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Previous Article | Next Article 
Molecular and Cellular Biology, September 2003, p. 6086-6102, Vol. 23, No. 17
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.17.6086-6102.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, 91191 Gif-sur-Yvette, France,1 Department of Microbiology, University of Virginia, Charlottesville, Virginia 229082
Received 11 February 2003/ Returned for modification 10 April 2003/ Accepted 2 June 2003
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ABSTRACT |
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mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9 mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The N-terminal lysines of the histones are crucial functional targets of HATs (56, 63). Histone acetylation is thought to affect chromatin structure and gene expression by neutralizing the highly basic N-terminal tails of the histones and thereby decompacting chromatin (14). Specific patterns of histone acetylation, along with other histone modifications, also serve as recognition sites for the recruitment of particular proteins to chromatin (31). Histone acetylation is important in gene expression, DNA repair (24), and kinetochore function (15, 67). In budding yeast, the NuA4 HAT complex is responsible for the bulk of histone H4 acetylation on lysines 5, 8, and 12 (1, 12, 39, 54), whereas lysine-16 is mainly acetylated by the Sas2 HAT complex (34, 66). The NuA4 HAT complex is composed of ca. 12 subunits (1, 19, 50). Nine of these have been identified: Esa1 is the catalytic subunit of the complex; Tra1 belongs to the phosphatidylinositol kinase family; Act1 is the conventional yeast actin; Arp4 is an actin-like protein; and Epl1, Yng2, Eaf2, Eaf3, and Eaf4 are subunits with unknown biochemical functions. NuA4 has been implicated in transcriptional activation (19, 54) and DNA double-strand break repair (4). Ynl107/Yaf9, a novel subunit of NuA4 (see below), has also been shown to be required for UV resistance (5). In the present study, we show that Ynl107/Yaf9, Yng2, and acetylated histone H4 are required for the cellular resistance to spindle stress. Complexes resembling NuA4 are found in animal cells, and they have been implicated in transcriptional regulation and DNA repair (28, 56, 63). Given these similarities, it is possible that these complexes also participate in the response of animal cells to spindle stress.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic lethality and complementation. Thermosensitive bbp1-1 and spc24-11 mutants were transformed by URA3 plasmids containing the wild-type alleles of each gene (pli36 and pli39, respectively). The entire YAF9 coding sequence was then replaced with the KanMX6 cassette (40) in each of the complemented strains. The double-mutant strains containing the wild-type allele of the thermosensitive mutants on a YCp-URA3 plasmid were then plated on medium lacking uracil or containing 5-fluoroorotic acid (5-FOA) (6) at the permissive temperature of 24°C in order to test for the ability of strains containing the double mutants to survive in the absence of a plasmid complementing the temperature-sensitive mutation.
Spindle sensitivity.
Exponentially growing wild-type (YPH499) and ILM162 (yaf9) strains in yeast extract-peptone-dextrose (YPD) medium containing 50 µg of adenine/ml were treated with nocodazole at 7.5 or 15 µg/ml. Once per hour for 6 h, aliquots were taken and analyzed to determine the percentage of rebudded cells and the cell viability. An aliquot of cells was also fixed with 70% ethanol for fluorescence-activated cell-sorting analysis (27).
Gel filtration analysis. A total of 50 ml of exponentially growing YPH499 cells at an optical density at 600 nm of 0.5 in YPD was washed with PBS, resuspended in 4 ml of PBS plus Complete protease inhibitor cocktail (Roche Biochemicals), and broken in an Eaton press. The lysate was then centrifuged for 15 min at 4°C in an Eppendorf centrifuge, and the supernatant was passed through a 0.45-µm (pore-size) Nalgene filter. Then, 100 µl of filtered protein extract containing 300 µg of protein was applied to a Superdex 200 gel filtration column on a Pharmacia SMART analytical chromatography apparatus. Next, 50-µl fractions were collected, and the elution positions of Yaf9 and Esa1 were determined by immunoblotting and compared to the elution positions of a standard set of marker proteins with known molecular masses.
