyses
to further analyse the kinetics of gene expression in the time-course experiment we applied the software package masigpro  <cit>  on the filtered data, which allowed a multiple comparison of all three time points.
there were  <dig> genes that significantly changed their expression levels during the time-course , and the expression of almost all  was changed in both control and uvc-irradiated cells.
fifteen genes were identified as specific for the irradiated cells.
there was a good correlation between the genes identified in this profile analysis and the differentially expressed genes determined in the statistical analysis above .
the expression levels of the  <dig> genes with expression that varied during the time-course were subjected to a clustering analysis, forming a two-dimensional map .
genes being up- or downregulated at the particular time point are represented by different colours, green indicating induction and blue repression.
the map shows that analysis of the two biological replicates revealed that they were indeed similar and assembled into the closest branches of the cluster.
this confirms a good reproducibility of the experiments.
furthermore, the profile analysis is also consistent with the conclusions of the pairwise statistical analyses , showing that the expression patterns in uv <dig> and uv <dig> were similar.
the map shows little difference between control and irradiated samples at all time points, and the least closely related branches of the cluster refer to changes occurring during progression in the cell cycle.
thus, this analysis corroborates our conclusions from the above pairwise comparisons, that the cell cycle progression affects transcription profiles more than the uvc treatment and there is only a weak transcriptional response to uvc irradiation.
pathway analyses
gene ontology  enrichment analysis was performed on the  <dig> uvc-induced genes in the restrictive-temperature experiment  using the david software after id-conversion.
ten unique genes  were members of enriched go groups, suggesting upregulation of genes involved in ion transport or ion homeostasis in the restrictive-temperature experiment .
analysis of over-represented go annotations amongst the  <dig> upregulated genes in the time-course experiment  showed that  <dig> unique genes  are members of the enriched go groups.
the nature of these groups indicates regulation of genes that affect protein biosynthesis or structural components of ribosomes .
further network analysis shows a concerted response involving direct protein-protein interactions between  <dig> of the  <dig> induced gene products.
thus, the analyses suggest a coordinated induction of rrna biogenesis, ribosome assembly and components of the 60s and 40s ribosomal subunits in the time-course experiment .
* count denotes the number of genes in our dataset in each cluster.
percent coverage of these genes relative to the numbers on the gene ontology clusters were calculated.
confirmation of the microarray data
rna blotting and hybridisation was used to confirm our microarray results for four selected transcripts.
the induction of spac2e1p <dig> c, fip <dig> and gst <dig> found in the time-course experiment was verified .
fip <dig>  an iron permease, and gst <dig>  a glutathione s-transferase, have also been shown to be induced after ir  <cit> .
on the other hand, rhp4b had no values in any of the  <dig> arrays in our time-course experiments and was also not detected after rna blotting and hybridisation.
however, in the restrictive-temperature experiment, rhp4b transcription was found to be induced by uvc irradiation and this finding was also confirmed by rna blotting .
therefore, rna-blotting experiments with all four selected genes verified the results from the microarray experiments.
uvc does not induce the unfolded protein response
the unfolded protein response  is activated by the accumulation of unfolded proteins in the endoplasmic reticulum .
the upr triggers a transcriptional response which serves to induce the production of er components and to increase the degradation capacity to dispose of the unfolded proteins  <cit> .
it is possible that uvc irradiation might stress the er and thus activate the upr.
furthermore, gcn <dig> has been shown to be required for the induction of a majority of upr target genes during er stress in s. cerevisiae  <cit> .
we therefore used the present data from both the restrictive-temperature and time-course experiments to investigate whether upr genes are induced by uvc in fission yeast.
we identified upr-genes in fission yeast as the homologues of the upr-induced budding yeast genes  <cit> .
there was little, if any, induction of the upr-genes identified by this method .
the lack of transcriptional response of these genes after uvc strongly argues that the g1/s checkpoint is not a manifestation of the upr.
discussion
here we have investigated gene expression in s. pombe cells traversing the g1/s border in a synchronous manner, both uvc-irradiated and unirradiated cells.
the transcriptional response after uvc-irradiation in g <dig> phase was surprisingly weak.
the vast majority of genes did not change their transcription pattern appreciably and the few that did increased or decreased their expression levels only two- to three-fold.
comparison of the data from the two experiments
<dig> genes were identified as specifically uvc-regulated in the time-course experiment and  <dig> in the restrictive-temperature experiment.
