ABSTRACT
The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Δ phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.
In eukaryotes, DNA is packaged into chromatin, a nucleoprotein structure consisting of DNA, histones, and nonhistone proteins. The assembly of DNA into chromatin modulates the access of cellular machinery to DNA and thus regulates transcription, replication, repair, and recombination. Chromatin structure can be regulated by the addition of posttranslational modifications to histones, including acetylation, methylation, phosphorylation, and ubiquitination. Consistent with this, several histone posttranslational modifications have been linked to the regulation of gene expression.
Numerous multiprotein complexes are involved in the posttranslational modification of histones. These complexes vary in both their protein components and the modifications they effect. The most well-studied group of modifying complexes is the histone acetyltransferases (HATs), which use acetyl coenzyme A as a substrate for the acetylation of lysine residues within both the tail and globular domains of histones (39, 43, 45, 73). In the budding yeast Saccharomyces cerevisiae, there are at least eight proteins that have been identified as having HAT activity in vitro, including Gcn5p, Hat1p, Esa1p, Elp3p, Nut1p, Hpa2p, Sas2p, and Sas3p (45, 73), although for many of these proteins, whether histones represent their true substrates in vivo is not known. Three of these proteins are found in complexes that specifically acetylate the lysines within the tail of histone H3, including the GCN5-dependent SAGA, SLIK/SALSA, ADA, and HAT-A2 complexes (12, 16, 49, 58, 60), the ELP3-dependent elongator complex (70), and the SAS3-dependent NuA3 complex (25). The acetylation of histone tails is associated with regions of transcriptional activity, and histone acetylation is thought to modulate chromatin structure through two different mechanisms. First, the neutralization of charge associated with histone acetylation is believed to relax the structure of chromatin, allowing access of other factors to nucleosomal DNA (11). Second, histone acetylation has been shown to regulate the binding of chromatin-modifying factors to nucleosomes (9).
In a manner analogous to acetylation, histones are also modified by the addition of methyl groups to lysine and arginine residues within the amino-terminal tails of histones H3 and H4 (76) and within the globular domain of histone H3 (14, 32, 65). Although histone methylation has been linked to transcriptional silencing in other organisms (18), in S. cerevisiae, methylation is found associated with regions of transcriptional activity (46). Consistent with this, two yeast histone methyltransferases, Set1p and Set2p, bearing specificity toward histone H3 lysine residues 4 and 36, respectively, are found associated with transcriptionally active regions of the genome (29, 41). Set1p and Set2p are found associated with RNA polymerase II (RNAPII), and these interactions are dependent on the phosphorylation of the carboxyl-terminal domain of Rpb1p, the largest subunit of RNAPII (20). The interaction of Set1p with the carboxyl-terminal domain requires the phosphorylation of serine 5 by Kin28p, a TFIIH-associated kinase that mediates the transition between transcription initiation and elongation (41). In contrast, Set2p interacts with RNAPII that has been phosphorylated on serine 2, which is performed primarily by Ctk1p at the later stages of elongation (29, 33, 34, 56, 72). Consequently, the level of H3 lysine 4 trimethylation peaks at the promoters and early coding regions of genes (40, 41, 46, 54), whereas H3 lysine 36 di- and trimethylations are spread throughout the open reading frames (29, 40, 46, 56, 72). Recently, methylation of the histone H3 tail has been implicated in mediating the interaction of several HAT complexes (40, 48) and one histone deacetylase complex (HDAC) (8, 26) with nucleosomes. Thus, it seems an attractive possibility that the methylation of specific lysine residues within histone H3 serves a universal role in mediating histone acetylation.
NuA3 is an approximately 400-kDa acetyltransferase complex containing the catalytic subunit Sas3p, in addition to at least four other subunits (22, 25). NuA3 was originally identified on the basis of its ability to acetylate histone H3 in vitro, but whether NuA3 is capable of acetylating histones in vivo is unknown. Deletion of SAS3 is synthetically lethal with the loss of Gcn5p, the catalytic subunit of several histone H3-specific HAT complexes. However, this synthetic lethality is not due to the loss of any of the known Gcn5p-dependent HATs, suggesting that Gcn5p may have a function that is unrelated to histone acetylation (21). To understand the essential function of NuA3 in the cell, we addressed whether this complex is truly acting as a HAT in vivo and asked how it is recruited to the histones that it modifies. With a chromatin pulldown assay, we were able to demonstrate an interaction between NuA3 and chromatin and found that this interaction is dependent on the H3 tail. Consistent with the importance of this interaction for NuA3 function, we found that loss of the H3 tail recapitulates a unique sas3Δ-associated phenotype. In addition, we provide evidence that SET1- and SET2-dependent methylation of histone H3 lysines 4 and 36, respectively, is required for NuA3 interaction with chromatin and for NuA3 function in vivo. These data, together with observations that histone H3 methylation mediates the acetylation of histones by other HAT complexes (40, 48), suggest that SET1- and SET2-dependent methylation may serve as universal marks for targeting histone acetylation to active regions of the genome.