Immunoblots with antibodies to acetyl-lysine-histone H4. Cultures (10 ml) of yeast cells were centrifuged and washed once with water, and the pellets were then resuspended in 300 µl of HSB lysis buffer (45 mM HEPES-KOH [pH 7.4], 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% NP-40, Complete protease inhibitor cocktail). Then, 250 µl of acid-washed glass beads was added, and the cells were lysed with a Bead-Beater. The extract was spun in an Eppendorf centrifuge for 10 min at 4°C, and the supernatant was recovered. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline plus 0.1% Tween (TBST) containing 5% nonfat powdered milk and then incubated with a 1/1,000 dilution of anti-hyperacetylated histone H4 (Penta) rabbit antibodies from Upstate Biotechnology (catalog no. 06-946) for 1 h at room temperature. Membranes were washed with TBST and then incubated with a 1/3,000 dilution of a goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase in TBS-5% milk for 1 h at room temperature. After a wash with TBST, the immunoblots were visualized with enhanced chemiluminescence (Amersham-Pharmacia).
Coimmunoprecipitation analyses. Exponentially growing yeast cell cultures (400 ml) in YPD at an optical density at 600 nm of 0.6 were centrifuged, washed once with cold water, and resuspended in 0.7 ml of immunoprecipitation (IP) buffer (50 mM Tris-Cl [pH 7.5], 100 mM NaCl, 10 mM EDTA, 15% glycerol, Complete protease inhibitor cocktail), and the cells were broken in an Eaton press. Triton X-100 was then added to 0.1%, and the lysate was centrifuged for 15 min at 4°C in an Eppendorf centrifuge. Next, 360 µg of protein extract in 100 µl of IP buffer plus 0.1% Triton X-100 were mixed with 40 µl of Dynal magnetic Dynabeads Pan-Mouse IgG saturated with either 12CA5 anti-HA monoclonal antibodies, 9E10 anti-myc monoclonal antibodies, or no antibody, followed by incubation overnight at 4°C. The beads were washed three times with 1 ml of IP buffer plus 0.1% Trition X-100 and then heated at 94°C for 5 min in SDS-PAGE sample buffer to release immunoprecipitated proteins.
Quantification of Western blots. Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes. After incubation with appropriate antibodies, the Western blots were developed by using enhanced chemiluminescence plus reagents (Amersham-Pharmacia); the chemiluminescent images were acquired and quantified with a Fluorochem Imager from Alpha Innotech, Inc. This imager uses a photographic lens and a cooled charge-coupled device camera to quantitatively capture and digitize the chemiluminescent signal.
Microarray analyses.
Exponentially growing wild-type (YPH499) and ILM162 (yaf9) strains in YPD media containing 50 µg of adenine/ml were treated with 15 µg of nocodazole/ml until >90% of cells accumulated with a large bud (ca. 2.5 h). Cells were then harvested, and RNA was prepared by using a Qiagen RNeasy kit after the cells were broken with an Eaton press. A 12.5-mg portion of total RNA was used as a template for double-stranded cDNA synthesis. Direct labeling of cDNA was performed during the synthesis as previously described (17). Labeled cDNA was hybridized to yeast genome arrays produced by the Service de Génomique Fonctionnelle-CEA/Evry. Hybridized arrays were scanned with a GenePix 4000A scanner (Axon Instruments, Inc.), and fluorescence ratio measurements were determined with the GenePix Pro 3.0 software (Axon Instruments). From five independent arrays, we chose open reading frames (ORFs) showing a modified signal on at least three of the five arrays, in the same direction and with a wild-type/mutant ratio differing by a factor of 2.5 or more. (These ORFs are listed in Fig. 8A.) The microarray results were confirmed by quantitative reverse transcription-PCR (RT-PCR) analysis of total RNA as described previously (22). The ACT1 mRNA was used as a normalization standard for each amplification reaction. Oligonucleotides were chosen for each ORF to give an amplification product of ca. 400 bp.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mutant is synthetically lethal with mutants of the mitotic apparatus and is hypersensitive to MT depolymerizing agents.
