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Yaf9, Taf14, and Sas5 comprise the YEATS domain family in Saccharomyces cerevisiae, which in humans includes proteins involved in acute leukemias. The YEATS domain family is essential, as a yaf9Δ taf14Δ sas5Δ triple mutant is nonviable. We verify that Yaf9 is a stable component of NuA4, an essential histone H4 acetyltransferase complex. Yaf9 is also associated with the SWR1 complex, which deposits the histone H2A variant Htz1. However, the functional contribution of Yaf9 to these complexes has not been determined. Strains lacking YAF9 are sensitive to DNA-damaging agents, cold, and caffeine, and the YEATS domain is required for full Yaf9 function. NuA4 lacking Yaf9 retains histone acetyltransferase activity in vitro, and Yaf9 does not markedly reduce bulk H4 acetylation levels, suggesting a role for Yaf9 in the targeting or regulation of NuA4. Interestingly, yaf9Δ strains display reduced transcription of genes near certain telomeres, and their repression is correlated with reduced H4 acetylation, reduced occupancy by Htz1, and increased occupancy by the silencing protein Sir3. Additionally, the spectra of phenotypes, genes, and telomeres affected in yaf9Δ and htz1Δ strains are significantly similar, further supporting a role for Yaf9 in Htz1 deposition. Taken together, these data indicate that Yaf9 may function in NuA4 and SWR1 complexes to help antagonize silencing near telomeres.
Chromatin structure is a central regulator of gene expression. Nucleosomes are the basic unit of chromatin structure, consisting of a histone octamer disk around which is wrapped 147 bp of DNA. The amino-terminal tails of the histone proteins are exposed on the surface of the nucleosome and serve as platforms to which additional proteins can bind and influence chromatin structure and transcriptional activity, either positively (to form active euchromatin) or negatively (to form repressive heterochromatin) (24). In Saccharomyces cerevisiae, the products of the SIR2, SIR3, and SIR4 (silent information regulator) genes can bind to histone tails and form repressive heterochromatin by polymerization down the chromatin template (19, 20, 48).
Sir2/3/4 polymerization is observed at telomeres and at the transcriptionally silent mating type loci (20) and is initiated by their recruitment to these loci by the specific DNA-binding protein Rap1. Histone acetylation is a central regulator of the chromatin state; hyperacetylation is associated with transcriptional activation, and hypoacetylation is associated with repression. Sir2/3/4 complexes prefer to bind to deacetylated tails (7), consistent with the observation that the histone tails of transcriptionally silent telomeres are largely deacetylated (50). Importantly, the Sir2 protein itself is an NAD-dependent histone deacetylase whose activity is essential for Sir2/3/4 spreading (23, 31, 47), suggesting that the deacetylation of neighboring nucleosomes by Sir2 enables Sir3/4 binding and subsequent spreading.
Clearly, the spreading distance must be regulated to ensure that important genes near telomeres are not improperly silenced. In principle, heterochromatin boundaries may be formed through the recruitment of factors that oppose heterochromatin spreading. For example, tRNA genes, which are robustly transcribed by RNA polymerase III, contribute to barrier formation at the silent mating type locus HMR (10, 11). In addition, certain subtelomeric regions contain binding sites for the transcription factors Reb1 and Tbf1, which help block the spreading of silencing (16, 17). As Sir proteins bind deacetylated histone tails and as Sir2 itself is a deacetylase, histone acetyltransferases (HATs) could provide a potent barrier to Sir propagation. Support for this proposal emerged from work on the SAS complex, which consists of the HAT Sas2, Sas4, and a third protein, Sas5 (33, 40). Sas5 is related to the human leukemogenic proteins AF9/ENL/Gas41 and to the yeast proteins Anc1/Tfg3/Swp29/Taf30/Taf14 (hereafter referred to as Taf14) and the protein characterized in this work, Yaf9 (40). These proteins all contain a conserved YEATS (Yaf9-ENL-AF9-Taf14-Sas5) domain, the function of which is unknown. Importantly, the HAT component Sas2 functions to prevent the spreading of Sir3 to telomere-proximal genes near the right end of chromosome VI (26, 49), although the role of the YEATS protein Sas5 in this process has not been determined. Sas2 acetylates lysine 16 on the histone H4 tail (H4K16 acetylation) (51), and the Sir2/3/4 complex binds poorly to H4 tails bearing this modification (7), providing one mechanism for the prevention of spreading. Consistent with these results, a K16R replacement in the histone H4 tail promotes Sir complex spreading at certain telomeres (49). Acetylation of histone H3 may also affect spreading, as the severe H3 hypoacetylation observed in elp3Δ gcn5Δ double mutants causes spreading at telomere 6R (28). In addition, the tethering of diverse HAT components (including SAS and NuA4 complex components) can block Sir spreading at silent mating type loci, further suggesting a role for HAT recruitment in antagonizing silencing (39).
