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Genetics. Jun 2008; 179(2): 757–771.
PMCID: PMC2429872

Schizosaccharomyces pombe Histone Acetyltransferase Mst1 (KAT5) Is an Essential Protein Required for Damage Response and Chromosome Segregation

Abstract

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.

MANY events in DNA metabolism, including replication and repair, are modulated by the pattern of different histone modifications in the chromatin, leading to the speculation that a “histone code” provides epigenetic information that facilitates numerous chromatin functions (reviewed in Jenuwein and Allis 2001; Kouzarides 2007). Histone acetylation is associated with diverse chromatin functions (reviewed in Kouzarides 2007; Lee and Workman 2007). Histone acetylation changes association of DNA with the underlying nucleosomes (Shogren-Knaak et al. 2006) and also creates specific binding sites for proteins involved in a variety of DNA transactions (reviewed in Kouzarides 2007; Lee and Workman 2007). Histone acetyltransferase (HAT) enzymes can be separated into several sequence-specific subfamilies that differ in their substrates and in the composition of their associated complexes. The MYST family of HATs (reviewed by Utley and Cote 2003) has been extensively characterized in budding yeast, which has three members (reviewed in Lafon et al. 2007). We have reported two MYST family proteins in fission yeast (Gómez et al. 2005): Mst2, the likely ortholog of Saccharomyces cerevisiae Sas2, is a nonessential gene required for normal telomere silencing (Gómez et al. 2005). Mst1, the likely ortholog of S. cerevisiae Esa1, is an essential gene and is the subject of this report.

Work from several experimental systems shows that ScEsa1 histone acetyltransferase is a catalytic subunit of a large protein complex called NuA4 and a smaller subcomplex, Piccolo, that is part of NuA4 (Allard et al. 1999; Boudreault et al. 2003; Eberharter et al. 2005; Selleck et al. 2005). The NuA4 complex acetylates histone H4 and H2A (Smith et al. 1998; Allard et al. 1999; Galarneau et al. 2000). It may also be associated with acetylation of the histone variant H2AZ (Keogh et al. 2006; Millar et al. 2006). Esa1 family HATs contain a chromodomain, a motif associated with binding to methylated histones (reviewed in de la Cruz et al. 2005). While this suggests that some aspect of Esa1 function is mediated by binding methylated histone(s), the target for such binding remains elusive. The mammalian homolog is Tip60 (reviewed in Squatrito et al. 2006). Under a new initiative to regularize nomenclature in the chromatin field, Mst1, Esa1, and Tip60 are defined as the KAT5 family of histone acetyltransferases (Allis et al. 2007).

ScESA1 is an essential gene, and loss of function results in cell-cycle-specific arrest after replication but before mitosis (Smith et al. 1998; Clarke et al. 1999), although a recent report suggests that it may also be required for cell cycle entry (Early et al. 2004). The roles of ScEsa1 are diverse, and recent studies have also linked it to the spindle assembly checkpoint (Le Masson et al. 2003) and the morphogenesis checkpoint (Le Masson et al. 2003; Ruault and Pillus 2006). Genome-level analyses in budding yeast indicate that ScEsa1 activity is associated with active gene expression (Galarneau et al. 2000; Vogelauer et al. 2002). Paradoxically, however, H4 acetylation is also associated with decreased gene expression (Deckert and Struhl 2001). Recent studies suggest that ScEsa1 may also repress activity of specific gene regions, particularly the heterochromatic domains of the telomeres and the rDNA (Clarke et al. 2006).

Histone acetylation by the NuA4 complex has a role in DNA double-strand break (DSB) repair (reviewed in van Attikum and Gasser 2005b; Squatrito et al. 2006). Increased histone acetylation near the damage site occurs (Yu et al. 2005) and acetylation of H4 is linked to activation of homologous recombination and nonhomologous end-joining (Bird et al. 2002; Tamburini and Tyler 2005). ScEsa1 is recruited to DNA breaks (Bird et al. 2002; Tamburini and Tyler 2005). One of the major responses to DNA double-strand breaks is phosphorylation of the histone variant H2AX by the damage checkpoint kinases ATM and ATR (reviewed in Foster and Downs 2005; Morrison and Shen 2005). H2AX phosphorylation and recruitment of NuA4 occur with similar timing (Downs et al. 2004). Interestingly, two chromatin-remodeling complexes, INO80 and SWR1, are also recruited to H2AX, at least in part through the common Arp4 subunit that they share with NuA4 (Downs et al. 2004; Robert et al. 2006). NuA4, INO80, and SWR1 are proposed to facilitate exchange of phospho-H2AX, ultimately downregulating the damage signal (Morrison et al. 2004; Tsukuda et al. 2005; van Attikum and Gasser 2005b; Papamichos-Chronakis et al. 2006). Recent studies have implicated the NuA4 complex in regulation and modification of an additional histone H2A variant called H2AZ (Babiarz et al. 2006; Keogh et al. 2006; Millar and Grunstein 2006). It has been suggested that the downregulation of the H2AX phosphorylation signal reflects exchange of phospho-H2AX with H2AZ (reviewed in van Attikum and Gasser 2005a).