YAF9 is a nonessential yeast gene, and the yaf9 mutant showed little or no growth defect relative to the wild type at 24 to 30°C but did grow more slowly than the wild type at 16°C and was temperature sensitive for growth at 37°C in the W303 strain background but not in the YPH or BY strain backgrounds (data not shown). Since Yaf9 interacted with Mps2 in our two-hybrid screen, we sought genetic interactions between yaf9 and mutants of the mitotic apparatus. The bbp1-1 mutant is unable to insert a newly synthesized SPB into the nuclear envelope at 37°C (60), whereas the spc24-11 mutant has a kinetochore defect at 37°C (36). The yaf9 mutant was found to be synthetically lethal when combined at the permissive temperature of 24°C with either the bbp1-1 mutant or the spc24-11 mutant (Fig. 3A and B) Synthetic lethality was thus observed with mutants affecting the function of both the SPB and the kinetochore. This result suggested that Yaf9 might be required for viability in conditions in which the structural integrity of the spindle was compromised. In support of this notion, we found that the yaf9 mutant was hypersensitive to growth on media containing the MT depolymerizing agent benomyl (Fig. 3C). We studied the response of the mutant in liquid cultures to another MT depolymerizing agent, nocodazole, in order to better characterize its defect. yaf9 cells lost viability more rapidly than the congenic wild type in the presence of nocodazole (Fig. 4A) and showed a slight increase in the frequency of multibudded cells (Fig. 4B). However, the loss of viability and the frequency of rebudding were less elevated than for spindle checkpoint mutants (data not shown) (64, 69). Flow cytometric analyses showed that the haploid yaf9 mutant accumulated with a 2C DNA content after 1 h of treatment with 7.5 µg of nocodazole/ml, whereas the wild-type strain showed only a slight and transient response at this concentration (Fig. 4C). The yaf9 mutant is thus hypersensitive to both nocodazole and benomyl. After 4 h of incubation in the presence of 7.5 µg of nocodazole/ml, the yaf9 cells escaped from the mitotic arrest, and the flow cytometry profiles suggested that some chromosome mis-segregation might have occurred to give rise to a small fraction of cells with a less than 1C and greater than 2C DNA content. When treated with 15 µg of nocodazole/ml, both the yaf9 mutant and the wild type remained arrested with a 2C DNA content over the 6-h period of the experiment (Fig. 4C). This result indicated that the nocodazole sensitivity of the yaf9 mutant was not due to a defect in the cell cycle arrest mediated by the spindle checkpoint but was likely due to a defect in the cellular response to MT depolymerization at the level of recovery from spindle stress. Further support of this interpretation was obtained by visualizing MTs in living cells expressing a GFP-Tub1 fusion protein that has previously been used to characterize spindle dynamics in living cells (65). Expression of GFP-Tub1 did not adversely affect the growth of the yaf9 mutant. Wild-type and yaf9 cells expressing GFP-Tub1 were treated with 3, 7.5, and 15 µg of nocodazole/ml for 5 h. At each hour, an aliquot of living cells was observed, and the percentage of large-budded cells with depolymerized MTs and no visible focus of tubulin staining at the spindle pole bodies was quantified (Fig. 5A) An example of wild-type and yaf9 cells expressing GFP-Tub1 and treated with 3 µg of nocodazole/ml for 3 h is shown in Fig. 5B. At each concentration of nocodazole, a higher percentage of the yaf9 cells contained disassembled spindles compared to the wild type. Moreover, at lower concentrations of nocodazole, a transient depolymerization of MTs was seen in the wild type, whereas the recovery of MTs was inhibited in the yaf9 mutant. Yaf9 is thus required for a normal level of resistance of yeast cells to MT depolymerizing agents.
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1-MDa complex and in low-molecular-weight fractions that may correspond to the monomeric protein (Fig. 6A). Esa1, the catalytic subunit of the NuA4 HAT complex, cofractionated with Yaf9 in the high-molecular-weight fractions. This result was consistent with Yaf9 being a subunit of the NuA4 complex with Esa1. We further verified the presence of Yaf9 in the NuA4 complex by testing for the coimmunoprecipitation of Yaf9 with Esa1, the catalytic subunit of the NuA4 complex, as well as with two other subunits of the complex: Yng2 (11, 39, 50) and Epl1 (19). Western blotting showed that both Yaf9 and Esa1 coprecipitated with Yng2-13myc and with Epl1-3HA (Fig. 6B). Likewise, Esa1 was found to coprecipitate with Yaf9-3HA. The combined data strongly suggest that Yaf9 is a previously unidentified subunit of NuA4.