MATERIALS AND METHODS
Yeast strains, plasmids, and genetic methods.All of the strains used in this study (Table 1) are isogenic to S288C. Yeast culture and genetic manipulations were carried out by standard protocols (2, 37, 50). Genomic deletions or epitope tag integrations were verified by PCR analysis. Mating assays were performed as described by others (59). Briefly, strains were patched onto yeast extract-peptone-dextrose medium and grown overnight at 30°C. Cells were replica plated onto a lawn of YLH101 on yeast extract-peptone-dextrose medium and allowed to mate overnight at 30°C. Finally, plates were replica plated onto medium lacking tryptophan and adenine to select for diploids and incubated at 30°C overnight. Strains carrying histone H3 mutations were derived by plasmid shuffle from FY2162 (YLH224) (10). This strain carries deletions of the HHT1-HHF1 and HHT2-HHF2 loci and carries HHT2-HHF2 on a URA3 plasmid (pHHT2). Because this strain and all of the strains derived from it carry a wild-type version of HHF2 on a plasmid, for simplicity, the genotypes of these strains will be referred to as hht1Δ hht2Δ in the figures and figure legends. Plasmid pHHT2 was constructed by ligation of the SpeI fragment from pDM18 (10) into the SpeI site of pRS414. phht2Δ3-29 was generated by ligating annealed phosphorylated oligonucleotides (5′-GATCCAAGCAAACACTCCACAATGGCCAGACCATCTA-3′ and 5′-CCGGTAGATGGTCTGGCCATTGTGGAGTGTTTGCTTG-3′) into the BamHI and AgeI sites of pHHT2. Plasmids containing mutations in HHT2 were generated with the Stratagene QuikChange site-directed mutagenesis kit. Plasmid pSAS3 (pLP640) consists of the SAS3 coding region, 1,300 bp of upstream and 100 bp of downstream sequences inserted into pRS316 (21).
Strains used in this study
Chromatin pulldown assays.All chromatin pulldown assays were performed with yeast whole-cell extracts prepared from mid-log-phase cells in IPP150 buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin A). Input protein amounts were normalized by Bradford protein assay, and 5 to 10 mg of protein was incubated with 10 μl of immunoglobulin G (IgG) Sepharose 6 Fast Flow resin (Amersham Biosciences, Piscataway, NJ). Samples were rotated at 4°C for 2 h, and the resin was washed five times with 20 volumes of cold IPP150 buffer. Bound proteins were eluted by boiling in sodium dodecyl sulfate sample buffer and analyzed by immunoblotting with antihemagglutinin (anti-HA)-peroxidase (Roche Diagnostics Canada, Laval, Quebec, Canada), peroxidase-antiperoxidase (Sigma Chemical Co., St. Louis, MO), or anti-diacetylated histone H3 (Upstate Biotechnology, Lake Placid, NY) antibodies.
RESULTS
NuA3 interacts with chromatin in vivo.Previously it was shown that the NuA3 acetyltransferase can acetylate histones in vitro (25); however, the possibility exists that NuA3 is acetylating a nonhistone substrate in vivo. To provide further support that NuA3 is acetylating histones, we asked whether NuA3 interacts with chromatin in vivo. To this end, we introduced a tandem affinity purification (TAP) tag to the carboxyl terminus of H2B (Htb1p) to enable us to purify native chromatin with IgG resin. Because of the presence of newly synthesized H2B in the cytoplasm, it was not expected for all of the TAP-tagged H2B that is bound by the IgG resin to be incorporated into nucleosomes. To ensure that at least some of the precipitated H2B is associated with nucleosomes, we immunoblotted precipitated material with anti-acetylated H3 antibodies. As shown in Fig. 1A, the Htb1TAP-tagged strain was capable of pulling down acetylated histone H3. Most of the published data support the hypothesis that nucleosome assembly occurs in a stepwise manner that involves the initial deposition of an H3-H4 tetramer on the newly synthesized DNA, followed by deposition of H2A-H2B dimers (19). If this is the case, then one would have to assume that histones H2B and H3 would not exist in a complex other than in the form of the nucleosome. The fact that we are able to copurify histone H3 with H2B indicates that our TAP-tagged Htb1p strain is capable of pulling down intact nucleosomes.
NuA3 interacts with chromatin in vivo. Chromatin pulldown assays were performed with strains YDM126 and YDM127 (A and B) and strains YDM126, YLH139 YDM127, and YDM210 (C), and the resulting samples were subjected to Western blotting with immunodetection with anti-diacetylated histone H3 antibodies (AcH3), anti-HA antibodies (Sas3HA), or peroxidase-antiperoxidase antibodies (Htb1TAP). Yeast whole-cell extract (1.4% of input) was also blotted for HA (INPUT).
To determine whether NuA3 is capable of binding chromatin, we HA tagged Sas3p, the catalytic subunit of NuA3, in our Htb1TAP strain. Chromatin pulldown experiments were performed with IgG resin, followed by anti-HA immunoblot analysis for Sas3p. Supporting previous in vitro evidence that NuA3 is capable of binding nucleosomes (22), we found that Sas3p coprecipitated with Htb1p in the TAP-tagged strain but not the untagged strain (Fig. 1B). Additionally, we were also able to coprecipitate Nto1p, another component of NuA3 (J. M. Dukowski et al., unpublished data), indicating that the entire NuA3 complex, and not just Sas3p, is capable of binding chromatin (Fig. 1C).