Tip60, the human ortholog of Mst1 and ScEsa1, acetylates nonhistone proteins independently of the NuA4 complex with substrates including Notch (Kim et al. 2007), ATM (Sun et al. 2005; Jiang et al. 2006), p53 (Sykes et al. 2006; Tang et al. 2006), and DNA-PKcs (Jiang et al. 2006). Tip60-deficient cells exposed to ionizing radiation have defects in chromosome repair and reduced apoptosis (Ikura et al. 2000). This leads to the important conclusion that HATs are not limited to histones as substrates. These data suggest that KAT5 family HATs contribute to DNA damage response in two ways: first, by affecting DNA repair and recovery (e.g., through histone modifications that recruit specific DNA damage response proteins) and, second, by affecting the checkpoint response (e.g., through direct modification of p53 or ATM).

To gain further insight into the diverse function of the KAT5 protein family in DNA metabolism, we investigated the role of Schizosaccharomyces pombe Mst1 in the tractable fission yeast system. In this report, we present initial genetic characterization of this gene. Mst1 is essential for viability, and mutants are sensitive to a variety of DNA-damaging agents, including hydroxyurea (HU), ultraviolet (UV) irradiation, methyl methanesulfonate (MMS), and bleomycin. Mutants are also sensitive to the spindle poison thiabendazole. Using a two-hybrid screen, we identify a number of potential interaction proteins involved in DNA dynamics, including chromatin proteins, transcription factors, and the recombination protein Rad22 (ScRad52). Analysis of the HU response suggests that Mst1 is required for normal recruitment of Rad22 in HU. Thus, Mst1 has multiple roles in maintenance of genome integrity in S. pombe.

MATERIALS AND METHODS

Strains, media, and manipulations:

Strains used in this study are listed in Table 1. Strains were grown and maintained on yeast extract plus supplements (YES) or Edinburgh minimal media with appropriate supplements, using standard techniques (Forsburg and Rhind 2006). Matings were performed on synthetic sporulation agar plates for 2–3 days at 25°. Temperature-sensitive (ts) cells were grown at 25° and non-ts cells at 32°. Transformations were carried out by electroporation. Double mutants were constructed by standard tetrad analysis or random spore analysis. G418 plates were YES supplemented with 100 μg/ml G418 (Sigma, St. Louis).

TABLE 1
Strains used in this study

Plasmids and constructions:

Disruption and tagging of mst1+ is described in Gómez et al. (2005). Site-directed mutants were constructed using the Quick-Change mutagenesis kit (Stratagene, La Jolla, CA).

Primers used were L-S/F (CATACAATTTTCTTATGAATCGACCAAGCGTGAGCACAAAC) and L-S/R GTTTGTGCTCACGCTTGGTCGATTCATAAGAAAATTGTATG and L-P/F GGTGTAGGAATATTTGTCTTCCGTCCAACCTTTTTCTGGATCAC and L-P/R GTGATCCAGAAAAAGTTTGGACGGAAGACAAATATTCCTACACC. Complementation and temperature sensitivity were assessed by transforming the expression plasmid into the heterozygous mst1+/Δmst::ura4+ diploid FY1754 and by isolating haploids that contain both Δmst1::ura4+ and the plasmid markers. An integrant was constructed by moving the Pnmt-mst1-L344S cassette from plasmid pEBG99 into plasmid pJK148 to create plasmid pEBG98 and integrating this construct at the leu1-32 locus in the diploid. Haploid temperature-sensitive segregants containing both Δmst1::ura4+ and leu1+::nmt-mst1-L344S were isolated by random spore analysis.

Silencing was assessed by examining growth in the absence of uracil and low adenine in a strain with ura4+ integrated into the centromere and ade6+ integrated into the telomere of a minichromosome.

FACS:

Samples were collected after 5 and 10 hr, ethanol fixed, and analyzed by flow cytometry as described previously (Gómez and Forsburg 2003).

Immunoprecipitation:

Samples were prepared as described in Gómez et al. (2002). For co-immunoprecipitation with MCM proteins, we used anti-HA antibody 12CA5 (Abcam) and antiV5 (Invitrogen, San Diego). For histone acetylation assays, the dried beads were treated with 20 μl of a histone acetylation mix containing 5 μm C14-Acetyl CoA (ICN), 0.7 μg of core histones, 50 mm HEPES (pH 7.9), 10% glycerol, and 1 mm DTT. The reaction was incubated 1 hr 30 min at 30°. Beads were spun down, and supernatant was recovered and boiled in SDS–Laemmli loading buffer before electrophoresis in 15% SDS–polyacrylamide gel electrophoresis (PAGE). The gel was fixed and treated with fluorography-enhancing solution (NEN Life Science) before drying and autoradiography.

For co-immunoprecipitation with Rad22-YFP or Cbh1-GFP (strains 3436, 3480), we prepared soluble protein lysates from fission yeast cells in B88 buffer as in Moreno et al. (1991) A total of 1.0 mg protein was used per immunoprecipitation. Lysates were precleared with sepharose A beads (Repligen) for 1 hr. One microliter of anti-GFP (Abcam 290) was added and left to rotate overnight at 4°. Proteins were analyzed by 8% SDS–PAGE, followed by immunoblotting with anti-V5 (Invitrogen) at 1:2500 dilution and with anti-GFP (living colors, Clontech) at a 1:1000 dilution. Crosslinking immunoprecipitations were done as previously described (Auger et al. 2008). Briefly, cells were crosslinked with 1% formaldehyde (Sigma) for 50 min on ice. Harvested cells were vortexed with glass beads in IP buffer (50 mm HEPES–KOH, pH 1.6, 500 mm NaCL, 1 mm EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). Extracts were treated with DNase I to reverse crosslink. A total of 1.0 mg protein was used per immunoprecipitation. Whole-cell extracts and immunoprecipitates were incubated with SDS sample buffer for 1 hr at 90° before Western blot analysis.