surprisingly, as few as  <dig> genes  were found to be common for the two datasets, and these  <dig> genes were not differently expressed in uv <dig> and uv <dig> in the time-course experiment , which is the time period when the irradiated cells were arrested in the g1/s checkpoint.
the uvc irradiation elicits a quite weak transcriptional response on the cells both when considering the number of genes affected and the level of the response for the affected genes.
thus, our assay must be considered to be rather sensitive and even a small change in the experimental setup might bring the marginal levels of gene expression over or under our threshold, which could be one reason for the poor overlap between the two experiments.
this further underlines our conclusion that uvc has only a marginal effect on gene transcription when given in g <dig> phase.
it is possible that the biological differences between the cells in our two types of experiments is dominating and that the dissimilar sets of regulated genes reflect a biological difference rather than an artefact of our data analysis.
in the restrictive-temperature experiment cells were arrested in g <dig> phase by the inactivation of cdc <dig>  which means that cell-cycle dependent responses were inhibited.
thus, this experiment reveals transcriptional regulation exclusively due to uvc irradiation, at a specific stage in the cell cycle.
in contrast, in the time-course experiment cells were allowed to progress into the cell cycle.
even though we have subtracted the cell-cycle-regulated genes when identifying the uvc-regulated genes, the cell cycle stage in which the cellular response is analysed was still different from that of cells held in the cdc <dig> block.
consistent with this line of reasoning, we have shown that little happened at the transcriptional level during the first  <dig> minutes after irradiation in the time-course experiment , and most of the  <dig> genes changed their transcription enough to satisfy the criteria of our analysis only by a later time point, as the cells entered s phase.
therefore these genes have not been identified as regulated in the restrictive-temperature experiment.
missing values
in the data for the time-course experiment values were missing for many data points.
this can be attributed to at least two reasons: lack of expression of the relevant open reading frames and technical problems with the microarrays.
at any one time there are genes that are poorly expressed, so it was expected that there would be some missing values in our datasets.
low expression is the most likely reason for the absent microarray signal for the rhp4b transcript, which could also not be detected after rna blotting.
however, lack of expression cannot explain all the missing values.
the  <dig> microarrays used in the time-course experiment comprised from 18% to 55% missing values , arguing that there were technical problems with at least some of the microarrays.
such a high level and difference in the number of missing values was not found for the two repeats of the restrictive-temperature experiment, which both had about 6% missing values, further suggesting technical problems with the arrays used in the time-course experiment.
it should be noted that the microarrays used for this experiment came from a different batch/production than those used for the restrictive-temperature experiment.
this problem did reduce the quality of the data for some of the genes in the time-course experiment, and we decided to remove all the data pertaining to genes where too many data points were missing .
however, the stringency of our analysis allows us to draw conclusions in spite of the missing data.
furthermore, any technical problem would have affected a random set of genes, and a strong transcriptional response would have been obvious even from the time-course experiment.
comparisons with other organisms
this is the first report about the global transcriptional response after uvc irradiation in fission s. pombe and there are only a few reports about similar experiments in other organisms.
the available data indicate that the weak transcriptional response to uvc stress we observed in fission yeast might be a conserved feature.
for example, in human cells exposed to uvc only  <dig> of more than  <dig> genes investigated changed their expression more than 2-fold  <cit> .
an early microarray-report using escherichia coli cells identified several differentially expressed genes after uvc-irradiation, but the response was generally not more than two-fold  <cit> .
many checkpoints, both in fission yeast and in other organisms, involve transcriptional regulation.
for instance, in multicellular organisms one of the best characterised checkpoint targets is p <dig>  a transcription factor which is mutated in over half of human cancers.
p <dig> stimulates transcription of cell-cycle inhibitors such as p <dig>  <cit>  and is essential for a persistent g <dig> arrest.
another tumour suppressor, prb, targets the e2f-driven transcriptional programme in the g1/s checkpoint  <cit> .
in budding yeast activation of the g1/s checkpoint also impinges on transcriptional regulation in that the transcription factor swi <dig> is phosphorylated and thereby inactivated by the checkpoint protein rad <dig>  leading to delayed transcription of cln <dig> and cln <dig> and a delayed entry into s phase  <cit> .
recent data show that in fission yeast the transcriptional response is important in the intra-s checkpoint after exposure to hydroxyurea  <cit> .
comparisons with other stress agents
we have observed no obvious change of the transcriptional programme that could be responsible for the g1/s checkpoint and also no strong induction of genes involved in dna repair or checkpoint function.