NuA3 function is dependent on the amino-terminal tail of histone H3.To provide further evidence that NuA3 is indeed interacting with chromatin in vivo, we mapped the region of the nucleosome that serves as an interaction site for NuA3 and asked whether loss of this site creates a sas3Δ-specific phenotype. In light of the fact that NuA3 is an H3-specific HAT, we surmised that a likely interacting target would be the H3 tail itself (25). To this end, we developed a mutant strain lacking amino acids 3 to 29 of the H3 tail (hht2Δ3-29) and performed chromatin pulldown assays. Samples were normalized for levels of Htb1TAP in the pulldown material, and Fig. 2A shows that coprecipitation of Sas3p with Htb1 TAP was abolished in the H3 tailless mutant. The loss of interaction seen in the H3 tailless mutant indicates that the H3 tail is required for NuA3 to bind chromatin. We next asked whether loss of the H3 tail recapitulates the phenotype of a sas3Δ mutant. It has been shown that deletion of SAS3 alone results in only minor phenotypes (51). However, if SAS3 is deleted in conjunction with deletion of GCN5, the result is a synthetic lethality (21). Gcn5p is present in multiple HAT complexes in yeast, including SAGA, SLIK/SALSA, ADA, and HAT-A2, each of which contains both unique and shared protein subunits (16, 47, 49, 52). In addition to Gcn5p, the SAGA, SLIK/SALSA, ADA, and HAT-A2 complexes also share Ada2p, and experimental evidence suggests that Ada2p is required to potentiate Gcn5p HAT activity (3, 63). Catalytically active ADA, SAGA, or HAT-A2 complexes fail to purify from strains in which ADA2 has been deleted (3, 16, 58). Furthermore, phenotypes associated with deletions of GCN5 are indistinguishable from those associated with deletions of ADA2 (15). Thus, both in vitro and in vivo evidence supports the fact that Ada2p is required for the function of Gcn5p as a HAT. An interesting characteristic of the gcn5Δ sas3Δ synthetic lethality is that loss of SAS3 is compatible with deletion of ADA2, indicating that the gcn5Δ sas3Δ synthetic lethality is not due to loss of SAGA, SLIK/SALSA, ADA, or HAT-A2 and thus may be due to failure of Gcn5p to acetylate a nonhistone substrate. To date, no genes have been identified, other than those that encode components of NuA3, which exhibit a genetic interaction with GCN5 but not ADA2 (22). It has been previously demonstrated that loss of the H3 tail is lethal in a gcn5Δ strain (75), so we tested whether loss of the H3 tail resulted in the same phenotype in an ada2Δ background. GCN5 or ADA2 was deleted from strains which also carried deletions of both the HHT1 and HHT2 loci and expressed histone H3 from a URA3 plasmid. TRP1-based plasmids expressing HHT2 or hht2Δ3-29 were introduced into these strains, and the resulting transformants were plated on 5-fluoroorotic acid (5-FOA). When cells were subjected to negative selection for the URA3-based plasmid, no growth was observed for gcn5Δ hht2Δ3-29 mutant cells, while ada2Δ hht2Δ3-29 mutant cells were viable (Fig. 2B). The fact that deletion of the H3 tail recapitulated phenotypes seen in sas3Δ mutants supports the hypothesis that the interaction of NuA3 with chromatin is important for NuA3 function. We also plated the cells on medium containing 3-aminotriazole (3-AT), which mimics histidine starvation. Strains carrying ADA2 or GCN5 deletions are defective for growth on medium containing 3-AT due to poor expression of the HIS3 gene (38, 44). Figure 2B shows that the gcn5Δ and ada2Δ strains used in this study grow poorly on 3-AT compared to the wild type, demonstrating that these strains are exhibiting previously published phenotypes.
NuA3 function is dependent on the H3 tail. (A) Chromatin pulldown assays were performed from the indicated strains (YDM141, YDM143, YDM142, and YDM144), and the resulting samples were subjected to Western blotting with immunodetection with anti-HA antibodies (Sas3HA) or peroxidase-antiperoxidase antibodies (Htb1TAP). Yeast whole-cell extract (1.4% of input) was also blotted for HA (INPUT). (B) Tenfold dilutions of yeast strains YLH170 (sas3Δ), YLH356 (gcn5Δ sas3Δ), YLH357 (ada2Δ sas3Δ), YLH347 (hht1Δ hht2Δ), YLH348 (gcn5Δ hht1Δ hht2Δ), and YLH349 (ada2Δ hht1Δ hht2Δ) containing the indicated plasmids were plated on synthetic complete medium (control), synthetic complete medium with 5-FOA, or synthetic histidine dropout medium with 50 mM 3-AT and incubated at 30°C for 3 days.