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mutant does not show a global reduction of acetylated histone H4.
We tested the effect of the yaf9 mutant on NuA4 function by immunoblotting protein extracts from wild-type and mutant strains with an antibody that recognizes hyperacetylated histone H4. This antibody recognizes tetra-acetylated histone H4, and it may recognize triacetylated forms. The yaf9 mutant did not show significantly reduced amounts of hyperacetylated histone H4 compared to the wild type at either 30°C or 37°C (Fig. 6C). In contrast, two temperature-sensitive mutants of the Esa1 catalytic subunit of NuA4 showed strong defects, as previously reported (4). The esa1-L357H mutant had normal levels of hyperacetylated histone H4 at 30°C but much less than the wild type after transfer to 37°C. The esa1-1851 mutant showed drastically reduced levels of hyperacetylated histone H4 at both 30 and 37°C. These results indicated that Yaf9 is not required for global H4 acetylation, but they did not exclude the possibility that it might be required to target the NuA4 complex to some specific genomic locations or for the acetylation of some other substrate. Yaf9 is a nuclear protein whose levels are increased when cells are treated with nocodazole. The intracellular localization of Yaf9 was determined in living cells by using a Yaf9-GFP fusion protein and in fixed cells by immunofluorescence by using a Yaf9-3HA epitope-tagged protein. The Yaf9-GFP and Yaf9-3HA proteins appeared to be functionally normal in that cells expressing these fusion proteins in place of the wild-type Yaf9 had the same resistance to benomyl as had the wild type (data not shown). Localization was observed in wild-type cells in the absence or the presence of nocodazole and in an mps2-2 mutant at the permissive and restrictive temperatures. The mps2-2 mutant is unable to insert a duplicated SPB into the nuclear envelope and thus gives rise to cells with monopolar spindles at 37°C (47). A weak intranuclear localization was seen for Yaf9-GFP in an mps2-2 mutant at the permissive temperature of 24°C (Fig. 7A) as well as in the isogenic wild type created by transforming the mps2-2 mutant with a plasmid containing the wild-type MPS2 gene (Fig. 7C). Yaf9-GFP remained nuclear in these cells at the restrictive temperature of 37°C; however, the intensity of the Yaf9-GFP signal increased in the mps2-2 mutant after incubation at 37°C for 3 h (Fig. 7B) but not in the isogenic wild type (Fig. 7D). The immunofluorescence localization of Yaf9-3HA was only weakly visible in wild-type cells (data not shown). However, when these cells were incubated in nocodazole for 3 h, the nuclear localization of Yaf9-3HA was readily apparent (Fig. 7E). The combined results on the intracellular localization of Yaf9 suggested that it was a nuclear protein whose levels increased in conditions in which the integrity of the mitotic spindle was affected. We determined more precisely the quantity of Yaf9 in nocodazole-treated cells by immunoblotting. Yaf9 levels increased 2.5-fold within 2 h of treatment with nocodazole (Fig. 7F). In contrast, the levels of the Esa1 catalytic subunit of the NuA4 complex remained constant in the presence of nocodazole (Fig. 7F). Thus, Yaf9 protein levels seem to be increased in response to spindle stress.
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mutant.
ESA1, the gene encoding the catalytic acetyltransferase subunit of NuA4, is essential for yeast cell viability (12). Analysis of esa1 conditional mutants has shown that it is required for the bulk of histone H4 N-terminal acetylation and for the correct expression of a subset of yeast genes (19, 39, 54). Since Yaf9 is a nonessential subunit of NuA4, it cannot be required for all NuA4 functions. We used yeast genome microarrays to explore any differences in the transcriptomes of yaf9 and wild-type cells that were treated with nocodazole for 3 h, since perturbations in gene expression could contribute to the phenotypes of the yaf9 mutant. This analysis identified 11 genes whose expression was reduced at least 2.5-fold in the yaf9 mutant compared to the wild type (including the YAF9 gene itself, as is expected since this sequence is deleted in the yaf9 strain) and 12 genes whose expression was elevated at least threefold in the yaf9 mutant compared to the wild type (Fig. 8A). We used the RT-PCR to verify the microarray results for 10 genes that were hypoexpressed in the yaf9 mutant relative to the wild type (Fig. 8B). RT-PCR analysis confirmed the results of the microarray results and showed that most of the genes that were underexpressed in yaf9 in the presence of nocodazole were also underexpressed in the mutant in the absence of nocodazole. However, one gene (AHP1/YLR109w encoding alkyl hydroxyperoxidase reductase) seemed to be expressed normally in the yaf9 mutant in the absence of nocodazole but was underexpressed in the presence of nocodazole. AHP1 thus seems to be induced in the wild type in the presence of nocodazole in a manner dependent on Yaf9.