Recently, a central role for the histone H2A variant Htz1 in preventing spreading was demonstrated, as Htz1 is present at certain telomeres and at the silent locus HMR and as the loss of Htz1 results in the spreading of Sir proteins at HMR and at a subset of telomeres (9, 29, 34). Importantly, the replacement of Htz1 for canonical H2A is an active ATP-dependent process performed by the SWR1 chromatin remodeling complex, and mutations in SWR1 components cause the repression of telomere-proximal genes (27, 35). Certain components of the SWR1 complex are also present in NuA4, raising the possibility that histone acetylation and Htz1 replacement may be coordinated to affect boundary formation. Consistent with this idea, the double-bromodomain protein Bdf1, which binds to acetylated H4 tails, interacts with SWR1 and has a role in establishing the heterochromatin boundary (27, 29, 30). Thus, diverse factors collaborate for boundary formation, and understanding their coordinated functions is an important current area of study.
Our interest in Yaf9 arose from the identification of a related factor, Taf14, as a member of transcription- and chromatin-regulating complexes, including SWI/SNF, TFIID, TFIIF, and NuA3 (6, 21, 25, 41). Together, these results strongly suggest that YEATS proteins are involved in transcription and chromatin structure, but their mechanistic role is not known. In addition, members of the YEATS protein family have an intriguing link to cancer, as they are common translocation partners of the human trithorax (HRX/MLL/ALL-1) gene, forming fusion proteins that cause acute leukemias (37, 52). Recently, Yaf9 was linked to the SWR1 and NuA4 complexes (27, 29, 32, 35). The NuA4 complex is a multiprotein complex that helps regulate ribosomal protein genes (2, 43) as well as PHO5 (38). The NuA4 component Esa1 is an essential histone H4 HAT (8, 46), and mutations that reduce HAT activity reduce target gene transcription (14, 18). NuA4 also contains approximately eight other proteins that appear to assist in either targeting or regulation and likely correspond to the p400/TIP60 HAT-DNA repair complex in human cells (5, 13, 22).
Here we provide a functional analysis of Yaf9 and show that it is important for proper DNA repair and metabolism and for the acetylation and expression of telomere-proximal genes. We also provide evidence for Yaf9 function in SWR1 complex and Htz1 functions, as yaf9Δ mutants are defective in Htz1 deposition at telomere-proximal genes and show extensive overlap with htz1Δ mutants in their transcriptional profiles and phenotypes. Additional genetic and genomic work with Yaf9, Taf14, and Sas5 reveals the importance of the YEATS domain and the YEATS protein family to yeast viability and proper gene expression through transcription and chromatin structure.
Preparation of media, genetic methods, and strain isolation followed standard procedures. Strain genotypes are shown in Table Table1.1. All gene deletions and epitope tagging were verified by PCR and/or immunoblot analysis; details are available on request.
The YAF9 gene was isolated from YBC605 genomic DNA by PCR and cloned into plasmid pRS316 (CEN6 URA3), and the sequence was verified. Plasmid p416.MET25 (36) was used to construct plasmids directing the expression of Flag-tagged Yaf9 (pFlag-YAF9, pFlag-yaf91-186, and pFlag-yaf9SDM) from the MET25 promoter. A site-directed mutation of YAF9 was generated by the Quick Change method (Stratagene). All clones were verified by sequencing; detailed construction information is available on request.