NuA3 function is dependent on lysine 14 of histone H3.Although chromatin binding is required for NuA3 function, it is still formally possible that NuA3 is recruited to nucleosomes but is acetylating a nonhistone substrate. To deal with this possibility, we asked whether loss of the site targeted by NuA3 phenocopies a sas3Δ phenotype. NuA3 has been shown to preferentially acetylate lysine 14, and to a lesser extent lysine 23, on the H3 tail (21). Thus, we once again took advantage of the differential gcn5Δ and ada2Δ genetic interactions with SAS3 mutants to test the importance of histone H3 lysine 14 for NuA3 function. As shown previously, mutation of lysine 14 to arginine creates a severe growth defect in gcn5Δ strains (75). However, we found that this mutation was tolerated in ada2Δ strains (Fig. 3A). To verify that the sas3Δ phenotype seen in lysine 14 mutants was not due to loss of NuA3-nucleosome interaction, we performed a chromatin pulldown assay on the lysine 14 mutant. Samples were normalized for levels of Htb1TAP in the pulldown assays, and Fig. 3B shows that mutation of lysine 14 to arginine did not affect Sas3p binding to chromatin, suggesting that lysine 14 is important for NuA3 function at a step downstream of nucleosome binding. Thus, the fact that mutation of lysine 14 recreated a sas3Δ-specific phenotype supports our hypothesis that NuA3 modifies this residue in vivo. Interestingly, the data obtained thus far indicate that in the absence of GCN5, Sas3p is essential for histone acetylation, while in the absence of SAS3, Gcn5p may be required for acetylation of a nonhistone substrate.
NuA3 function is dependent on lysine 14. (A) Tenfold dilutions of yeast strains YLH170 (sas3Δ), YLH356 (gcn5Δ sas3Δ), YLH357 (ada2Δ sas3Δ), YLH347 (hht1Δ hht2Δ), YLH348 (gcn5Δ hht1Δ hht2Δ), and YLH349 (ada2Δ hht1Δ hht2Δ) containing the indicated plasmids were plated on synthetic complete medium (control), synthetic complete medium with 5-FOA, or synthetic histidine dropout medium with 50 mM 3-AT and incubated at 30°C for 3 days. (B) Chromatin pulldown assays were performed with the indicated Sas3HA3-expressing strains (YDM141, YDM142, YDM193), and the resulting samples were subjected to Western blotting with immunodetection with anti-HA antibodies (Sas3HA) or peroxidase-antiperoxidase antibodies (Htb1TAP). Yeast whole-cell extract (1.4% of input) was also blotted for HA (INPUT).
Histone H3 modifiers, Set1p and Set2p, are required for NuA3 interaction with chromatin.Our results obtained thus far support the hypothesis that the NuA3 HAT complex binds and acetylates nucleosomes in vivo. It has been previously shown that the binding of many chromatin-modifying complexes to nucleosomes is regulated by histone posttranslational modifications. For example, the SAGA HAT complex has been shown to preferentially modify histones that are methylated at H3 lysine 4 and phosphorylated at H3 serine 10 (36, 48). To determine whether the binding of NuA3 to the H3 tail is regulated by other histone modifications, we once again employed the chromatin pulldown assay with mutants that lacked various posttranslational modifications of the H3 tail, including snf1Δ, gcn5Δ, set1Δ, and set2Δ strains. Samples were normalized for levels of Htb1TAP in the pulldown assays, and Fig. 4A shows that deletion of SNF1 or GCN5, which, respectively, phosphorylates or acetylates specific residues within the H3 tail (16, 17, 30, 35, 49), did not affect the interaction of Sas3p with chromatin. Similarly, the level of Sas3p precipitated with Htb1TAP did not appear to be dependent on SET1, which is required for methylation of lysine 4 on histone H3. In contrast, deletion of SET2 had a significant impact on this interaction. Set2p methylates lysine 36 within the H3 tail, suggesting that this modification may facilitate the binding of NuA3 to histone H3. Interestingly, although deletion of SET1 did not seem to have a significant impact on Sas3p binding, set1Δ strains consistently showed increased levels of Sas3HA in the yeast whole-cell extracts (Fig. 4A, INPUT). This may be due to either increased expression of the SAS3 gene or increased solubility of the Sas3 protein upon extract preparation. Because the latter may be suggestive of a role for SET1 in the interaction of Sas3p with chromatin, we decided to investigate the involvement of SET1 in NuA3-chromatin interaction further. To this end, we tested the effect of a SET1 deletion on Sas3p binding to chromatin in a set2Δ background. We found that, despite increased levels of Sas3p in the input whole-cell extract, concomitant deletion of SET1 and SET2 had an even greater effect than a SET2 deletion alone, suggesting that the binding of NuA3 to chromatin is dependent on both SET1 and SET2 (Fig. 4A). This result suggests that methylation of lysines 4 and 36 mediates the interaction of NuA3 with chromatin.
Histone H3 modifiers, Set1p and Set2p, are required for NuA3 function. (A) Chromatin pulldown assays were performed from the indicated Sas3HA3-expressing strains (YDM126, YDM127, YDM151, YDM155, YDM153, YDM163, and YDM187), and the resulting samples were subjected to Western blotting with immunodetection with anti-HA antibodies (Sas3HA) or peroxidase-antiperoxidase antibodies (Htb1TAP). Yeast whole-cell extract (1.4% of input) was also blotted for HA (INPUT). (B) Deletion of SET2 restores silencing to strains with mutations in HMR-E. Wild-type (YLH164), sas3Δ (YLH165), set1Δ (YLH312), set2Δ (YLH324), and set1Δ set2Δ (YLH325) strains were assayed for mating efficiency with YLH101 as a tester strain.