None of the genes whose expression is affected in the yaf9 mutant encode proteins that are obviously related to MT stability. Nine of the ten genes that are hypoexpressed in the yaf9 mutant relative to the wild type are nonessential for viability, including AHP1. We thus tested the benomyl sensitivity of individual yeast strains from which each of these genes had been deleted. All nine mutant strains had a wild-type level of benomyl resistance (data not shown). Individual underexpression of these genes in the yaf9 mutant thus cannot be responsible for its benomyl sensitivity. The essential YLR424W gene that is also underexpressed in the yaf9 mutant encodes a protein that copurifies with a large mRNA splicing complex that contains the Cef1 protein (20). Interestingly, cef1 mutants are hypersensitive to benomyl at their permissive temperature and arrest preferentially in mitosis at their restrictive temperature principally due to defects in the splicing of the intron from the TUB1 gene encoding the major isoform of alpha tubulin (7). Replacing the wild-type TUB1 gene with an intronless version of the gene suppressed these phenotypes in the cef1 mutant. However, replacing the wild-type TUB1 gene with an intronless version of the gene did not suppress the benomyl sensitivity of the yaf9 mutant (data not shown). Thus, gene expression defects may not be responsible for this phenotype.
The yng2 mutant is sensitive to benomyl, and the benomyl resistance of the wild-type strain is increased by TSA, an inhibitor of HDACs.
The sensitivity of the yaf9 mutant might be due to a defect in NuA4 complex function, or it might be due to a function that is specific to Yaf9 and distinct from any possible function it might have as part of the NuA4 complex. We examined three other NuA4 mutants—two different esa1 temperature-sensitive mutants (4) and a yng2 mutant (11)—for their sensitivity to benomyl in order to test for a possible function of NuA4 in the cellular response to MT depolymerization. The esa1-1851 mutant was hypersensitive to benomyl at 30°C, whereas the esa1-L357H mutant was not (Fig. 9A and B). Thus, reduced acetylated histone H4 levels in the esa1-1851 mutant (Fig. 6C) is correlated with hypersensitivity to benomyl. The yng2 mutant was also sensitive to benomyl (Fig. 9C and E). Global histone H4 acetylation is reduced in the yng2 mutant, and these cells grow more poorly than in the wild type, but Yng2 is not required for viability (11, 39, 50). The sensitivity of the esa1-1851 and yng2 mutants to benomyl suggests that NuA4 acetylation of some substrate is required for normal cellular resistance to benomyl. This interpretation is supported by the effects of TSA, an inhibitor of histone deacetylases (HDACs) (71), on the resistance of yeast cells to benomyl. The resistance of wild-type yeast cells to benomyl was increased when cells were grown in the presence of 30 µg of TSA/ml (Fig. 9E to G). This result suggests that the hyperacetylation of some cellular protein or proteins increases the resistance of wild-type yeast cells to benomyl. TSA has been shown to partially suppress the slow growth phenotype, the G2/M accumulation, and the histone H4 hypoacetylation of the yng2 mutant (11). We thus examined the effect of TSA on the benomyl sensitivity of the yng2 and yaf9 mutants. As for its other known phenotypes, TSA suppressed the benomyl hypersensitivity of the yng2 mutant (Fig. 9E and F). Remarkably, however, the opposite effect was observed with regards to the yaf9 mutant: TSA inhibited its growth in the absence of benomyl and did not suppress its sensitivity to benomyl (Fig. 9C to F). The benomyl sensitivity of the yaf9, yng2, and esa1-1851 mutants suggests that they have common defects that cause this phenotype, but the opposing response of the yaf9 and yng2 mutants to TSA indicates that they must also have some distinct biochemical functions.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/yaf9 mutant has little or no growth defect at 24 to 30°C. Thus, Ynl107/Yaf9 is not necessary for the essential functions of NuA4. Moreover, the global levels of histone H4 acetylation were not significantly diminished in the yaf9 mutant (Fig. 6C). One interesting possibility for Yaf9 function is that it may target the NuA4 complex to acetylate histone H4 at some specific genomic locations. This idea could by tested by genome-wide chromatin IP experiments (55). Ynl107 was named Yaf9 because of its similarity to human AF9, a protein that induces acute leukemia when fused to the N-terminal portion of the MLL gene product (13). However, this similarity is restricted to the YEATS domain of the two proteins, and our sequence analysis and the predicted secondary structure of Ynl107 indicates that the human Gas41 and its metazoan homologs are more similar to Ynl107 than are the other YEATS domain proteins. Gas41 is a sequence that was identified by its amplification in human gliomas (18, 46). It will be of great interest to determine whether Gas41 is a subunit of a NuA4-like HAT complex in animal cells and whether YEATS domain proteins are generally associated with HAT complexes.