it is interesting to note that other dna-damaging agents, such as h2o <dig> and ir, also do not lead totranscriptional induction of many dna repair- or checkpoint-related genes  <cit> .
therefore, it is likely that there is no need to specifically induce transcription of dna repair-related genes, suggesting that the dna repair capacity is high already before the uvc exposure, as the case is for budding yeast  <cit> .
we have compared our data from synchronised cells to data based on h2o2- and ir-treated asynchronous cells  <cit> , and there is little overlap in the spectrum of non-cesr genes differentially expressed after exposure to the three agents.
as discussed above, it seems that the cell-cycle position is important for the transcriptional profile obtained when exposing the cells to a stress treatment.
however, when comparing the differentially expressed genes in asynchronously growing cells exposed to uvc  to cells exposed to h2o <dig>  <cit>  and ir  <cit>  there is little overlap.
pathway analyses
dna-damaging agents, heat and other forms of stress give overlapping responses, described as the cesr, that involves 14% of the genome in s. cerevisiae  <cit> .
few uvc-specific expression changes have been reported  <cit>  and the fold-changes observed are low  <cit> .
it is therefore possible that transcriptional responses to heat stress combined with stringent statistical analyses and exclusion of the cesr-genes mask uvc-relevant gene expression changes in the restrictive-temperature experiment.
similarly, the absence of significant gene-expression changes after  <dig> minutes recovery in the time-course experiment indicates that uvc-relevant transcriptional responses are masked by the small fold-changes and extensive overlap between the uvc-specific response and the cesr.
this is supported by appearance of differences on the transcriptional level first after  <dig> minutes.
our network analyses of gene-expression changes indicate that pathways involved in recovery after dna damage are induced after  <dig> minutes .
the processes involved probably reflect the importance of the translation machinery in recovery after the insult.
these recovery processes are likely to be important for survival after uvc treatment, as was shown in mms-treated s. cerevisiae  <cit> , although they may not be components of the g1/s checkpoint per se.
regulation of the g1/s checkpoint
in the g1/s checkpoint the gcn <dig> kinase is activated to phosphorylate the eukaryotic initiation factor 2α, eif2α, thereby inhibiting translation  <cit> .
there is a good correlation between eif2α phosphorylation and checkpoint activation  <cit> , but it is still unclear whether and, if so, how the checkpoint is dependent upon this phosphorylation and on the ensuing downregulation of translation.
gcn <dig> is best known for its role in the starvation response, where eif2α phosphorylation leads to induction of the transcription factor gcn <dig> both in budding yeast and higher eukaryotes.
fission yeast does not have a gcn <dig> homologue, and it remains to be seen whether the gcn2-dependent g1/s checkpoint in budding yeast  <cit>  involves activation of gcn <dig>  one might expect a transcriptional response after gcn <dig> activation also in s. pombe, but the finding that no such transcriptional response could be identified argues that in fission yeast the g1/s checkpoint does not operate like it does in budding yeast or higher eukaryotes and is not dependent upon the strong transcriptional induction of one or a set of genes.
we have shown that the g1/s checkpoint in s. pombe is associated with a strong downregulation of translation and it might be relevant that among the few genes that are affected at the transcription level, 15% affect the translation machinery.
importantly, amongst the transcriptionally regulated genes none were detected that are likely to be directly involved in the g1/s checkpoint according to their annotations.
induction or inhibition of transcription is a fairly slow response and regulation of the cell cycle in s. pombe should preferably occur rapidly in order to be efficient and meaningful.
therefore, it intuitively makes sense that the present data suggest that the g1/s checkpoint is regulated at the level of protein modification and/or translation, which is rapid, rather than a slower regulation of gene expression.
CONCLUSIONS
the transcriptional response to uvc irradiation of fission yeast cells in g <dig> phase was shown to be weak.
we conclude that the novel g1/s checkpoint is not regulated by changing the transcriptional programme.
this is supported by an examination of the few genes that are induced or repressed by uvc, and none of them appears to have any relationship to cell-cycle regulation.