Microscopy:

Whole fixed cells were stained as described for DAPI and Calofluor (Gómez and Forsburg 2003). Cells were visualized with a Leica DMR microscope. Objectives used were Leica 100X/1.30, PLFL, NA = 1.30, and Leica 63X/1.32, PLApo, NA = 1.32. Images were captured with a Hamamatsu digital camera and Improvision Openlab software (Improvision, Lexington, MA). Images were assembled using Canvas (ACD/Deneba) and adjusted for contrast. Nuclear spreads were performed as in Pankratz and Forsburg (2005) with the following modifications. Rad22YFP was detected using Abcam 290-50 rabbit anti-GFP, 1:2000 in 5% BSA (Sigma), and secondary donkey anti-rabbit cy3 1:500 DNA was stained with 1× Hoechst (Invitrogen). The objective used was Leica 63X/1.32, PLApo, NA = 1.32. Images were captured and assembled as above.

Damage assays:

Cells were grown to A595 = 1, serially diluted fivefold, and spotted onto YES plates or YES plates containing 10 μg/ml thiabendazole (TBZ), 5 mm HU, or 0.005, 0.007, or 0.01% MMS, and incubated for 3–5 days at 25°, 29°, or 32°, as indicated. For UV irradiation assay, 1000 cells were plated in duplicate onto YES and immediately treated with the indicated doses of UV irradiation using a Stratalinker (Stratagene). Control plates were left untreated. Plates were incubated at 32° for 2–3 days. Colonies were counted and the percentage survival was obtained relative to the number of colonies grown on the untreated control plates.

Two-hybrid strains:

The mst1+ open reading frame was isolated by PCR from plasmid pEBG72 with primers HAT-3 forward (AGATCTCGCCAAAAACTTGCTCAATCTTCTTCC) and HAT-3 reverse (GGTACCCGACATAGACTGGCACATTACATCC) and cloned into the two-hybrid vector pDBLeu cleaved with NcoI and NotI to create plasmid pEBG91. Budding-yeast two-hybrid strain AH109 (Clontech) with HIS3, ADE2, and lacZ reporters downstream of heterologous GAL4-responsive promoter elements was transformed with an S. pombe Matchmaker cDNA library (Clontech) and screened for interactions as described in Leverson et al. (2002). Plasmids from positive clones were isolated and verified by retransformation, and the identity of the interacting open reading frame was determined by sequence.

RESULTS

Δmst1 cells undergo disordered mitosis:

Previously, we identified two members of the MYST family of histone acetyltransferases in fission yeast. Our analysis indicated that Mst1 is the likely ortholog of S. cerevisiae Esa1, and like ScEsa1, is a chromatin-bound protein that is essential for viability (Gómez et al. 2005). Data from metazoans suggest that the Hbo1 histone acetyltransferase, which is also a MYST family protein, may function in DNA replication (Iizuka and Stillman 1999; Burke et al. 2001; Iizuka et al. 2006). To investigate whether S. pombe Mst1 contributes to DNA replication, we examined the phenotype of cells containing the Δmst1 allele. Mutant diploids heterozygous for Δmst1::ura4+ and wild-type diploids heterozygous for ura4+ were induced to sporulate. We inoculated purified spores from both diploids into medium lacking uracil to carry out bulk spore germination. Only spores containing the ura4+ gene are able to germinate in these conditions, and their DNA content was determined by flow cytometry. Spores containing Δmst1::ura4+ synthesized DNA to an approximately 2C DNA content, with timing similar to that of wild type (Figure 1). Half of each population contains nongerminating ura4-D18 spores, which persist as a 1C population. Upon microscopic examination, we observed that many of the Δmst1::ura4+ cells showed highly disordered mitosis, including a high fraction of cut phenotypes. This suggests defects in chromosome segregation, which might result from mitotic defects or from replication defects and checkpoint disruption.

Figure 1.
Spore germination. (A) Wild-type diploid strain (ura4+/ura4-D18) and (B) heterozygous disruption mutant (mst1+/Δmst1::ura4+) were sporulated and processed for random spore analysis. Spores were inoculated into media ...

Isolation and characterization of a temperature-sensitive allele:

To better characterize Mst1, we constructed a temperature-sensitive allele based on temperature-sensitive alleles in S. cerevisiae ESA1 (Clarke et al. 1999). We tested two mutations: L271P (corresponding to L254P in ScEsa1) and L344S (corresponding to L267S in ScEsa1). Constructs containing these were placed in a LEU2 plasmid under control of the nmt1 promoter and transformed into the Δmst1::ura4+ diploid. Following sporulation, we assessed recovery of spores at 25° containing both the ura4+ marker (marking Δmst1) and the LEU2 marker (marking the plasmid). Both mutants were able to complement the disruption at 25°. However, upon shift to 36°, only the mst1-L344S strain was temperature sensitive.

We attempted to replace the endogenous mst1+ allele with the L344S allele, without success. We reasoned that, when integrated under the native promoter, a single copy of mst1-L344S might be insufficient for viability. Therefore, we integrated mst1-L344S under control of the nmt1+ promoter into the leu1-32 locus of the heterozygous diploid and sporulated to create the haploid strain Δmst1::ura4+ leu1+::Pnmt1-mst1-L344S. Upon shift to 36° and addition of thiamine to shut off the nmt1 promoter, this strain was unable to grow. However, cells were able to grow in the presence of thiamine at 25° or 32°, indicating that the protein is functional at lower temperatures. There is sufficient background expression of the nmt1+ promoter even the presence of thiamine for stable proteins to be maintained (Figure 2, A–D). An equivalent strain with a kanMX marker at Δmst1 behaved similarly (data not shown).