The NuA4 complex was purified from YBC1093 as follows. Eighteen liters of culture was grown to an optical density at 600 nm (OD600) of 9.0 in 2× yeast extract-peptone-dextrose (YPD), and cells were harvested by centrifugation and washed with 0.5× phosphate-buffered saline-5% glycerol. About 320 g of the cell pellet was resuspended in 160 ml of 3× lysis buffer (150 mM HEPES-OH [pH 7.5], 750 mM potassium acetate [pH 7.5], 60% glycerol, 30 mM EDTA, 1.5 mM dithiothreitol [DTT], protease inhibitor [PI] cocktail [2 μg of chymostatin/ml, 2 μM pepstatin A, 0.6 μM leupeptin, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride]). Cell breakage and protein recovery were carried out as described by Sayre et al. (45). The supernatant (310 ml) was augmented with 15.5 ml (1/20 volume) of 3 M potassium acetate and 10.16 g of ammonium sulfate (to 250 mM). After the mixture was stirred for 30 min, 4.65 ml (1/66.7 volume) of 10% (vol/vol) polyethylenimine (pH 8.0) was added. The mixture was stirred for 10 min and spun in a Beckman 45Ti rotor at 45,000 rpm for 2 h. The supernatant was recovered and supplemented to 0.1% NP-40 with stirring.
NuA4 was purified as described by Puig et al. (42) with the following modifications. A whole-cell extract (297.5 ml at 29 mg/ml) was used directly for immunoglobulin G (IgG) binding; IgG beads were washed four times with wash buffer supplemented with 400 mM NaCl, 0.5 mM DTT, and PIs and washed three times with tobacco etch virus protease (TEV) cleavage buffer. TEV cleavage was performed for 3 h at 15°C; calmodulin binding was performed for 2 h at 4°C. Calmodulin-bound proteins were eluted with supplemented calmodulin elution buffer (25 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM imidazole, 4 mM EGTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, PIs). The calmodulin elution fractions were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by silver staining and Western analysis. Peak fractions were pooled, and about 4.4 ml of final purified NuA4 product (at an estimated concentration of 20 μg/ml) was obtained. Details regarding the purification of hemagglutinin (HA) epitope-tagged Yaf9 and protein identification of NuA4 complex members are in the supplemental data.
Reaction mixtures (40 μl) contained free recombinant yeast core histones (800 ng), 2 μl (~40 ng) of purified NuA4 complex (or buffer as a control), and 0.1 μCi of 3H-acetyl coenzyme A (3.8 Ci/mmol; Sigma) in HAT buffer (50 mM Tris-HCl [pH 8], 50 mM NaCl, 0.1 mM EDTA, 1 mM MgCl2, 25 μg of bovine serum albumin/ml, 1 mM DTT) and were incubated at 30°C for 30 min. Samples were processed essentially as described previously (2). The reaction mixtures were separated on an SDS-10 to 20% gradient acrylamide gel, followed by Coomassie blue staining, destaining, and fluorography with Amplify (Amersham).
Relevant strains were grown in YPD at 30°C to an OD600 of 0.8. Cells were collected and frozen in liquid nitrogen. Total RNA was isolated by acid phenol-chloroform extraction. Total RNA was treated with RNase-free DNase I and purified with a Qiagen RNeasy kit. Antisense RNA (aRNA) was amplified with an Arcturus RiboAmp RNA amplification kit. aRNA (2 μg) was subjected to microarray analysis as described by Angus-Hill et al. (3). Cy3 and Cy5 fluorescence readings were quantified and normalized such that the average Cy5/Cy3 intensity ratio was equal to 1. All experiments were performed in triplicate, and the log2 average ratios are reported. Significantly affected genes in the mutants were identified by a change in expression of twofold or more compared to the expression in the relevant wild-type (WT) strains.
Sir3 was tagged at its C terminus with 13 copies of the Myc epitope (Myc13) and Htz1 was tagged at its N terminus with 3 copies of the HA epitope (HA3) in the WT and yaf9Δ backgrounds, respectively. Isogenic untagged strains were used in control experiments. Chromatin immunoprecipitation (ChIP) experiments were performed as described by Roberts et al. (44) with the following modifications. Cells were cross-linked with 1% formaldehyde for 45 min at room temperature. For Sir3-Myc ChIP, 2 μl of anti-Myc antibody (9E11; Abcam) was prebound to 50 μl of pan-mouse IgG Dynabeads (~2.0 × 107 beads; Dynal Biotech catalog no. 110.23) and then incubated with 500 μg (protein) of supernatant from the sheared chromatin for 5 h at 4°C. For HA-Htz1 ChIP, 2 μg of anti-HA antibody (12CA5) and 800 μg of supernatant from the sheared chromatin were incubated overnight at 4°C. For histone H4 tetra-acetylation ChIP, 2 μl of anti-acetylated histone H4 antibody (Upstate Biotechnology catalog no. 06-866) was prebound to 35 μl of sheep anti-rabbit IgG M-280 Dynabeads (~2.0 × 107 beads; Dynal Biotech catalog no. 112.04) and then incubated with 500 μg of protein overnight at 4°C. A total of 6.25 μg of poly(dA)-poly(dT) (Amersham Pharmacia catalog no. 27-7860-02) was used in all ChIP experiments to reduce the nonspecific background. All experimental ChIPs were performed in triplicate, and untagged control ChIPs were performed in duplicate. The purified ChIP samples were used in quantitative PCR (qPCR) analysis and genome-wide ChIP (G-ChIP) analysis.
