Fission yeast shares several conserved histone acetyltransferases with other eukaryotes. In the GNAT family, the Elp3 (KAT9) and Gcn5 (KAT2) enzymes are highly conserved . In the MYST family, the Kat5 HAT Esa1/Tip60/Mst1 protein is also highly conserved. However, the relationships between the other MYST family members are less clear-cut. In budding yeast, two additional MYST proteins Sc Sas2 and Sc Sas3 have been characterized. Sc Sas2 antagonizes Sc Sir2 in telomere silencing as a component of the SAS complex. Sc Sas3, a component of NuA3 , overlaps with Gcn5 such that a double mutant Sc gcn5 sas3 is lethal . By contrast, there is only one additional MYST protein in S. pombe, Mst2, which resembles both S. cerevisiae proteins in primary sequence. Like Sc Sas2, Sp Mst2 antagonizes Sp Sir2 in telomere silencing , while work from this study suggests that like Sc Sas3, Sp Mst2 acetylates histone H3K14. This works shows that Sp Mst2 functions similarly to both Sc Sas2 and Sc Sas3. To dissect out the relationship between these potentially overlapping HAT enzymes Mst2, Elp3 and Gcn5, we compared their phenotypic and transcriptional interactions in S. pombe.
First, we performed the initial characterization of the catalytic HAT, Elp3, which is the likely catalytic subunit of the Elongator complex in S. pombe. The strain elp3 is viable, although it suffers defects in overall cell growth. It shows a delay in entry into the cell cycle, and premature cell cycle exit (Fig. 1B). It should be noted that some of the phenotypes seen in Elp3 defective strains could result from defects in other functions that have been reported for the Elongator complex in addition to its role as a HAT [39, 40]. We next investigated whether Δ elp3 shows genetic interactions with mutations in other non-essential HAT genes encoded by gcn5
+ and mst2
+. Double and triple mutants are all viable, although they show increasingly severe growth defects as more HAT genes are mutated. The viability of Sp Δ mst2 Δ gcn5 contrasts with the lethality of Sc Δ sas3 Δ gcn5 and suggests that Mst2 is not a simple equivalent of Sc Sas3. Alternatively, there may be another enzyme in fission yeast, which provides an additional degree of redundancy. We suggest that Mst2 fulfills many of the functions associated with both Sc Sas2 and Sc Sas3 (see below).
Phenotypic analysis suggests distinct effects of each HAT. For example, as shown previously Δ gcn5 mutants are de-repressed for mating and meiosis functions, and are sensitive to certain salt-stress conditions [27, 28]. Δ mst2 mutants show modest sensitivity to DNA damaging agents such as HU and MMS. Δ gcn5 Δ mst2 double mutants show significantly increased salt sensitivity, and a modest increase in DNA damage sensitivity relative to single mutants. Thus, we conclude that in response to salt stress, Gcn5 and Mst2 have a substantial functional overlap. In contrast, Δ gcn5 Δ elp3 and Δ mst2 Δ elp3 showed less evidence for synthetic phenotypes, with only a modest increase in TBZ sensitivity in Δ gcn5 Δ elp3 relative to the parents (Fig. 1C & 1D). In fact, Elp3 appears to be antagonistic to Mst2, because Δ elp3 suppresses Δ mst2 mutant sensitivity to high salt concentrations and HU. We speculate this could be due to slowing down the growth of Δ mst2 cells, allowing time for the cells to repair any damage that may have occurred. Together, these data suggest that Gcn5 affects multiple pathways, some overlapping with Elp3, and others overlapping with Mst2. However, since the double mutants and even the triple mutants are viable, either additional HAT enzymes play a role, or the acetylation modifications controlled by these enzymes are not strictly essential for viability.
To determine whether the functional redundancy between HATs is at the level of gene expression, we performed transcriptional profiling using DNA microarrays of Δ elp3, Δ gcn5, and Δ mst2. Similar to previous results with Δ gcn5 [27, 28] we found that few genes were affected by deletion of any single HAT gene under favorable growth conditions. Although HATs are presumed to be gene activators that facilitate transcriptional initiation or elongation , we found that gene expression was as likely to be increased as reduced in our mutant strains. Up-regulated genes could represent indirect targets, although many Gcn5-repressed genes are known to be bound by Gcn5 . This suggests that Gcn5 may play a direct role in gene repression, which has been described previously . It is thus possible that the other HATs studied here also play direct roles in transcriptional repression.
When we examined the double mutants, we found that their gene expression profiles were not simply the combination of the profiles from the two single mutants, but affected additional genes. This supports the hypothesis that the HATs play partly redundant roles in gene regulation, with multiple enzymes contributing to expression of common targets. Importantly, although they may affect common genes, the regulatory mechanisms may be distinct. In budding yeast, although Sc Gcn5 and Sc Elp3 affect transcription of the Sc Hsp70 genes Sc SSA3 and Sc SSA4, this occurs by different mechanisms because Gcn5 is required for transcription factor binding while Elp3 is required for proper Pol II elongation . However, we have shown that in fission yeast Sp Gcn5 also has a role in transcriptional elongation  which could suggest a more direct mechanistic overlap for Sp Gcn5 and Sp Elp3 in this species.