To provide further support for the involvement of SET1 and SET2 in NuA3 function, we asked whether deletion of these genes generated a sas3Δ phenotype. We initially looked for synthetic interactions of mutations of SET1 and/or SET2 with mutations in GCN5 and ADA2. However, we found that unlike ada2Δ sas3Δ mutants, ada2Δ set1Δ mutants did show a synthetic growth defect (data not shown), which suggested that Set1p has functions overlapping those of one of the Gcn5p-dependent HAT complexes. This may be due to loss of histone H3 lysine 4 methylation or loss of another function of Set1p, such as the recently demonstrated role for Set1p in methylation of the kinetochore protein Dam1p (74). Therefore, to further address the role of SET1 and SET2 in NuA3 function, we looked for a different sas3Δ phenotype in set1Δ and set2Δ mutants. We and others have previously shown that deletion of genes encoding components of NuA3 rescues the silencing defect resulting from mutations of the Rap1p and Abf1p binding sites at the HMR locus (22, 51). Although the molecular basis for this phenotype in sas3Δ mutants has not been confirmed, it is most likely due to decreased levels of acetylation spreading past the impaired HMR-E silencer into the heterochromatic mating type locus (27, 61). Figure 4B shows that deletion of SET2 also rescues this silencing defect, suggesting that Set2p is required for the function of the NuA3 complex. In contrast, a set1Δ mutant did not show this phenotype, and in fact SET1 has been previously shown to be required for the maintenance of silencing at the telomeres, the rRNA genes, and the HML locus (6, 29, 42). It was later demonstrated that the role of Set1p in silencing was indirect and was due to the fact that H3 lysine 4 methylation blocks the binding of the silence information regulator Sir3p to transcriptionally active regions of the genome (53). Loss of lysine 4 methylation therefore results in a titration of Sir3p away from heterochromatic regions, leading to a loss of silencing. A dual function for lysine 4 methylation in blocking the binding of Sir3p, while enhancing the recruitment of NuA3, may explain why SET1 deletions did not rescue HMR silencing defects. The loss of NuA3 recruitment in a set1Δ strain may be compensated for by a concomitant decrease in the levels of Sir3p present at HMR. Consistent with this hypothesis, deletion of SET2 does not rescue silencing in a set1Δ strain (Fig. 4B). Consequently, these results are consistent with a role for SET2, and possibly SET1, in the function of NuA3.
Histone H3 lysine residues 4 and 36 are required for NuA3 function.Although it appears that methylation of H3 lysines 4 and 36 mediates NuA3 interaction with chromatin, the possibility exists that Set1p and Set2p are methylating nonhistone substrates, and it is this methylation that mediates NuA3 function. Set1p has recently been implicated in the methylation of the kinetochore protein Dam1p (74), and the possibility that Set2p also methylates nonhistone substrates has not been ruled out. To clarify this issue, three histone H3 substitution mutants were created by site-directed mutagenesis, resulting in amino acid changes from lysine to arginine at lysines 4, 36, and both 4 and 36. Chromatin pulldown assays were then performed on all mutants after epitope tagging the C termini of H2B with a TAP tag and Sas3p with a triple HA tag. The results of subsequent immunoblot analysis closely mirrored the results obtained with the set1Δ and set2Δ mutants shown in Fig. 4A. Mutation of lysine 4 to arginine resulted in increased levels of Sas3HA in the whole-cell extract and an accompanying minor increase in the pulldown of Sas3p with Htb1p (Fig. 5A). Mutation of lysine 36 to arginine resulted in a modest decrease in the binding of Sas3p to chromatin, but when the higher levels of Sas3p present in these cells is taken into account, this level represents a substantial decrease in the fraction of Sas3p that is binding chromatin. Mutation of both lysines 4 and 36 resulted in almost total disruption of Sas3p-chromatin interactions. These results suggest that lysines 4 and 36 are required for Sas3p, and consequently NuA3, to bind chromatin. To determine whether NuA3 directly interacts with methylated histones, we examined the level of NuA3 acetylation of histone H3 tail peptides that were dimethylated on either lysine 4 or 36. The results showed no difference in the levels of histone acetylation between these substrates and unmodified peptides (data not shown). This may be indicative of a dependency for histone trimethylation for NuA3 acetylation, or the fact that the recruitment of NuA3 by histone methylation is mediated by an intermediary factor.