qPCR was performed as described by Roberts et al. (44), except that all PCRs were performed in triplicate and the average was calculated as the relative abundance of each target of interest. ACT1, ARS317, and PRP8 were used as the normalization controls to determine the enrichment of targets in Sir3, acetyl-histone H4, and Htz1 occupancy studies, respectively. Primer sequences are available on request.
Our interest in Yaf9 was based on a previous identification of its paralog, Taf14, as a member of the SWI/SNF complex and also on its sequence similarity to proteins such as AF9 and ENL, which are involved in human leukemias. Searches using the Basic Local Alignment Search Tool (BLAST) algorithm showed that Yaf9 was significantly more similar to AF9 (BLAST E-value of 1e −16) (and also to ENL) than was Taf14 (BLAST E-value of 1e −9); therefore, we named the protein Yaf9 (yeast AF9). However, more recent searches have revealed a much larger family, including proteins in Schizosaccharomyces pombe and Caenorhabditis elegans and additional human proteins, such as Gas41 (BLAST E-value of 1e −29) (Fig. 1A and B). All members bear a defining N-terminal YEATS domain and therefore have been termed YEATS family members (Fig. (Fig.1A).1A). However, close inspection of these protein sequences and analysis by BLAST suggest the presence of three additional sequence elements, which we termed the A, B, and C boxes, that we believe are useful for classifying the members (Fig. (Fig.1B).1B). The A box is present in many members but is absent in Taf14 and Sas5. The B box defines the C terminus of Yaf9, Gas41, and related proteins and is predicted to form a coiled coil (Fig. (Fig.1B1B and data not shown). Although the B box is absent in AF9 and ENL, these two proteins share homology at their C termini, and this region is also predicted to form a coiled coil. The C box is present only at the C termini of Taf14 and Sas5. Taken together, these results indicate that the family appears to be comprised of three sequence classes: a Yaf9/Gas41 class (A box, with a B box at the C terminus), an AF9/ENL class (A box, with a coiled coil at the C terminus), and a Sas5/Taf14 class (C box at the C terminus).
To determine the importance of the YEATS family in yeast cells, we isolated strains bearing combinations of null mutations in the three family members (Yaf9, Sas5, and Taf14). Although no individual member was essential, strains lacking two of the three members showed reduced growth ability, and a strain lacking all three was nonviable (Table (Table2).2). These results establish the YEATS family as being essential in yeast cells.
Initially, we determined that Yaf9 localizes to the nucleus (Fig. (Fig.1C),1C), consistent with a role in transcription. To identify associated proteins, Yaf9 was tagged at the C terminus with one of two different tandem affinity tags. One consisted of a tandem HA3-His7 tag, and the other consisted of a tandem protein A-calmodulin-binding protein (TAP) tag (see Materials and Methods). DNA encoding each tag was targeted to the 3′ end of chromosomal YAF9 by homologous recombination. The tags did not affect function, as strains bearing these tags lacked the phenotypes displayed by the null allele (described below). For the strain with the HA3-His7 tag, the final anti-HA affinity column afforded nine proteins, which were identified by mass spectrometry to be Eaf1, Eaf2/God1, Eaf3, Eaf5, Eaf6, Epl1, Esa1, Act1, and Nbn1/Yng2, all members of the NuA4 complex.
To assess the stability of the association, we performed coimmunoprecipitation analyses with anti-HA beads and the eluate from the Resource Q column (Fig. (Fig.2B).2B). NuA4 members could be depleted from the fraction with anti-HA beads (Fig. (Fig.2B,2B, lane 4) and recovered in the eluate (lane 6). Yaf9-NuA4 complexes resisted extensive washing with 400 mM NaCl, showing that Yaf9 is stably associated with NuA4.