 cell cycle progression is fundamental for all proliferation. transition from one cell-cycle phase to the next is often brought about by a changed transcriptional pattern: repression of specific genes and/or expression of new genes promote progression from one phase into the next. the regulation of transcriptional patterns during the cell cycle is conserved from yeast to humans, although the actual genes and transcriptional factors involved are not necessarily conserved. in addition to transcriptional regulation the key components of the cell cycle are also frequently regulated at the translational and the post-translational levels. regulation of transcription is important in the cellular response to environmental stress. exposure to radiation, toxic chemicals, fluctuations in temperature, osmolarity or nutrient availability profoundly affect cell growth and the genomic expression programme is adjusted to adapt to the different challenges. microarray technology has been used to characterise global gene expression profiles for several different stress conditions in the model organism schizosaccharomyces pombe. a common set of genes responding to many different forms of stress has been identified in both fission and budding yeast. these genes are known as core environmental stress response genes, cesr in s. pombe and environmental stress response genes or the common environmental response genes in saccharomyces cerevisiae. in addition to this common pattern there are genes that are specifically expressed in response to each individual stress treatment. in general, stress represents a threat to genome stability. depending on the type of damage inflicted and the position in the cell cycle different strategies are used for handling a stress situation. checkpoint mechanisms delay the cell cycle to allow the cells to repair dna damage and to ensure stable inheritance of the genome. several checkpoint pathways target the transcription machinery to ensure the appropriate expression levels of genes involved in the response to the insult. in s. pombe there are separate checkpoints that inhibit mitosis when the dna is damaged or when s phase has not been completed and that inhibits dna replication when the dna is damaged. these checkpoints have at least two features in common: they all operate through the five so-called checkpoint rad proteins and they all bring about the cell-cycle delay via inhibition of the cdc protein kinase, the key regulator of cell-cycle progression. in addition, they also include rad3-dependent transcriptional responses. in g phase the cell decides whether to commit to a new round of the cell cycle or to enter stationary phase or meiosis. the g1/s dna damage checkpoint regulates the transition into s phase, and insensitivity to growth-inhibitory signals, especially in the g phase, is one of the hallmarks of cancer. we have recently discovered and partly characterised a novel checkpoint mechanism in s. pombe which delays s-phase entry after uvc irradiation in a gcn2-dependent manner. in the present work we have investigated whether the response to uvc in g phase involves a specific transcriptional response and searched for possible genes to be involved in the g1/s checkpoint. furthermore, we compare the genes differentially expressed after uvc irradiation with the transcriptional response to oxidative stress and ionising radiation.  we have performed genome-wide expression analyses of uvc-irradiated g1-phase fission yeast cells to further characterise the g1/s checkpoint. we have investigated what kind of transcriptional response uvc irradiation imposes on the cells and searched for potential candidate genes involved in the regulatory process of the g1/s checkpoint. the cells were synchronised by employing a temperature-sensitive version of cdc a transcription factor required for progression from g into s phase. this method gives good synchrony and allows convenient detection of the g1/s checkpoint. during a four-hour shift to 36°c the cells were arrested in g phase and could be released synchronously into the cell cycle or kept in g phase. total rna was isolated from both uvc-irradiated cells and unirradiated control cells. the rna was subjected to total genomic microarray analysis. the two experiments two distinct experiments were performed: first, g1-phase cells were irradiated and kept at the restrictive temperature. thirty minutes after the time of irradiation the uvc-irradiated and unirradiated control cells were collected. these cells were still in early g phase due to the continued inactivation of cdc second, g1-cells were released into the cell cycle after synchronisation by reducing the temperature to 25°c, thus reactivating cdc allowing the cells to continue in the cell cycle. cell samples were collected at and min after irradiation. samples of irradiated and unirradiated cells were analyzed on microarrays and compared to a common reference pool. flow cytometry demonstrated that the control cells had entered s phase by minutes after release, whereas the uvc-irradiated cells delayed in g phase and moved from g to s phase around minutes after release, in agreement with previous data. we have previously shown the existence of the g1/s checkpoint using several synchronisation methods, but none of the other methods provided good enough synchrony to perform similar analyses on global transcription. we have looked at our data for all known cdc targets in the arrays from the time-course experiment and observed no trend showing that uvc delays the occurrence of these transcripts. this is consistent with our rna blots of the two selected transcripts cdc and cig which are not delayed by uvc in block-and-release experiments. the restrictive-temperature experiment in this experiment we searched for genes that altered their expression more than twofold as a consequence of uvc irradiation in g phase. of almost genes represented on the microarrays as few as genes were induced twofold or