Figure 2.
Characterization of temperature-sensitive allele. (A–D) Comparison of growth rate and FACS for wild-type (FY11) and mst1-L344S cells (FY 2396) grown in the presence of thiamine at 25° or 36°. (Top) Colony formation on plates, where ...

We examined the phenotype of cells containing mst1-L344S by shifting asynchronous cells to the restrictive temperature. The increase of cell number was blocked within 2 hr. However, cell mass measured by A595 continued to increase for ~8 hr. DNA content, measured by flow cytometry, showed no change from the 2C peak characteristic of exponentially growing wild-type fission yeast, although later time points showed a flattening of the profile and an increase of cells with higher DNA content. This is common in heterogeneous, elongated cells and is typical of most cdc mutants.

We examined cells for nuclear and septum morphology by staining with DAPI and Calcofluor, respectively, and observing under fluorescence (Figure 2E, quantified in Figure 2F). Strikingly, the number of cells staining with Calcofluor significantly increased in the mst1-L344S strain at restrictive temperature, with nearly 50% of cells arrested with septa by 6 hr. Some of these were mononucleate, showing asymmetric nuclear segregation. Additionally, there was an increase in the overall number of binucleate cells relative to wild type. S. pombe has an unusual cell cycle in which cytokinesis is completed during the subsequent nuclear cell cycle. Thus, cells with a 2C DNA content by FACS may be mononucleate cells in G2 or M phase or binucleated cells in which the nuclei are in G1 or S phase and cytokinesis is not complete. Therefore, arrest as a binucleate septated cell may represent a defect entering DNA replication in the ensuing cell cycle. Alternatively, it could represent a defect in cytokinesis that delays entry into the next cell cycle.

This phenotype is different from the disordered mitosis seen in the Δmst1 mutant spores. Because germinating spores arise from stationary or G1 cells, while exponentially growing S. pombe are predominantly G2 with a 2C DNA content, we reasoned that the different phenotypes could represent the effect of inactivating Mst1 at different cell cycle stages. Alternatively, they could suggest that the mst1-L344S allele creates a hypomorph rather than a complete inactivation of the gene. We attempted to resolve whether the mutation affects S phase by synchronizing the mst1-L344S cells in G1, using nitrogen starvation (supplemental Figure 1). When released from nitrogen starvation into complete medium, mst-L344S cells completed replication with similar timing to wild type, also suggesting that inactivation of mst1 by the mst1-L344S allele or by the Δmst1 allele is not sufficient for cells to block DNA replication in the first cell cycle.

mst1-L344S genetically interacts with replication, recombination, and heterochromatin mutants:

We examined the phenotypes of double mutants, which contained mst1-L344S as well as mutations in genes required for DNA replication, double-strand break repair, and heterochromatin assembly (Table 2, Figure 3). We tested the double-mutant strains for their temperature sensitivity. We defined a genetic interaction as occurring when a double-mutant strain was unable to grow at temperatures where both parents were growth proficient (“reduced growth temperature”). There were modest genetic interactions with many replication initiation mutants. Additionally, we observed that the maximum growth temperature was reduced when mst1 was combined with mutations Δrhp51 (ScRad51), Δrad50, and Δrad22, which affect homologous DNA recombination. The Δrad22 mst1-L344S strain grew extremely slowly even at 25°; however, because of the high frequency of spontaneous suppressors in Δrad22 backgrounds (Osman et al. 2005), we cannot be confident that this strain is suppressor free.

TABLE 2
Genetic interactions between mst1-L344S and other mutants
Figure 3.
Genetic interactions. Double mutants were constructed and serial dilutions were performed at different temperatures to characterize any synthetic interactions. Equivalent numbers of exponentially growing cells were diluted fivefold and incubated at the ...

Because mst1-L344S cells show defects in chromosome segregation and are sensitive to thiabendazole (see Figure 5), we examined interactions with genes required for centromere heterochromatin assembly (Table 2, centromere). We observed that maximum-growth temperature was strikingly lower than that of the parents when mst1-L344S was combined with Δswi6, Δclr3, and Δclr4, all of which define genes required to establish and maintain centromeric heterochromatin (reviewed in Ekwall 2007). However, we observed no defects in centromere or telomere silencing in mst1-L344S cells (data not shown). This suggests that the heterochromatin itself is intact. We examined additional chromatin-related proteins. No effect is observed when mst1-L344S is combined with mutation of histone acetyltransferase Δmst2 or the telomere-binding protein Δtaz1.

Figure 5.
Damage sensitivity of mst1-L344S. (A) mst1 sensitivity relative to DNA-damaging agents. Exponentially growing cells were diluted fivefold on YES plates with the indicated drugs and grown 4 days at 29°. (B) mst1 is sensitive to bleomycin. Exponentially ...

Identification of potential Mst1 interaction partners by two-hybrid screening:

SpMst1 interacts with the actin-related protein SpAlp5/ScArp4, a known subunit of the NuA4 complex (Minoda et al. 2005). Because evidence suggests that Tip60 may function independently of the NuA4 complex (see Introduction), we sought to identify additional proteins that interact with Mst1, using a two-hybrid screen. We isolated a number of interacting gene products, summarized in Table 3.