The alternative TAP tag procedure enabled the rapid preparation of a pure Yaf9-NuA4 complex (Fig. (Fig.2C),2C), which was confirmed by mass spectrometric sequencing and immunoblot analysis (data not shown). TAP-Yaf9 itself stains poorly with silver and migrates at approximately the same mass as Yng2 (36 kDa). However, mass spectrometric analysis of NuA4 prepared by conventional chromatographic methods (18) clearly identified peptides corresponding to Yaf9 (data not shown). Taken together, these data establish Yaf9 as a stable component of NuA4.
NuA4 bearing Yaf9 (purified by the TAP procedure) displayed robust acetyltransferase activity that was highly specific for histone H4 (Fig. (Fig.2D).2D). However, activity was essentially equal to that displayed by NuA4 lacking Yaf9 (isolated from a yaf9Δ strain bearing Epl1-TAP) (data not shown), suggesting that Yaf9 is not required for HAT activity or for H4 specificity. In addition, SDS-PAGE and Western analysis revealed the presence of all other NuA4 subunits in purified preparations from yaf9Δ strains (data not shown), indicating that Yaf9 is not required for complex assembly. Finally, strains lacking YAF9 showed little or no reduction in their bulk levels of H4 acetylation (Fig. (Fig.2E),2E), further suggesting that Yaf9 does not have a great impact on NuA4 H4 HAT activity or that its impact is limited to certain loci (such as telomeres; see below).
Clear evidence for the presence of Yaf9 in the SWR1 complex was recently demonstrated by others (27, 29, 35). Although SWR1 complex members were not clearly detectable by SDS-PAGE analysis in our preparations, components of SWR1 could be detected by immunoblot analysis of preparations from Yaf9 immunoprecipitations performed at a low stringency, and the Ruv helicases could be detected by mass spectrometric analysis at low levels (data not shown). Consistent with stringency affecting the Yaf9-Swr1 association, preparations of the SWR1 complex purified at a low stringency by others contained only substoichiometric amounts of Yaf9, although its presence was definitive. In addition, the placement of a tag on Yaf9 could, in principle, have slightly compromised its association with the SWR1 complex selectively.
A strain lacking YAF9 was viable but was unable to grow at 15°C (Cs−), on medium containing 0.03% methyl methanesulfonate (MMS; a DNA-damaging agent) or caffeine (5 to 15 mM; elicits a stress response), or after exposure to UV radiation (80 J/m2) and grew very slowly on 50 mM hydroxyurea (HU; an inhibitor of deoxynucleoside triphosphate synthesis) (Fig. (Fig.3A).3A). nua4 and swr1 complex mutants share most of these phenotypes (MMS−, caffeine negative, and HU−) (12), making it impossible to attribute these phenotypes in the yaf9Δ strain to a function in one particular complex (Table (Table2).2). However, we found that swr1Δ and htz1Δ strains were also Cs−, while NuA4 mutants (esa1, eaf3Δ, epl1, or arp4) were not (data not shown), suggesting that Yaf9 promotes growth in the cold through its function exclusively through SWR1/Htz1. Furthermore, we found that yaf9Δ esa1-L327S double mutants were nonviable (Table (Table2),2), with the combination of these two mutations possibly reducing NuA4 function below a critical threshold. Taken together, these results are consistent with functions for Yaf9 in both the SWR1 complex and the NuA4 complex.
The Yaf9 C terminus contains a B box that is predicted to form a coiled coil. The function of this domain is not known, although leukemogenic MLL fusions to YEATS family members all contain their extreme C terminus, suggesting a crucial role for this domain. Deletion of the B box (Yaf91-186) conferred a null phenotype (Fig. (Fig.3A),3A), although the derivative was produced well (Fig. (Fig.3C).3C). Interestingly, full-length Yaf9 coprecipitated Esa1, whereas Yaf91-186 did not, showing that the Yaf9 C terminus is critical for assembly into NuA4 (Fig. (Fig.3B3B).