more and of these were non-cesr genes. most of the genes were induced two- to threefold, and only three genes were induced more than fivefold. most of the induced non-cesr genes are likely to be uvc-specific and not cell-cycle related, since the cells did not move into s phase during the time of the experiment. no non-cesr genes were found to be induced by both uvc and h2o, but two genes, spcc c and spbc16a c were induced after both uvc and ir treatment. we categorised the non-cesr genes into eight different groups according to the functions of their products. these genes are involved in a variety of functions such as signalling and stress response, ribosome biogenesis and translation, dna/rna binding, and as many as of the genes are involved in transport mechanisms. there were no obvious candidates for genes involved in the g1/s checkpoint amongst the upregulated genes. surprisingly, only one of the induced genes, rhp4b, encoding a nucleotide excision repair factor, is involved in dna repair of uvc-induced lesions, strongly suggesting that the capacity to perform dna repair is not regulated at the transcriptional level. another possibly interesting induced gene is pyp a protein-tyrosine phosphatase that acts on sty the mapk that regulates various stress responses in s. pombe. we have shown that the g1/s checkpoint does not require sty, but it is possible that pyp has additional targets in the cell besides sty gene annotations are from genedb http://www.genedb.org/genedb/pombe/index.jsp. we also identified genes that were repressed more than twofold and almost all of them were non-cesr genes. these genes were categorised according to the function of their products, and, like for the proteins encoded by the induced genes, were known to be involved in a wide variety of activities including transport, metabolism, mating and mitochondrial activities. amongst the repressed genes no obvious candidate genes for checkpoint regulators were found. the time-course experiment in the unirradiated control cells only genes from the whole dataset were upregulated and were downregulated in either the c and/or the c sample relative to the situation in cells at the start of the experiment. in comparison, altogether genes were upregulated and downregulated in either the uv uv or the uv sample relative to c during the time-course. like in the restrictive-temperature experiment, most of the induced genes were only upregulated two- or threefold. only four genes in either c or uv were induced more than fivefold in both repeats of the experiment and this was only found for the c sample. it is striking that only about 1% of around genes has a changed expression after cell cycle progression and uvc irradiation. furthermore, we searched for genes differentially expressed between 0/ 30/ and 0/ minutes in both the c and uv samples. to this end we used moderated t-statistics with a p-value cut-off of. in these experiments each gene could potentially be assigned expression values. about 35% of the values were missing in the entire dataset. the reasons for the missing values will be discussed below. we decided to remove the data for a gene if four or more of the possible data values were missing. this action reduced the dataset from to genes, and data for the resulting 54% of the genes was considered more reliable and was used for further analysis. cell-cycle-regulated genes the rna samples from irradiated cells reflect gene expression changes that occur for two separate reasons: first, the cells are progressing through the cell cycle and will necessarily change gene expression and, second, the cells have been exposed to uvc light and will display stress-related and uvc-specific changes. in contrast, differences between the samples in the restrictive-temperature experiment should reflect only the stress-related and uvc-specific changes. in the present analysis, we attempt to identify the genes specifically affected by uvc exposure. for this reason we identified the genes in unirradiated control cells whose expression varied after release into the cell cycle and these genes were classified as not specifically responding to uvc. in the unirradiated cells altogether genes were found to be differentially expressed between g phase and s/g phase. only genes were differentially expressed at minutes after release into the cell cycle. in comparing c with c genes were found to be differentially expressed. finally, genes had a changed transcriptional level when comparing c to c. this means that, not surprisingly, most of the induced genes were found in cells entering s phase. well-known g1-specific genes, like cdc cig cdc and cdt were induced normally during g phase in our study, serving as convenient controls. uvc-regulated genes using the p-value cut-off of, we identified as many as genes in total that were differentially expressed in the uvc-irradiated cells after release into the cell cycle. of all the genes only were different between uv and uv so little was happening at the transcriptional level during the first minutes after irradiation. no genes were determined to be differentially expressed when comparing uv and uv. however, as many as genes were defined to be different between uv and uv. the apparent discrepancy between the numbers of regulated genes between uv0/uv and uv30/uv on the one hand and between uv0/uv on the other can be explained as follows: expression of a number of genes is different between uv and uv but only genes were significantly different. similarly, a number of genes changed their expression between uv and uv but no difference passed the threshold we had set. however, when comparing uv and uv a number of genes had altered their expression sufficiently during the total interval. it follows that the level of change for all of these genes was low. some of the genes that were up- or downregulated after uvc in this experiment are regulated as a consequence of the cell-cycle progression and not of the uvc irradiation. to