Yaf9, the NuA4 HAT complex, and histone H4 acetylation are required for normal cellular resistance to spindle stress.
We isolated Yaf9 in a two-hybrid screen with Mps2 as bait (36). Mps2 is a component of the SPB and the nuclear envelope that is required for insertion of the newly duplicated SPB into the nuclear envelope (47). It is thus necessary for establishing a normal bipolar spindle. We sought to test the physiological significance of this interaction and discovered that the yaf9 mutant is synthetically lethal with two mitotic mutants that we tested, i.e., the bbp1-1 and spc24-11 mutants. Remarkably, these mutants affect two distinct mitotic functions: Bbp1 is required along with Mps2 for insertion of the SPB into the nuclear envelope (60), whereas Spc24 is a kinetochore subunit that is required for stable binding of kinetochores to spindle MTs (30, 36, 70). Furthermore, we discovered that the yaf9 mutant is hypersensitive to MT depolymerizing agents such as nocodazole and benomyl. These results show that Yaf9 is required for viability under conditions in which the integrity of the mitotic spindle is compromised.
Interestingly, Gas41, the human protein that is most similar to Yaf9, has been found to interact with the NumA (23) and Tacc1 (35) proteins. In animal cells in mitosis, NumA is concentrated near centrosomes and helps organize the minus ends of spindle MTs (44). Tacc1 was also implicated in spindle structure (21). Given the intriguing similarities in the interactions between Yaf9-Mps2 and Gas41-NumA+Tacc1, it would be worthwhile to test for a possible role for Gas41 in responding to spindle stress in animal cells.
Phenotypic analysis of the yaf9 mutant indicates that its sensitivity to MT depolymerizing agents is not due to an obvious defect in the mitotic spindle checkpoint pathways. The yaf9 mutant does not exit mitosis prematurely in the presence of nocodazole as do the spindle checkpoint mutants. Instead, the MTs of the yaf9 mutant disassemble more readily than those of the wild type at the same concentration of nocodazole, and reassembly of MTs in the presence of limiting concentrations of nocodazole is inhibited in the yaf9 mutant. Furthermore, the yaf9 mutant is blocked in mitosis at lower concentrations of nocodazole than the wild type and recovers from this mitotic arrest less efficiently. Since Yaf9 is a subunit of the NuA4 histone acetylase complex, we tested the need of this complex for benomyl resistance by examining other NuA4 mutants. We found that the esa1-1851 and yng2 mutants are also hypersensitive to benomyl, and these mutants are known to have reduced levels of histone H4 acetylation (4, 11, 39, 50). Furthermore, we found that nonacetylatable histone H4 mutants were also hypersensitive to benomyl. In these mutants, the four N-terminal lysines of histone H4 were mutated to glutamine or to glutamine plus arginine. The similarity of these mutants to the NuA4 mutants strongly suggests that histone H4 is the relevant substrate that must be acetylated by NuA4 in order to confer a cellular resistance to benomyl. Yaf9 may target the NuA4 complex to specific genomic sites where histone H4 acetylation is important for responding to mitotic stress.