A Yaf9 derivative lacking the YEATS domain was unstable and almost undetectable in extracts by immunoblot analysis. Therefore, we prepared a Yaf9 derivative bearing site-directed mutations (SDM) that replaced three consecutive conserved residues in the YEATS domain (PPF, residues 80 to 82) with alanines (Fig. 1A and B). This derivative (Yaf9SDM) was stably produced (Fig. (Fig.3C)3C) and behaved as a moderate hypomorph; it fully complemented yaf9Δ phenotypes related to caffeine and HU sensitivity, partially complemented phenotypes related to cold and UV, but only weakly complemented MMS sensitivity (Fig. (Fig.3A).3A). To determine whether the YEATS domain is required for the functions of other YEATS proteins in yeast cells, we created an identical site-directed mutation in Taf14 (termed taf14SDM) and tested it for complementation of taf14Δ phenotypes. We found that the taf14Δ mutation conferred sensitivity to temperature (Ts−), caffeine (5 mM), HU (50 mM), UV (80 J/m2), and MMS (0.03%). Although expressed at WT levels (data not shown), the taf14SDM mutation only partially complemented UV and caffeine sensitivity and failed to complement Ts−, MMS−, and HU− phenotypes (Table (Table2).2). Taken together, these results show that the YEATS domain is important for many of the functions of Yaf9 and Taf14, providing the first evidence for a function for the YEATS domain in vivo.
To better understand the impact of Yaf9 on transcription, we compared the transcription profiles for yaf9Δ and WT strains. Overall, 224 genes are downregulated more than twofold, whereas 48 genes are upregulated more than twofold. These changes could largely be reversed by transforming the strain with a plasmid bearing WT YAF9 (data not shown). The majority of the changes observed did not fall into clear gene classes or pathways (the results obtained with the entire microarray are available on request). However, an examination of the changes relative to the physical chromosomal map revealed a striking position effect; a significant fraction of the genes (65 of 224) that were downregulated more than twofold (defined as “affected ” genes) in the yaf9Δ strain were positioned within 20 kb of the telomere (Fig. (Fig.44).
To depict this relationship, we first parsed the 40 kb of DNA proximal to telomeres into eight successive intervals of 5 kb and considered the remainder of the genome (distances of >40 kb) as the final interval (Fig. (Fig.4,4, abscissa). Affected genes were represented on the ordinate in terms of either total number (Fig. (Fig.4A)4A) or the fraction of genes affected in the particular interval (Fig. (Fig.4B).4B). Although the majority of the genes affected were not within 20 kb of the telomere (Fig. (Fig.4A),4A), the density of the affected genes was exceptionally high within 20 kb of the telomere (~25%) compared to the bulk of the genome (~2%). Stated differently, although the telomere-proximal 20-kb region contains only ~5% of all genes, it contains ~29% of the affected genes. This overlap is highly significant; the χ2 value generated by 65 genes affected within 20 kb of a telomere is 184.9, corresponding to a P value of <0.001. Affected genes were not lost from the telomere due to shortening; genomic DNA isolated from yaf9Δ strains was labeled and hybridized to the array and revealed no loss of telomere-proximal genes (data not shown).
Interestingly, only a subset of telomeres is affected in yaf9Δ strains. We define affected telomeres as those bearing within 20 kb three or more genes that are downregulated more than twofold. Certain telomeres are highly homologous to telomeres on other chromosomes. For this subset, it is difficult to attribute the downregulation of a transcript observed to gene silencing at a particular gene or telomere, as the transcript (labeled cDNA) derived from one telomere will hybridize to the spot on the microarray representing both homologs. Therefore, we apply the additional criterion that the telomere must contain unique affected genes, which may lead to an underestimation of the number of telomeres affected. According to these stringent criteria, affected telomeres in yaf9Δ strains are 2R, 3R, 4L, 4R, 6L, 11L 13R, 14R, and 15L (see Table S1 in the supplemental data), with several other telomeres being moderately affected. Affected telomeres do not appear to share an obvious common element (such as Y′) or attribute that would distinguish them clearly from other telomeres.
Our results show a striking overlap with telomeres affected in strains lacking Htz1 (see below and Table S1 in the supplemental data) (34). In keeping with the results and the focus of studies by others on Htz1 and Swr1, we chose to focus our detailed analysis on telomere 14R and the region between silent mating type locus HMR and telomere 3R (Fig. (Fig.5A).5A). At telomere 14R, the loss of Yaf9 causes a significant downregulation of proximal genes (Fig. (Fig.5B).5B). Likewise, several genes in the region between telomere 3R and HMR (YCR099C/100C/101C/104W) are affected (Fig. (Fig.5C5C and data not shown). Consistent with a role for NuA4 in this process, a mutation in the catalytic subunit of NuA4 that reduces H4 acetylation, esa1-L254P (8), confers significant downregulation of genes at 14R (Fig. (Fig.4A4A and and5B),5B), although it has only a slight impact at 3R (data not shown). The impact of esa1-L254P on telomeres is not as extensive as that observed with the yaf9Δ strain (see Table S1 in the supplemental data). However, we observed a significant overlap in the genes affected in esa1-L254P and yaf9Δ strains, and this overlap was quite pronounced when we considered the subset of affected genes located near telomeres (Fig. (Fig.5D5D).