identify the uvc-specific transcripts, we excluded the cell-cycle-regulated genes identified above, resulting in uvc-regulated genes, of which are non-cesr genes. of these genes as many as genes are dedicated to the translational machinery. nine of the genes were specifically upregulated in uv, which is at a time when cells are arrested in g phase by the g1/s checkpoint. these genes were compared to the set of non-cesr genes induced by h2o and ir. no genes were found to be induced by both uvc and h2o treatment. only two genes, gst and fip were induced after both uvc and ir treatment. none of the uvc-specific genes are likely to be regulators of the g1/s checkpoint, judging from their annotations. spac23h c gene annotations are from genedb http://www.genedb.org/genedb/pombe/index.jsp. profile analyses to further analyse the kinetics of gene expression in the time-course experiment we applied the software package masigpro on the filtered data, which allowed a multiple comparison of all three time points. there were genes that significantly changed their expression levels during the time-course, and the expression of almost all was changed in both control and uvc-irradiated cells. fifteen genes were identified as specific for the irradiated cells. there was a good correlation between the genes identified in this profile analysis and the differentially expressed genes determined in the statistical analysis above. the expression levels of the genes with expression that varied during the time-course were subjected to a clustering analysis, forming a two-dimensional map. genes being up- or downregulated at the particular time point are represented by different colours, green indicating induction and blue repression. the map shows that analysis of the two biological replicates revealed that they were indeed similar and assembled into the closest branches of the cluster. this confirms a good reproducibility of the experiments. furthermore, the profile analysis is also consistent with the conclusions of the pairwise statistical analyses, showing that the expression patterns in uv and uv were similar. the map shows little difference between control and irradiated samples at all time points, and the least closely related branches of the cluster refer to changes occurring during progression in the cell cycle. thus, this analysis corroborates our conclusions from the above pairwise comparisons, that the cell cycle progression affects transcription profiles more than the uvc treatment and there is only a weak transcriptional response to uvc irradiation. pathway analyses gene ontology enrichment analysis was performed on the uvc-induced genes in the restrictive-temperature experiment using the david software after id-conversion. ten unique genes were members of enriched go groups, suggesting upregulation of genes involved in ion transport or ion homeostasis in the restrictive-temperature experiment. analysis of over-represented go annotations amongst the upregulated genes in the time-course experiment showed that unique genes are members of the enriched go groups. the nature of these groups indicates regulation of genes that affect protein biosynthesis or structural components of ribosomes. further network analysis shows a concerted response involving direct protein-protein interactions between of the induced gene products. thus, the analyses suggest a coordinated induction of rrna biogenesis, ribosome assembly and components of the 60s and 40s ribosomal subunits in the time-course experiment. * count denotes the number of genes in our dataset in each cluster. percent coverage of these genes relative to the numbers on the gene ontology clusters were calculated. confirmation of the microarray data rna blotting and hybridisation was used to confirm our microarray results for four selected transcripts. the induction of spac2e1p c, fip and gst found in the time-course experiment was verified. fip an iron permease, and gst a glutathione s-transferase, have also been shown to be induced after ir. on the other hand, rhp4b had no values in any of the arrays in our time-course experiments and was also not detected after rna blotting and hybridisation. however, in the restrictive-temperature experiment, rhp4b transcription was found to be induced by uvc irradiation and this finding was also confirmed by rna blotting. therefore, rna-blotting experiments with all four selected genes verified the results from the microarray experiments. uvc does not induce the unfolded protein response the unfolded protein response is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. the upr triggers a transcriptional response which serves to induce the production of er components and to increase the degradation capacity to dispose of the unfolded proteins. it is possible that uvc irradiation might stress the er and thus activate the upr. furthermore, gcn has been shown to be required for the induction of a majority of upr target genes during er stress in s. cerevisiae. we therefore used the present data from both the restrictive-temperature and time-course experiments to investigate whether upr genes are induced by uvc in fission yeast. we identified upr-genes in fission yeast as the homologues of the upr-induced budding yeast genes. there was little, if any, induction of the upr-genes identified by this method. the lack of transcriptional response of these genes after uvc strongly argues that the g1/s checkpoint is not a manifestation of the upr. discussion here we have investigated gene expression in s. pombe cells traversing the g1/s border in a synchronous manner, both uvc-irradiated and unirradiated cells. the transcriptional response after uvc-irradiation in g phase was surprisingly weak. the vast majority of genes did not change their transcription pattern appreciably and the few that did increased or decreased their expression levels only two- to three-fold. comparison of the data from the two experiments genes were