There are several possible ways to explain the sensitivity of NuA4 mutants and nonacetylatable histone H4 mutants to spindle stress. One possibility is through defects in the expression of genes encoding proteins involved in MT assembly or structure, since histone H4 acetylation is implicated in transcriptional regulation (56, 63). We identified 10 genes that were underexpressed in the yaf9 mutant relative to the wild type and 12 genes that were overexpressed. None of these genes are obviously involved in spindle functions, and we directly tested and ruled out a phenotypic contribution for the 10 genes that are hypoexpressed in the yaf9 mutant. Although we cannot exclude an effect of perturbed gene expression on the benomyl hypersensitivity of yaf9, our current data do not favor this model.
Another possibility is that the acetylation of histone H4 is required for the recruitment of specific proteins to chromatin (31), some of which may be necessary to form a structurally resistant spindle. Recruitment of the Ran GTPase and its GDP/GTP exchange factor Rcc1 to chromatin is important for proper mitotic spindle assembly in Xenopus extracts and human cells (10, 45). Ran and its regulators also influence spindle structure and function in budding yeast (52, 53), fission yeast (57), and worms (2). Ran binds chromatin directly through interactions with histones H3 and H4 and indirectly through binding to Rcc1 that interacts directly with histones H2a and H2b (3, 49). However, docking of Ran to chromatin does not appear to require the N-terminal extremities of histones H3 and H4 (3), which suggests that its chromatin binding might not be affected in NuA4 mutants or the nonacetylatable histone H4 mutant. Another possibility is that centromeric histone acetylation directly or indirectly affects kinetochore-MT interactions. In budding yeast, we found that treatment of wild-type yeast cells with TSA, an HDAC inhibitor, increases their resistance to benomyl. Thus, the degree of acetylation of some protein in budding yeast, presumably histone H4, is limiting its resistance to benomyl. This result is in contrast to the effect of TSA in fission yeast (15) and animal (67) cells. In the latter two cases, TSA treatment perturbs the heterochromatic centromeric chromatin and leads to increased rates of chromosome loss. Budding yeast centromeres are smaller and simpler than those of fission yeast and animal cells. The absence of extensive heterochromatin around the budding yeast centromeres may have allowed us to observe a positive effect of protein acetylation on spindle stability. In eukaryotes containing extensive centromeric heterochromatin, kinetochore perturbation by histone hyperacetylation of centromeric heterochromatin may be dominant to any potential beneficial effects of TSA treatment on spindle stability. Determining the exact mechanism by which protein acetylation increases spindle stability in budding yeast will allow us to test for its conservation in other eukaryotes.
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ACKNOWLEDGMENTS |
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I.L.M. was supported by an allocation from the French Ministère de l'Education Nationale, de la Recherche, et de la Technologie and by stipend support from the Association pour la Recherche sur le Cancer (ARC). M.M.S. was supported by NIH grant GM28920, and C.M. received funding from the Association pour la Recherche sur le Cancer (ARC 4470) and from a collaborative program between the CEA and the Curie Institute on epigenetic parameters in the response to genotoxic agents and the control of the cell cycle.
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FOOTNOTES |
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" and "ß" indicate the position of alpha-helical and ß-sheet secondary structures for Yaf9 as predicted by the HCA analysis. Blue brackets show the positions of predicted coiled-coil sequences in the C-terminal regions of Yaf9, Gas41, and AF9. The global analysis of the sequences shows that these proteins are composed of two domains: a similar N-terminal region that corresponds to the YEATS domain and a C-terminal region that is more divergent. For Yaf9, Taf14, and AF9, a linker region could be clearly seen between these two domains. Examination of the HCA plots of human Gas41, AF9 and S. cerevisiae Yaf9, Taf14, and Sas5 sequences reveals the presence of similar hydrophobic motifs in the YEATS domain. The boundaries of this domain in the different proteins are shown with a dotted green line. Residues 55 to 100 of the Yaf9 sequence indicate an extremely conserved region shown within the dotted red lines. This central part of the first domain of the proteins was found to be characteristic of the YEATS domain family. The CLUSTALW (Fig. 