In keeping with a role for Yaf9 in the SWR1 complex, about one-third of the genes downregulated in htz1Δ strains are identical to those downregulated in yaf9Δ strains (Fig. (Fig.5D).5D). As with esa1-L254P, the overlap was even more extensive when we considered the subset of affected genes within 20 kb of the telomere; in that situation, the overlap approached 50%. Taken together, these results suggest that Yaf9 assists both SWR1/Htz1 and NuA4 complexes in telomere-proximal gene expression.
Strains lacking Sas2 show extensive downregulation of genes near certain telomeres, and their downregulation is correlated with a reduction in histone H4 acetylation (26, 49). However, the impact of the associated YEATS protein Sas5 on this process has not been determined. We found that a loss of Sas5 affects telomeres 1L, 2R, 3R, 4R, 5L, 6L, 7L, 9R,11L, 12R, 13R, 14R, 15L, and 16R (using the criteria established above), although many additional telomeres are moderately affected (see Table S1 in the supplemental data). The work of others on sas2Δ focused on selected telomeres, primarily 6R, which is one of the two telomeres for which gene(s) are lacking on our array and which therefore could not be analyzed. These results extend the importance of the YEATS family in the process of gene expression or antisilencing at telomeres in vivo. These results also suggest that certain telomeres, such as 14R, rely on multiple complexes for antisilencing, whereas others depend primarily on the SAS complex.
Downregulation of genes at telomere-proximal loci might be a consequence of the spreading of Sir3 into these regions, a reduction in histone H4 acetylation in these regions, a reduction in Htz1 replacement in these regions, or a combination of these factors. To help discern among these possibilities, we determined whether downregulated telomere-proximal genes acquire Sir3 and the extent to which their downregulation depends on Sir3. Interestingly, the removal of Sir3 from strains lacking Yaf9 restores the transcription (either partially or fully) of certain genes (PAU6 and YNR074 for 14R; GIT1 and YCR099C/100C for 3R), whereas the transcription of other loci in these regions is not restored. We suggest that genes whose transcription is restored by the removal of Sir3 are those silenced by the Sir complex (in yaf9Δ strains), whereas those that remain silent in the yaf9Δ sir3Δ double mutant are deficient in histone acetylation and/or Htz1 replacement due to the absence of Yaf9 (see below).
As the loss of SIR3 partially restores the profiles of expression of telomere-proximal genes in yaf9Δ, the loss of SIR3 might suppress certain yaf9Δ phenotypes. Compared to yaf9Δ mutants, sir3Δ yaf9Δ double mutants show slightly improved growth at low temperatures and WT growth on medium containing caffeine (Fig. (Fig.3D),3D), suggesting that these defects in yaf9Δ strains are related to telomere-proximal gene repression caused by the spreading of Sir proteins. However, no suppression of phenotypes related to DNA repair and metabolism was observed, suggesting that these defects are not restricted to functions at telomeres and are not related to Sir protein spreading. These results further suggest that Yaf9, as a member of either the SWR1 complex or the NuA4 complex, plays roles both at telomeres and at other loci.
To directly determine the extent of Sir3 spreading in yaf9Δ strains, we performed ChIP of Sir3 in WT and yaf9Δ strains, each bearing a SIR3 allele encoding a Myc13-tagged Sir3 derivative integrated at the SIR3 locus. The levels of Myc13-tagged Sir3 protein were virtually identical in WT and yaf9Δ strains (Fig. (Fig.6A).6A). We then assessed the enrichment of particular loci by two methods: (i) genome-wide occupancy determinations involving ChIP combined with an immobilized array of the entire yeast genome parsed into open reading frame and intergenic fragments (G-ChIP) and (ii) qPCR. For telomere14R, telomere-proximal PAU6 is highly occupied by Sir3 in both WT and yaf9Δ strains, as indicated by both G-ChIP (Fig. (Fig.6B)6B) and qPCR (Fig. (Fig.6D).6D). Note that Fig. Fig.6D6D depicts the relative enrichment of Sir3 in the yaf9Δ strain compared to the WT strain, whereas the intensity of Cy5 (red) in the G-ChIP assay (Fig. 6B and C) reflects the absolute levels of Sir3. In the yaf9Δ strain, a redistribution of Sir3 is clearly observed at telomere 14R, consistent with spreading from the telomere end toward internal genes (Fig. 6B and D). Likewise, genes interposed between HMR and telomere 3R display increased Sir3 occupancy. Also, a reduction in occupancy at HMR itself was revealed by qPCR, suggesting that the spreading of Sir3 to the intervening region may come from (or at the expense of) HMR; however, a very significant amount of Sir3 remains (Fig. (Fig.6C6C and data not shown).