Histone H3 residues lysine 4 and 36 are required for NuA3 function. (A) Chromatin pulldown assays were performed with the indicated Sas3HA3-expressing strains (YDM141, YDM142, YDM192, YDM207, and YDM208), and the resulting samples were subjected to Western blotting with immunodetection with anti-HA antibodies (Sas3HA) or peroxidase-antiperoxidase antibodies (Htb1TAP). Yeast whole-cell extract (1.4% of input) was also blotted for HA (INPUT). (B) Tenfold dilutions of yeast strains YLH347 (hht1Δ hht2Δ), YLH348 (gcn5Δ hht1Δ hht2Δ), and YLH349 (ada2Δ hht1Δ hht2Δ) containing the indicated plasmids were plated on synthetic complete medium (control), synthetic complete medium with 5-FOA, or synthetic histidine dropout medium with 50 mM 3-AT and incubated at 30°C for 3 days. (C) Tenfold dilutions of yeast strains YLH224 (hht1Δ hht2Δ) and YLH315 (sas3Δ hht1Δ hht2Δ) containing the indicated plasmids were plated on yeast-peptone-dextrose medium and incubated at 20°C, 30°C, and 37°C for 3 days.
While histone H3 lysines 4 and 36 are required by NuA3 to interact with chromatin, we were unsure as to whether these residues are essential for NuA3 function. If these residues are indeed required by NuA3 for its function, it therefore follows that mutations of these residues should result in sas3Δ phenotypes. With this question in mind, plasmids encoding wild-type and mutant versions of histone H3 were transformed into wild-type, gcn5Δ, and ada2Δ strains that carried a wild-type histone H3 gene on a URA3-based plasmid. Transformants were plated on 5-FOA to select for loss of the URA3 plasmid, and Fig. 5B shows that mutation of both lysines 4 and 36 to arginine resulted in sensitivity to 5-FOA in a gcn5Δ mutant but not in an ada2Δ mutant. A similar result was seen upon mutation of lysines 4 and 36 to alanines (data not shown). The strength of these phenotypes correlated closely with the effect of SET1 and SET2 deletion on the binding of Sas3p to chromatin. Taken together, these results confirm that the binding and acetylation of histone H3 by NuA3 are dependent on prior methylation of histones by Set1p and Set2p.
To verify that NuA3 functions in the same pathway as lysine 4 and 36 methylation, we examined the phenotypes of wild-type and sas3Δ strains carrying arginine substitutions of lysines 4 and 36. If NuA3 functions in the same pathway as methylation of these residues, then we would expect that deletion of SAS3 from K4R, K36R, and K4R K36R strains would not result in any additional phenotypes. Figure 5C shows that the growth of K4R, K36R, and K4R K36R mutants was indistinguishable from that of wild-type histone H3 strains at 20°C and 30°C, and deletion of SAS3 did not affect this. However, mutation of lysine 36 to arginine resulted in a mild temperature sensitivity, as indicated by poor growth at 37°C. This temperature sensitivity was further pronounced in the K4R K36R mutant. This synthetic phenotype is not surprising when one considers the accumulating number of factors that are recruited by methylation of histone H3 in yeast. These include the Isw1p ATPase (55) and the SAGA and SLIK/SALSA HATs (48), which bind methylated lysine 4, the Rpd3S HDAC (8, 26), which is targeted by methylation of lysine 36, and the NuA3 and Esa1p HATs (40, 57), which are targeted by methylation of both lysines 4 and 36. Interestingly, deletion of SAS3 partially rescued the temperature sensitivity of the K36R mutant, as well as the K4R K36R mutant, albeit to a lesser extent. Because lysine 36 methylation is also required for targeting of Esa1p and the Rpd3S HDAC to transcribed genes (8, 26, 40), it is difficult to interpret these results. One possibility is that NuA3 and Rpd3S have opposing functions in the cell. NuA3 may acetylate lysine 14 of histone H3 to facilitate the passage of RNA polymerase, whereas the Rpd3S HDAC removes this modification. It has been proposed that removal of transcription elongation-associated acetylation is required to suppress subsequent intragenic transcription initiation, and consistent with this, loss of the Rpd3S HDAC or mutation of lysine 36 results in cryptic initiation from intragenic sites within multiple genes (8). This may then result in a growth defect at higher temperatures. In the K36R mutant, however, NuA3 would still be recruited by lysine 4 methylation, and thus a lysine 36 mutant would maintain higher histone acetylation levels than a wild-type strain. This may be rescued by deletion of SAS3, which removes the acetylation in the absence of Rpd3S recruitment.