identified as specifically uvc-regulated in the time-course experiment and in the restrictive-temperature experiment. surprisingly, as few as genes were found to be common for the two datasets, and these genes were not differently expressed in uv and uv in the time-course experiment, which is the time period when the irradiated cells were arrested in the g1/s checkpoint. the uvc irradiation elicits a quite weak transcriptional response on the cells both when considering the number of genes affected and the level of the response for the affected genes. thus, our assay must be considered to be rather sensitive and even a small change in the experimental setup might bring the marginal levels of gene expression over or under our threshold, which could be one reason for the poor overlap between the two experiments. this further underlines our conclusion that uvc has only a marginal effect on gene transcription when given in g phase. it is possible that the biological differences between the cells in our two types of experiments is dominating and that the dissimilar sets of regulated genes reflect a biological difference rather than an artefact of our data analysis. in the restrictive-temperature experiment cells were arrested in g phase by the inactivation of cdc which means that cell-cycle dependent responses were inhibited. thus, this experiment reveals transcriptional regulation exclusively due to uvc irradiation, at a specific stage in the cell cycle. in contrast, in the time-course experiment cells were allowed to progress into the cell cycle. even though we have subtracted the cell-cycle-regulated genes when identifying the uvc-regulated genes, the cell cycle stage in which the cellular response is analysed was still different from that of cells held in the cdc block. consistent with this line of reasoning, we have shown that little happened at the transcriptional level during the first minutes after irradiation in the time-course experiment, and most of the genes changed their transcription enough to satisfy the criteria of our analysis only by a later time point, as the cells entered s phase. therefore these genes have not been identified as regulated in the restrictive-temperature experiment. missing values in the data for the time-course experiment values were missing for many data points. this can be attributed to at least two reasons: lack of expression of the relevant open reading frames and technical problems with the microarrays. at any one time there are genes that are poorly expressed, so it was expected that there would be some missing values in our datasets. low expression is the most likely reason for the absent microarray signal for the rhp4b transcript, which could also not be detected after rna blotting. however, lack of expression cannot explain all the missing values. the microarrays used in the time-course experiment comprised from 18% to 55% missing values, arguing that there were technical problems with at least some of the microarrays. such a high level and difference in the number of missing values was not found for the two repeats of the restrictive-temperature experiment, which both had about 6% missing values, further suggesting technical problems with the arrays used in the time-course experiment. it should be noted that the microarrays used for this experiment came from a different batch/production than those used for the restrictive-temperature experiment. this problem did reduce the quality of the data for some of the genes in the time-course experiment, and we decided to remove all the data pertaining to genes where too many data points were missing. however, the stringency of our analysis allows us to draw conclusions in spite of the missing data. furthermore, any technical problem would have affected a random set of genes, and a strong transcriptional response would have been obvious even from the time-course experiment. comparisons with other organisms this is the first report about the global transcriptional response after uvc irradiation in fission s. pombe and there are only a few reports about similar experiments in other organisms. the available data indicate that the weak transcriptional response to uvc stress we observed in fission yeast might be a conserved feature. for example, in human cells exposed to uvc only of more than genes investigated changed their expression more than 2-fold. an early microarray-report using escherichia coli cells identified several differentially expressed genes after uvc-irradiation, but the response was generally not more than two-fold. many checkpoints, both in fission yeast and in other organisms, involve transcriptional regulation. for instance, in multicellular organisms one of the best characterised checkpoint targets is p a transcription factor which is mutated in over half of human cancers. p stimulates transcription of cell-cycle inhibitors such as p and is essential for a persistent g arrest. another tumour suppressor, prb, targets the e2f-driven transcriptional programme in the g1/s checkpoint. in budding yeast activation of the g1/s checkpoint also impinges on transcriptional regulation in that the transcription factor swi is phosphorylated and thereby inactivated by the checkpoint protein rad leading to delayed transcription of cln and cln and a delayed entry into s phase. recent data show that in fission yeast the transcriptional response is important in the intra-s checkpoint after exposure to hydroxyurea. comparisons with other stress agents we have observed no obvious change of the transcriptional programme that could be responsible for the g1/s checkpoint and also no strong induction of genes involved in dna repair or checkpoint function. it is interesting to note that other dna-damaging agents, such as h2o and ir, also do not lead totranscriptional induction of many dna repair- or checkpoint-related genes. therefore, it is likely that there is no need to specifically induce transcription of dna repair-related