TABLE 3
Genes isolated by two-hybrid screening

We isolated Rad22, the S. pombe ortholog of homologous DNA recombination protein Rad52. Rad22 promotes the strand-invasion step of homologous recombination. Fission yeast has an additional Rad22 family member, called Rti1. We constructed plasmids to test whether Mst1 interacted in the two-hybrid system with Rti1 or with Rhp51. Although Rhp51 and Rad22 themselves interact in two-hybrid screens (e.g., Catlett and Forsburg 2003), we observed no evidence for interaction between Mst1 and Rhp51 or between Mst1 and Rti1, suggesting that this interaction is specific (Figure 4A). Immunoprecipitation of Rad22YFP using a cross-reacting anti-GFP antibody precipitates Mst1-V5 (Figure 4B); however, immunoprecipitation of Mst1-V5 does not precipitate Rad22-YFP (data not shown).

Figure 4.
Two-hybrid interactions. (A) A prey construct containing Mst1 was tested for interaction with full-length Rad22, Rti1, Rhp51, Cbh1, Cbh2, and Abp1 (see materials and methods). S. cerevisiae cells containing the indicated plasmids were tested for growth ...

We isolated three chromatin-related proteins as potential Mst1 partners. The first, Hip1, is a putative histone chaperone most closely related to ScHir1 and human HIRA (Blackwell et al. 2004). The second, Cbh1, is one of the three CenpB homologs in fission yeast associated with centromere assembly (Baum and Clarke 2000; Nakagawa et al. 2002). We tested all three CenpB homologs for interaction with Mst1. Only Cbh1 showed a specific interaction (Figure 4). We observed no co-immunoprecipitation between Cbh1 and Mst1 from soluble or crosslinked lysates (data not shown).The third gene that we isolated was Msc1. Msc1 was identified as a high-copy suppressor of the checkpoint mutation Δchk1; it has E3 ligase activity and homology to a family of demethylases, and mutation disrupts histone acetylation (Ahmed et al. 2004; Dul and Walworth 2007). We constructed double mutants containing mst1-L344S with Δhip1, Δcbh1, or Δmsc1 and tested growth at different temperatures. There was no difference in the growth characteristics of the double mutants compared to mst1-L344S cells alone. We were unable to detect Msc1 in soluble extracts and our crosslinking protocol was unsuccessful on this protein (data not shown).

The most frequently isolated two-hybrid clone was skb1+, which encodes an arginine methyltransferase, an ortholog of S. cerevisiae Hsl7 (Fujita et al. 1999; Shulewitz et al. 1999; Lee et al. 2000). We observed no obvious genetic interactions between Δskb1 and mst1-L344S, in contrast to budding yeast where hsl7 and esa1 mutants have been shown to have synthetic interactions (Ruault and Pillus 2006). Two putative transcription factors were also isolated. The res2/pct1+ gene has been studied extensively and it is required for G1/S-specific transcription (Tanaka et al. 1992; Zhu et al. 1994). taf111+ (also known as taf1+) is a putative TFIID subunit required for induction of genes during nitrogen starvation and sexual development (Ueno et al. 2001). The remaining two-hybrid isolates included bud6+, which is also associated with actin function (Glynn et al. 2001), and a SEC18 homolog likely involved in vesicular transport (SPAC1834.11c). The only previously described fission yeast Mst1 interactor, Alp5 (Minoda et al. 2005), was not isolated in our screen, nor were any other subunits of the NuA4 complex.

We observed very little Mst1 (or Mst2) protein in the soluble fraction (supplemental Figure 2a). This may explain why our attempts to confirm the two-hybrid interactions using co-immunoprecipitation in fission yeast were for the most part unsuccessful. Upon overproduction of Mst1, it is possible to observe some interactions with other proteins; for example, we found that Mcm2 can interact with Mst1 or Mst2 but only when both partners are overproduced (supplemental Figure 2b). However, we observed no interactions with endogenous levels of MCM proteins (data not shown), even though MCMs themselves can be coprecipitated under those conditions (e.g., Sherman et al. 1998; Gómez et al. 2002). We completed our initial characterization of Mst1 protein by showing that acetylation of the four core histones can be observed in an IP–acetylation assay (supplemental Figure 3).

mst1-L344S cells are damage sensitive:

Data suggest that the NuA4 complex in other species is recruited to double-strand breaks through association with phosphorylated histone H2AX (Downs et al. 2004; Robert et al. 2006). Our data showing an interaction both genetically and physically with Rad22 suggested that Mst1 may contribute to repair of DSBs by influencing homologous recombination. Rad22 has also been implicated in recovery from HU (Meister et al. 2005; Bailis et al. 2008). Therefore, we examined mst1-L344S mutants for evidence of a role in the damage response.

We first examined growth of mst1-L344S in response to chronic exposure to low levels of DNA-damaging agents (Figure 5A). We examined MMS (alkylating agent delays replication), HU (inhibits ribonucleotide reductase and causes fork stalling), and camptothecin (CPT; blocks topoisomerase and causes DSBs). mst1-L344S cells were sensitive to HU and MMS, but not to CPT at 29°. mst1-L344S was also sensitive to UV irradiation (200 J/m2), but not to γ-irradiation (400 Gy) at 25° (not shown). We observed that sensitivity to these agents increased with temperature (data not shown). The mst1-L344S mutant was extremely sensitive to bleomycin at 32° (radio-mimetic; Figure 5B). These results are consistent with known roles for Mst1 family acetyltransferases in some forms of DSB repair. We also observed substantial sensitivity to thiabendazole, a microtubule-destabilizing drug (Figure 5C). Mutations affecting centromere or kinetochore function are often TBZ sensitive; this may relate to the synthetic interactions that we observed with the heterochromatin mutants (Table 2) and to the physical interactions with Cbh1 and Msc1, which affect centromere function (Table 3).