The Set1p and Set2p methyltransferases mediate steady-state levels of histone acetylation on bulk histones.The fact that NuA3 binding to chromatin is dependent on SET1 and SET2 suggests that these methyltransferases may mediate histone acetylation. To address this, we asked whether loss of SET1 and/or SET2 results in a loss of steady-state histone acetylation in vivo. This experiment was complicated by the fact that lysine 4 methylation has recently been shown to enhance histone H3 acetylation by the GCN5-dependent SAGA and SLIK/SALSA HAT complexes (48). To rule out the possibility that any observed loss of histone acetylation in set1Δ mutants was due to a failure to recruit these HATs, we examined the histone acetylation of bulk histones in wild-type, set1Δ, set2Δ, and set1Δ set2Δ strains that had been generated in a gcn5Δ background. We normalized for levels of histone H3 by immunoblotting with an antibody specific for the carboxyl terminus of histone H3 and probed an identical blot with an antibody that is specific for acetylated lysine 14 of histone H3 (62). Figure 6A shows that there was no detectable difference in the immunoblot signals seen in the wild type versus the set1Δ, set2Δ, or set1Δ set2Δ mutants when whole-cell extracts were probed with this αAcK14 antibody. However, this antibody also failed to detect a difference in the acetylation signals seen in the wild type versus a K14R mutant (Fig. 6B), suggesting that the signals observed do not accurately represent the levels of lysine 14 acetylation seen in the cell. To circumvent this problem, we made use of a second antibody that was raised against histone H3 that was diacetylated on lysines 9 and 14. This antibody also failed to detect a difference in histone H3 acetylation in the wild type versus a K14R mutant (Fig. 6B); however, this was expected since this antibody also detects histone H3 acetylated at lysine 9, which has been shown by others to be the predominant epitope recognized by this antibody (75). However, since of all our strains were generated in a gcn5Δ background, we did not anticipate this being a problem since deletion of GCN5 has been shown to result in the loss of the majority of histone H3 lysine 9 acetylation in vivo (75). When this antibody was used to probe normalized levels of histone H3 from wild-type, set1Δ, set2Δ, and set1Δ set2Δ strains, the level of histone acetylation in set1Δ and set2Δ strains was seen to modestly decrease. However, concomitant deletion of SET1 and SET2 resulted in a more substantial decrease in histone acetylation (Fig. 6A), suggesting that SET1 and SET2 mediate bulk Gcn5p-independent histone acetylation in vivo. This, taken together with the decreased interaction of NuA3 with chromatin in set1Δ and set2Δ mutants, suggests that NuA3 function is dependent on the methylation of lysine 4 or 36 within the H3 tail.
The Set1p and Set2p methyltransferases mediate the steady-state levels of histone acetylation of bulk histones. Whole-cell extracts from the indicated strains (YLH326, YLH327, YLH328, YLH329 [A], and Y224 transformed with pHHT2, phht2K14R, and phht2K9,14,18,23R [B]) were subjected to Western blotting and immunodetection with anti-acetylated histone H3 lysine 14 antibodies (αAcK14) and anti-diacetylated histone H3 lysine 9 and 14 antibodies (αAcK9,14). Samples were normalized for levels of histone H3 by immunoblotting with anti-carboxyl terminus of histone H3 antibodies (αH3).
DISCUSSION
NuA3 acetylates histones in vivo.The NuA3 HAT complex was originally identified on the basis of its ability to acetylate histone H3 in vitro (25). However, the possibility existed that histone H3 was not the main substrate for NuA3 in vivo. In this study, we have shown that Sas3p is capable of interacting with chromatin in vivo and that this interaction is dependent on the H3 tail. Moreover, deletion of the H3 tail, or mutation of H3 lysine 14 to arginine, is synthetically lethal with gcn5Δ, indicating that the gcn5Δ sas3Δ synthetic lethality is due to loss of lysine 14 acetylation by NuA3. Finally, we have previously shown that a temperature-sensitive mutation of SAS3 in a gcn5Δ background results in a decrease in H3 acetylation levels seen in bulk histones (21). Although we cannot rule out the possibility that Sas3p is also acetylating another substrate, this finding indicates that NuA3 is a physiological histone H3 acetyltransferase.
The synthetic lethality between gcn5Δ and histone H3 Δ3-29 and K14R mutants has been reported previously (75). The same work showed that mutation of H3 lysine 9 in a gcn5Δ background does not result in an enhanced phenotype over gcn5Δ alone, which is consistent with the fact that Gcn5p is functioning in the same pathway as lysine 9 acetylation. Others have shown that Gcn5p acetylates H3 lysines 9, 18, 23, and 27 but not lysine 14 in vivo (28, 62). This, taken together with the gcn5Δ K14R synthetic phenotype, suggests that H3 lysine 14 is acetylated by a HAT other than Gcn5p. The fact that deletion of SAS3 recapitulates phenotypes seen upon mutation of lysine 14 suggests that this HAT is NuA3. The evidence that Gcn5p does not acetylate lysine 14 in vivo is puzzling when one considers that, in vitro, native Gcn5p HAT complexes target primarily lysine 14 when nucleosomal histones are used as substrates (17). A similar result has been seen with the elongator complex, which shows a preference for acetylating lysine 14 of peptides in vitro but a different substrate specificity in vivo (28, 68). One simple explanation for these observations is that Gcn5p and elongator acetylate lysine 14 in vivo, but in the absence of these HATs, NuA3 is able to compensate such that no net loss of acetylation is observed. Alternatively, lysine 14 may be acetylated by NuA3 before other HATs have the opportunity to do so. NuA3 has been shown to interact with FACT, a complex with a role in DNA replication (25). It is possible that this interaction allows for acetylation of lysine 14 by NuA3 immediately following DNA replication, before HATs that are targeted by other mechanisms can access this residue. However, this would argue that acetylation by NuA3 precedes transcription, which due to the link between transcription and histone H3 methylation, is not supported by the results of this study. A third hypothesis is that, in vivo, H3 lysine 14 is presented in such a way that it cannot serve as a good substrate for Gcn5p complexes or elongator.