genes, suggesting that the dna repair capacity is high already before the uvc exposure, as the case is for budding yeast. we have compared our data from synchronised cells to data based on h2o2- and ir-treated asynchronous cells, and there is little overlap in the spectrum of non-cesr genes differentially expressed after exposure to the three agents. as discussed above, it seems that the cell-cycle position is important for the transcriptional profile obtained when exposing the cells to a stress treatment. however, when comparing the differentially expressed genes in asynchronously growing cells exposed to uvc to cells exposed to h2o and ir there is little overlap. pathway analyses dna-damaging agents, heat and other forms of stress give overlapping responses, described as the cesr, that involves 14% of the genome in s. cerevisiae. few uvc-specific expression changes have been reported and the fold-changes observed are low. it is therefore possible that transcriptional responses to heat stress combined with stringent statistical analyses and exclusion of the cesr-genes mask uvc-relevant gene expression changes in the restrictive-temperature experiment. similarly, the absence of significant gene-expression changes after minutes recovery in the time-course experiment indicates that uvc-relevant transcriptional responses are masked by the small fold-changes and extensive overlap between the uvc-specific response and the cesr. this is supported by appearance of differences on the transcriptional level first after minutes. our network analyses of gene-expression changes indicate that pathways involved in recovery after dna damage are induced after minutes. the processes involved probably reflect the importance of the translation machinery in recovery after the insult. these recovery processes are likely to be important for survival after uvc treatment, as was shown in mms-treated s. cerevisiae, although they may not be components of the g1/s checkpoint per se. regulation of the g1/s checkpoint in the g1/s checkpoint the gcn kinase is activated to phosphorylate the eukaryotic initiation factor 2α, eif2α, thereby inhibiting translation. there is a good correlation between eif2α phosphorylation and checkpoint activation, but it is still unclear whether and, if so, how the checkpoint is dependent upon this phosphorylation and on the ensuing downregulation of translation. gcn is best known for its role in the starvation response, where eif2α phosphorylation leads to induction of the transcription factor gcn both in budding yeast and higher eukaryotes. fission yeast does not have a gcn homologue, and it remains to be seen whether the gcn2-dependent g1/s checkpoint in budding yeast involves activation of gcn one might expect a transcriptional response after gcn activation also in s. pombe, but the finding that no such transcriptional response could be identified argues that in fission yeast the g1/s checkpoint does not operate like it does in budding yeast or higher eukaryotes and is not dependent upon the strong transcriptional induction of one or a set of genes. we have shown that the g1/s checkpoint in s. pombe is associated with a strong downregulation of translation and it might be relevant that among the few genes that are affected at the transcription level, 15% affect the translation machinery. importantly, amongst the transcriptionally regulated genes none were detected that are likely to be directly involved in the g1/s checkpoint according to their annotations. induction or inhibition of transcription is a fairly slow response and regulation of the cell cycle in s. pombe should preferably occur rapidly in order to be efficient and meaningful. therefore, it intuitively makes sense that the present data suggest that the g1/s checkpoint is regulated at the level of protein modification and/or translation, which is rapid, rather than a slower regulation of gene expression.  the transcriptional response to uvc irradiation of fission yeast cells in g phase was shown to be weak. we conclude that the novel g1/s checkpoint is not regulated by changing the transcriptional programme. this is supported by an examination of the few genes that are induced or repressed by uvc, and none of them appears to have any relationship to cell-cycle regulation. 
in many cell types, including the fission yeast schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by dna damage. as part of characterising a novel g1/s checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light. microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after nm ultraviolet irradiation, which is known to induce a g1/s checkpoint. amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the g1/s checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation. pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from g to s phase. we discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. there is little response in fission yeast to ultraviolet light at the transcriptional level. many of the checkpoint responses include a substantial change of the transcriptional pattern. interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress.
 in many cell types, including the fission yeast schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by dna damage. many of the checkpoint responses include a substantial change of the transcriptional pattern. as part of characterising a novel g1/s checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light.  microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after nm ultraviolet irradiation, which is known to induce a g1/s checkpoint. we discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress. pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from g to s phase.  there is little response in fission yeast to ultraviolet light at the transcriptional level. amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the g1/s checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation. 