Mst1 is required for recovery from HU arrest:

HU causes nucleotide depletion, which results in replication fork stalling early in S phase. Defects in HU response may reflect a failure to arrest the cell cycle during the acute treatment or an ability to restore replication and recover after HU is removed (reviewed in Branzei and Foiani 2007). As shown in Figure 6, A, B, and D, mst1-L344S mutants arrest with a 1C DNA content and modestly elongated morphology, as do wild-type and Δcds1 cells, indicating that checkpoint-mediated cell cycle arrest is intact. As seen in the time course in Figure 6, A and B, wild-type cells complete S phase within 30 min of removing HU, while Δcds1 mutants are severely delayed, consistent with their recovery defect. Wild-type cells also proceed to division and enter the next cell cycle, as seen in the increase of 4C population (reflecting binucleate cells that complete S phase before completing the previous cytokinesis). This population is not seen in the Δcds1 mutants, which are defective for recovery and show a broad, heterogeneous peak of DNA content at the later time points. The mst1 cells resemble wild type, suggesting that the mutants maintain and restart the replication fork normally. This suggests that the sensitivity that we observed in mst1 cells in HU reflects defects in a later stage of recovery. Similar results have been seen for Δrad22 and Δrhp51 (Meister et al. 2005; Bailis et al. 2008). Interestingly, although mst1-L344S cells were sensitive to HU during chronic treatment (Figure 5), viability was relatively unaffected by an acute treatment in the HU block-and-release protocol (Figure 6C). This suggests that prolonged exposure to HU leads to an accumulation of growth defects, whereas transient exposure for a single generation causes relatively little problem.

Figure 6.
mst1-L344S has HU recovery defect. (A and B) Time course of DNA content following release from HU at 32° (A) and 36° (B) for cells in YES. Strains: wild type (FY11), mst1-L344S (FY2396), Δcds1 (FY865). (C) Viability of cells following ...

One measure of DNA damage is provided by recruitment of recombination proteins to repair centers (Caspari et al. 2002; Du et al. 2003; Meister et al. 2005). During HU treatment, Rad22 focus formation can distinguish two functions. In wild-type cells, Rad22 is recruited in a burst during the recovery phase after cells are released from HU. In contrast, in checkpoint mutants that fail to respond properly to HU, collapsed replication forks cause DNA breaks and Rad22 is recruited during HU treatment (Caspari et al. 2002; Du et al. 2003; Meister et al. 2005; Bailis et al. 2008). To determine whether reduced Mst1 function causes DNA damage, we examined Rad22YFP focus formation in wild-type and mst1-L344S cells at 32° (Figure 6, E and F). Due to background fluorescence observed in mst1-L344S mutant cells, we had to detect Rad22YFP using indirect immunofluorescence on nuclear spreads. We observed a modest increase in Rad22 focus formation in mst1-L344S cells without HU treatment, relative to wild-type cells (compare 0-hr time point of mst1 and wild type in Figure 6F). This suggests that the cells suffer some amount of endogenous damage. During acute HU treatment, Δcds1 cells show a dramatic increase in Rad22YFP focus formation, consistent with replication fork collapse and double-strand breaks (Meister et al. 2005; Bailis et al. 2008). There is no change in the Rad22YFP foci in mst1-L344S, again consistent with the model that replication forks are intact.

DISCUSSION

The MYST family of histone acetyltransferases is associated with diverse functions (reviewed in Utley and Cote 2003). Previously, we showed that S. pombe mst1+ is an essential gene that encodes a protein constitutively bound to the chromatin (Gómez et al. 2005). In this report, we have characterized the phenotypes associated with deletion of mst1+ and a temperature-sensitive allele.

S. pombe Mst1 is the ortholog of S. cerevisiae Esa1 and human Tip60, the catalytic subunit of the NuA4 family of histone acetyltransferases, which is now referred to as the KAT5 family (reviewed in Squatrito et al. 2006; Allis et al. 2007). In addition to its presumed effects in transcriptional regulation (Smith et al. 1998; Allard et al. 1999; Clarke et al. 1999, 2006; Durant and Pugh 2006), this family is implicated in checkpoint control by direct acetylation of ATM and p53 (Sun et al. 2005; Jiang et al. 2006; Sykes et al. 2006; Tang et al. 2006), as well as in repair by direct recruitment to phosphorylated histone H2AX at double-strand breaks (Bird et al. 2002; Downs et al. 2004; Tamburini and Tyler 2005; Robert et al. 2006). Data also suggest that this family of HAT proteins acetylates histone H2AZ (Babiarz et al. 2006; Keogh et al. 2006; Millar et al. 2006); this histone variant is paradoxically associated with both regions of active gene transcription, as well as with centromeres and silent heterochromatin (reviewed in Guillemette and Gaudreau 2006).