Histone methylation mediates global histone acetylation.Histone acetylation can be targeted to active regions of the genome through a number of different mechanisms. The SAGA and NuA4 HAT complexes specifically interact with DNA-bound transcriptional activators, thus directing histone acetylation to the promoter region of genes (7, 23, 24, 64, 66, 67). The elongator complex interacts with the elongating form of RNAPII and thus would be expected to be targeted to regions of transcription activity (70). The NuA3 HAT complex has been shown to interact with FACT, a protein complex which modulates chromatin structure (25). However, the abundance of FACT in the cell approaches that of the nucleosome (5, 69), and we estimate that NuA3 is present in less than 5% of this amount (unpublished data). Thus, if NuA3 is targeted to specific regions of the genome, another mechanism must exist to control NuA3 recruitment. In support of this, we find that the ability of NuA3 to bind chromatin is dependent on the Set1p and Set2p histone methyltransferases and on histone H3 lysines 4 and 36, the substrates for these methyltransferases, respectively. Consistent with their role in mediating histone acetylation, loss of SET1 and SET2 results in a moderate decrease in steady-state levels of histone acetylation, as shown by Western blot assays of bulk histones. These data provide support for a hypothesis whereby methylation of histones provides a signal for the recruitment of NuA3 and subsequence histone acetylation.
The recruitment of Set1p and Set2p to genes is dependent on an association with RNAPII (20). Therefore, the ability of these methylation marks to mediate histone acetylation by NuA3 suggests that histone acetylation is dependent on transcription. A number of studies have examined the correlation between transcription and histone H3 acetylation in yeast. However, the majority of these studies employed an antibody that was raised against H3 peptide tails that were diacetylated at lysines 9 and 14, and work with histone mutants suggests that these antibodies recognize primarily acetylated lysine 9 (46, 75). Two studies using antibodies directed specifically against acetylated H3 lysine 14 to examine genome-wide acetylation profiles of open reading frames showed a positive correlation between H3 lysine 14 acetylation and transcription (31, 46). Interestingly, a similar correlation was seen between transcription and acetylation of H3 lysine 23, which can also be acetylated by NuA3 in vitro (31, 46).
The ability of histone methylation to mediate histone acetylation has also been observed by others. Methylation of H3 lysine 4 has been shown to enhance histone acetylation by the SAGA and SLIK/SALSA HATs (48). Additionally, lysine 36 methylation mediates the interaction of the Rpd3S HDAC with transcribed genes (8, 26), suggesting that histone acetylation can be positively and negatively regulated by histone H3 methylation. SET1 and SET2 are also required for the association of the NuA4 complex with the MET16 and RPS11B promoters and the subsequent acetylation of lysine 8 on histone H4 (40). Interestingly, genome-wide histone acetylation studies indicate that there is a strong positive correlation between histone H4 lysine 8 and histone H3 lysine 14 acetylation, suggesting that NuA3 and NuA4 may be recruited through a shared mechanism (31).
The recruitment of SAGA, SLIK/SALSA, Rpd3S, and NuA4 by methylated histones is not an unexpected result, as all three complexes contain protein subunits with chromodomains (8, 13, 26, 40, 48). A subset of chromodomain proteins have been shown to bind specifically to methylated lysines (4), but none of the known subunits of NuA3 contain this motif. Recent studies, however, have identified novel protein domains which are capable of binding methylated histones. These include the WD40 repeat protein WDR5, which is a common component of multiple H3 K4 methyltransferase complexes in higher eukaryotes (71). Thus, it is possible that another protein motif within NuA3 is responsible for recognizing methylated histones. One candidate would be the PHD finger within the Yng1p subunit of NuA3. This zinc finger-like motif occurs in various proteins thought to be involved in chromatin-mediated gene regulation (1). Despite the intriguing possibility that this motif mediates the interaction of NuA3 with chromatin, we find that Yng1p lacking the PHD domain is still able to rescue the gcn5Δ yng1Δ growth defect, suggesting that this region of the protein is not required for the function of the NuA3 complex (22). It is possible that NuA3 binding to methylated histones is mediated by intermediary factors such as an unidentified protein or another histone modification that is dependent on histone methylation. A similar result was seen for the yeast Isw1p ATPase, which requires SET1 to bind chromatin but cannot bind methylated peptides in a purified system (55). Regardless of the mechanism, our work clearly indicates that NuA3 binding to methylated histones is an important means for directing the HAT activity of NuA3 to chromatin and explains how histone H3 methylation can mediate transcription.
ACKNOWLEDGMENTS
Support for this work was provided by a grant to L.H. from the Cancer Research Institute of the Canadian Institute for Health Research. L.H. is a scholar of the Michael Smith Foundation for Health Research.
We gratefully acknowledge the valuable comments provided by Jacques Côté, Jerry Workman, and members of the Molecular Epigenetics Group of the Life Sciences Institute at the University of British Columbia. We are also grateful to Fred Winston and Jasper Rine for providing yeast strains and plasmids.
FOOTNOTES
- Received 30 August 2005.
- Returned for modification 13 October 2005.
- Accepted 2 February 2006.
- Copyright © 2006 American Society for Microbiology