Only one gene was significantly increased in all strains: the RecQ-type DNA helicase SPBCPT2R1.08c, located in the sub-telomere domain (Tlh2; [32, 33]). We showed previously that Δ mst2 increases silencing at the telomere and in telomere associated regions, consistent with a role for Mst2 in antagonizing Sir2. Loss of Mst2 results in a loss of overall H4 acetylation  and H3K9 acetylation in Δ mst2 in a very limited region adjacent to the telomere 18 kb from the end. Interestingly, this region is telomere-distal to the tlh2 helicase gene located 13 kb from the end of the telomere chromosome II, which is highly up-regulated in the mutant. In contrast, genes at 27.5, 29 and 31 kb away from the telomere are down-regulated in Δ mst2 (SPAC186.06, SPAC186.05 and SPBCPT2R1.02 respectively), while at 47 kb telomere distal (SPAC869.01), there is no change in expression levels. This suggests a gradient effect on transcription of genes in these regions.
The few genes that are down-regulated in Δ gcn5 are localized near the ends of chromosome I and II which suggest that the repression might be due to spreading of heterochromatin by loss of H3K9ac. The combined effect of the double mutant may indicate a combination of effects associated with loss of both histone H3K9 and H3K14 acetylation. Clearly, there are specific regions at the telomeres that are regulated by specific HATs but more work needs to be done to fully understand the boundaries of the telomeres and the regulation between the highly heterochromatic region and the rest of the chromosome.
We used gene ontology (GO) classification to define the functional roles of the few genes that were differentially expressed in single mutants. We found that those in Δ elp3 and Δ gcn5, but not Δ mst2 associated with a few distinct classes. Consistent with previous results , Δ gcn5 mutants showed increased expression of specific sexual differentiation genes as well as enrichment for GO terms  related to mating and meiosis.
Gcn5 represses transcription of ste11
+ , which is an important regulator of the mating pathway in response to nutrient limitation. Although we find very similar functional groups that are positively regulated in the absence of Δ gcn5, we did not see evidence for induction of ste11 above our threshold level (Table 1).
Consistent with prior work, our study also found that mei2
+ was up-regulated in Δ gcn5 ; this was also seen for mst1
and to a lesser extent Δ mst2 (Fig 3), indicating multiple inputs into this essential regulator of sexual development. The Δ gcn5 Δ mst2 mutant increased mei2
+ and ste11+ mRNA levels above those observed in the single mutants, suggesting these two HATs function redundantly in repression. It is possible that they might also have non-histone targets in common since the repression of mei2
+ expression by Gcn5 was suggested to be mediated by a histone independent mechanism .
In contrast, deletion of Δ elp3 results in loss of mei2
+ transcription and in the Δ gcn5 Δ elp3 and Δ mst2 Δ elp3 double mutants there is also a reduction of mei2
+ transcription. Even the triple mutant shows lower levels of mei2
+ than the Δ gcn5 Δ mst2 double mutant, suggesting that Δ elp3 has an opposite effect on mei2
+ transcription than Δ gcn5. Interestingly transcription of SPAC1039.02, a putative phosphoesterase, shows a similar, but opposite, regulation pattern as it is down-regulated in Δ gcn5, mst1
and Δ mst2 while being up-regulated in Δ elp3. Thus, for some targets, Gcn5 and Mst2 appear to overlap, while Elp3 appears to be antagonistic. This finding suggests that the common model linking HAT enzymes with gene activation is to simplistic. Dissecting the contribution of each HAT to gene expression or other effects will require future molecular studies mapping the histone acetylation and physical binding of HATs at different gene regions or their association with the transcription machinery.
To determine the molecular basis for the overlap of HAT targets, we examined histone H3 acetylation. Three acetylation sites on histone H3 are associated with gene expression: K9, K14, and K18 . H3K9 is acetylated in euchromatin, but methylated in heterochromatin; consistent with other species, our data indicate that Gcn5 is the primary contributor to H3K9Ac because acetylation of this residue is strongly reduced in Δ gcn5 but relatively unperturbed by the other HAT mutations.
H3K14 acetylation is also associated with gene expression . Mutations that change the H3K14 residue display hypersensitivity to KCl and CaCl2 induced stress in Δ gcn5 cells . Little or no H3K14 acetylation is observed in the Δ gcn5 Δ mst2 double mutant. Consistent with this, we observed that Δ gcn5 Δ mst2 is significantly more salt-sensitive than either single mutation. We conclude that these two HATs both contribute to H3K14 acetylation in response to stress. Importantly, this acetylation is not essential for viability in S. pombe. Data from budding yeast also indicates an overlap between Sc Sas3 and Sc Gcn5 in H3K14 acetylation . Interestingly, the budding yeast data also suggest that Δ sas3 is not lethal in combination with disruption of other SAGA subunits that are essential for Gcn5 activity against histones, and deletion of histone N-terminal H3 tails entirely is not lethal in budding yeast . Thus, the essential redundant function of these enzymes may not be in histone modification. It is now well-established that HAT proteins acetylate substrates other than histones . Perhaps it is one such substrate that relies on Gcn5 or Sas3 in budding yeast, but on a different enzyme in fission yeast. These results indicate that Mst2 has functions in common with both budding yeast enzymes.
Finally, we found that as expected there was little overlap between expression profiles in the non-essential HAT mutant strains, and cells with a temperature sensitive mutation of the essential HAT mst1
+. Mst1 is known to be required for DNA damage response and chromosome segregation . However, we did not see a significant increase in GO terms related to these functions, which is consistent with data suggesting its effects are not mediated through the transcriptional program . There was a significant overlap of regulated genes between Mst1 and the triple mutant Δ gcn5 Δ mst2 Δ elp3, as half of the genes regulated in the triple mutant were also regulated in mst1
. When we compared the regulated GO terms from mst1
and Δ gcn5 Δ elp3 Δ mst2 we found that these mutants shared terms related to metal and ion homeostasis, metal transports as well as regulation of meiosis. Since both strains show severe growth defects under the conditions employed, we suggest that these overlaps may represent the effects of general cellular stress rather than a particular transcriptional program.