We characterized two mutations affecting mst1+ function. As we reported previously, the Δmst1 mutation causes lethality (Gómez et al. 2005). In this report, we show that spores bearing the disruption are able to germinate and complete DNA replication. However, they show a highly disordered mitosis. We also constructed the temperature-sensitive mutant mst1-L344S, which contains the same lesion as a temperature-sensitive budding-yeast mutant. At the restrictive temperature, this strain is also proficient for replication, whether it is shifted asynchronously (Figure 2), released from nitrogen starvation in G1 (supplemental Figure 1), or released from hydroxyurea in S phase (Figure 6). We observed pleiotropic phenotypes, including disordered mitosis, irregular segregation, and a high fraction of cell cycle arrest with septa. Cells arrest within one to two cell cycles with this mixed phenotype.

Our genetic analysis and subsequent two-hybrid screen suggest that Mst1 impacts three pathways (Table 4): damage response, which is consistent with the physiological analysis of the mst1-L344S mutant and data from other systems; centromere function; and control of mitotic entry.

TABLE 4
Summary of processes associated with mst1+ function

Even at permissive temperatures, mst1-L344S cells are sensitive to a variety of agents that disrupt the cell cycle, including bleomycin and MMS, which cause DNA damage and double-strand breaks; hydroxyurea, which causes replication fork arrest; and thiabendazole, which causes spindle depolarization. The damage sensitivity was expected because data from numerous sources link members of this family to the double-strand break response. The recruitment of KAT5 orthologs to double-strand breaks is thought to modulate the break response and perhaps downregulate the signal associated with H2AX phosphorylation by recruiting the remodelers INO80 and SWR1 and the alternative histone H2AZ (Morrison et al. 2004; Tsukuda et al. 2005; van Attikum and Gasser 2005b; Papamichos-Chronakis et al. 2006).

When mst1-L344S is combined with the mutations Δrhp51 (Rad51), Δrad50, or Δrad22, cells have a severe synthetic growth phenotype in which they grow more poorly than either parent. This is particularly apparent in Δrad22 mst1-L344S; however, because Δrad22 alone is prone to accumulate suppressors in the fbh1+ gene (Osman et al. 2005), we cannot be sure that our double mutant is not also carrying a suppressor. It is possible that Δrad22 mst1-L344S will be synthetically lethal but we have not been able to test this convincingly. These genetic data suggest that mst1-L344S requires a functional recombination pathway for viability. To assess whether mst1-L344S causes damage itself, we examined focus formation of Rad22YFP in mst1-L344S cells. There is an increase in foci in mst1 compared to wild type at 32°, even in the absence of any damaging treatment (Figure 6F). This suggests that attenuation of Mst1 function results in some form of damage that requires Rad22 for repair, but also suggests that Rad22-YFP can be recruited to sites of damage in the absence of Mst1.

We observed a physical association between Mst1 and Rad22 in two-hybrid analysis and in co-immunoprecipitation. This interaction is specific to Rad22, as neither the Rad22 homolog Rti1 nor the Rad51 homolog Rhp51 associate with Mst1 in two-hybrid tests (Figure 4). This could reflect a role in recruitment. Another possibility is that Mst1 acetylates Rad22 itself, as Tip60 does to ATM or p53, to modulate its behavior, and we will investigate this in the future.

Unexpectedly, mst1-L344S cells are also sensitive to hydroxyurea, which causes replication fork arrest. The mutant cells arrest properly with a 1C content without Rad22-YFP foci and appear to complete S phase following release, indicating that the checkpoint functions and the replication fork are intact; instead, they have modestly reduced viability reminiscent of Δrhp51 or Δrad22 mutants (Meister et al. 2005; Bailis et al. 2008). We observe that HU sensitivity of mst1-L344S to HU differs if cells are exposed to low levels over a prolonged time, which causes cell death (chronic exposure, shown in Figure 5), or whether they are exposed to higher levels over a single generation (acute exposure, shown in Figure 6). Similar results have been observed for the fork protection mutant Δswi1 (ScTof1), which survives acute exposure but dies during chronic exposure (Sommariva et al. 2005). It is also possible that this HU sensitivity is related to the interaction between mst1 and mutations in recombination genes; however, Rad22-YFP recruitment appears largely normal in mst1 cells treated with HU.

A second broad functional area defined by our two-hybrid and genetic interactions is centromere assembly. Fission yeast centromeres are large and complex elements, with a central core surrounded by flanking repeats that form peri-centromeric heterochromatin (reviewed in Ekwall 2007; Grewal and Jia 2007). Mutations disrupting the heterochromatin, the central core, or the kinetochore typically show sensitivity to spindle poisons such as TBZ, and a high fraction of chromosome loss and disordered mitoses, as we observed (Figures 1, ,2,2, and and5).5). Our genetic analysis suggests that mutations that disrupt normal pericentromeric heterochromatin function such as Δclr3 (histone deacetylase), Δclr4 (methyltransferase), or Δswi6 (heterochromatin protein 1) reduce the growth temperature of mst1-L344S quite substantially. The simplest interpretation of this epistasis experiment is that mst1+ affects a separate pathway from Clr4 and Swi6 in centromere function, leading to a synthetic effect in the double mutant. We see no loss of silencing at centromeres or telomeres in a mst1 mutant, which suggests that Swi6-formed heterochromatin is largely intact, and some other aspect of centromere assembly and function is disrupted. A good candidate is the central core. Three of the proteins that we identified in the two-hybrid tests are linked to central core and kinetochore function:

  1. We isolated Msc1, which was first identified as a high-copy suppressor of a strain lacking the checkpoint kinase Chk1 (Ahmed et al. 2004). A recent study shows that overexpression of Msc1 rescues mutations of cnp1 (the specialized H3-Cen/CenpA of the centromere central core) in addition to Δchk1 and that this suppression requires the H2AZ histone variant encoded by pht1+ (Ahmed et al. 2007). H2AZ is linked to heterochromatin assembly and centromere function (reviewed in Guillemette and Gaudreau 2006) and H2AZ is a known substrate of KAT5 family HATs (Babiarz et al. 2006; Keogh et al. 2006; Millar et al. 2006; Auger et al. 2008).
  2. We isolated Cbh1, which is one of the three CenpB homologs in fission yeast (Baum and Clarke 2000; Nakagawa et al. 2002). The CenpB homologs are proposed to affect centromere structure (Nakagawa et al. 2002) and, in metazoans, CenpB is involved in recruitment of H3-Cen/CenpA (reviewed in Mellone and Allshire 2003).
  3. We isolated Hip1, the HIRA/Hir1 homolog. HIR proteins are histone chaperones required for assembly of chromatin outside of S phase (reviewed in Gunjan et al. 2005). In fission yeast, deletion of Hip1 causes chromosome loss, TBZ sensitivity, and disruption of centromere silencing (Blackwell et al. 2004).

HIR proteins in S. cerevisiae are implicated in normal kinetochore function (Sharp et al. 2002). In contrast to the heterochromatin mutants, we see no synthetic growth phenotypes when mst1 is combined with mutants Δcbh1, Δmsc1, or Δhip1. This would be consistent with mst1+ acting in a pathway with these genes. Together with the segregation defects of mst1-L344S, these two hybrid interactions suggest that Mst1 may affect the centromere central core. We are examining mitotic phenotypes of mst1 in more detail to determine how this may occur and the role of H2AZ in this process.

There is indirect evidence that Mst1 may affect mitotic entry. We isolated the Skb1 methyltransferase in our two-hybrid screen. Budding yeast Hsl7, the ortholog of Skb1, was identified because mutations of hsl7 are synthetically lethal with histone tail mutants (Ma et al. 1996); genetic interactions between ScΔhsl7 and ScEsa1 suggest that histone tail modifications are important for normal mitosis when Swe1 kinase is activated (Ruault and Pillus 2006). While we observed no genetic interaction between SpΔskb1 and our temperature-sensitive allele of mst1, the physical interaction that we observed, together with the genetic data from budding yeast, suggests that there is a role for histone tail modification in the proper regulation of mitotic entry in both species. Another link to CDK activation is provided by our isolation of Hip1 as a putative interactor. The Δhip1 mutation is synthetically lethality with cdc25-22, which is a temperature-sensitive mutation in the mitotic inducer Cdc25 that opposes Wee1 (ScSwe1) (Blackwell et al. 2004; reviewed in Karlsson-Rosenthal and Millar 2006).

We did not isolate any of the NuA4 complex proteins in our two-hybrid screen, although the previously characterized SpAlp5/ScArp4 is known to interact with Mst1 by co-immunoprecipitation (Minoda et al. 2005). This is not surprising as the two-hybrid screen identifies binary associations, and many of the interactions in the large NuA4 complex are likely to be indirect (Auger et al. 2008). We speculate that the structure of the two-hybrid bait construct, in which the amino-terminus of Mst1 is fused to the DNA-binding domain of Gal4, may interfere with the assembly of NuA4 subunits with Mst1. Conversely, we did not observe co-immunoprecipitation of epitope-tagged Mst1 with most of the two-hybrid clones. This could reflect chromatin association of the proteins involved, temporally or spatially limiting association, or could indicate that the C-terminal epitope tag on Mst1 is not available when the protein is in a complex with other factors. A more detailed structure–function analysis and localization studies are in progress to resolve these observations.

The interactions that we observed, in addition to the phenotypes associated with the mst1 mutants, suggest that Mst1 affects a variety of cell functions. Importantly, the recent discovery that Tip60 can acetylate nonhistone proteins in the absence of the NuA4 complex (e.g., Sun et al. 2005; Sykes et al. 2006; Tang et al. 2006; Kim et al. 2007) indicates that the identification of Mst1 as a histone acetyltransferase may be too limiting a definition. We speculate that the pleiotropic effects of mst1 mutants indicate a variety of functions that may be directly modulated by Mst1 catalytic activity, not necessarily linked to histone acetylation. Our study suggests that Mst1 may have functions in replication fork stability and in centromere assembly, and experiments are underway to determine whether these effects are mediated by histone modifications or by other aspects of Mst1 function.

Acknowledgments

We thank Louise Clarke, Greg Freyer, Stevan Marcus, Paul Russell, Nancy Walworth, and Simon Whitehall for strains. We thank Vanessa Angeles and Paula Tran for assistance with genetics and growth assays; Su Lam for help with the two-hybrid analysis; Marc Green for microscopy assistance; and Jeff Hodson, John Marlett, and Ji-Ping Yuan for lab support. We thank Joaquin Espinosa for assistance with histone acetyltransferase assays. We are grateful to Ruben Petreaca for careful reading of the manuscript and to members of the Forsburg lab for many helpful comments throughout the course of the study. We thank Lorraine Pillus and the Pillus lab at the University of California, San Diego, for their hospitality to E.B.G. during part of this work. R.L.N. was supported by Department of Defense W81XWH-05-1-0391 (X. Chen). This work was supported by National Institutes of Health grant R01 GM059321 to S.L.F.

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