To test whether downregulation near telomeres is correlated with reductions in H4 acetylation, we performed ChIP analyses with an antibody raised against the hyperacetylated (at K5, K8, K12, and K16) tail of histone H4. For WT cells, we observed high levels of H4 acetylation at particular loci in both regions. Interestingly, these loci are either near or at the boundary of Sir3 spreading in WT cells (COS10 for 14R and GIT1 and RDS1 for 3R) (Fig. (Fig.7A).7A). However, RDS1 is both occupied by Sir3 and significantly acetylated, suggesting that H4 acetylation may not entirely prevent Sir3 binding or that the region is dynamic with respect to acetylation and deacetylation or Sir binding. For yaf9Δ cells, we observed a significant reduction in the acetylation of these loci but not of several other control loci. However, as similar reductions at these loci have been observed in strains lacking Htz1 (34), we cannot directly attribute the entire effect to Yaf9 assisting acetylation by NuA4 (see Discussion).
Our genetic and genomic results all suggest a close functional connection between Yaf9 and Htz1, raising the possibility that Yaf9 may assist the SWR1 complex in the deposition of Htz1 in vivo. To test this possibility, we used ChIP and qPCR to assess the occupancy of Htz1 in WT and yaf9Δ strains. To this end, we deleted YAF9 from a strain bearing an HTZ1 allele encoding an HA3-tagged version of Htz1 integrated at its genomic locus. Initially, we performed ChIP for both strains and quantified the enrichment of loci at telomeres 14R and 3R by qPCR. As observed with our H4 ChIP analyses, only certain loci are occupied by Htz1. Interestingly, although the sample size is small, there appears to be a significant correlation between loci showing H4 hyperacetylation and loci showing Htz1 occupancy, raising the possibility that these two functions are linked. Remarkably, Htz1 occupancy is significantly reduced at the telomeric loci tested in the yaf9Δ strain (Fig. (Fig.7B).7B). Interestingly, the reduction in Htz1 occupancy in the yaf9Δ strain may not be restricted to telomeres, as Htz1 occupancy is also reduced at GAL7.
To determine whether deletion of YAF9 affected the assembly of the SWR1 complex, we performed gel filtration analyses with WT and yaf9Δ strains bearing a Myc-tagged Swr1 derivative. For both strains, the SWR1 complex eluted at a molecular mass of 700 to 850 kDa (data not shown), suggesting that the complex remains intact in the yaf9Δ strain.
Chromosomes contain regions of specialized chromatin to help regulate chromosomal processes and transcriptional programs. For example, centromeres and telomeres represent loci where special chromatin structures are initiated to assist in processes such as kinetochore formation, sister chromatid cohesion, and DNA end protection and replication processes (for telomeres). RNA polymerase II transcription in these regions may interfere with the fidelity of these processes; this activity likely underlies the paucity of polymerase II genes in these regions. The heterochromatin of telomeres includes Sir proteins, which have the capacity to silence telomere-proximal genes via histone deacetylation. As Sir proteins bind to deacetylated histone tails, cycles of deacetylation and binding allow the Sir complex to spread and polymerize down the chromatin, extending silencing to neighboring regions. However, the spreading process must be limited (or regulated) to avoid improper silencing of neighboring polymerase II genes. This work provides evidence that YEATS family transcription factors help limit the propagation of silent heterochromatin through their functions in histone modifying and remodeling complexes.
YEATS proteins were originally discovered through the cloning of reciprocal chromosomal translocations involving the MLL/HRX/ALL-1 gene. These translocations encoded MLL-YEATS fusion proteins, which are commonly found in acute leukemias. Studies with yeast cells have firmly connected the YEATS protein family to transcription and chromatin (see the introduction). Here we show that the YEATS protein family in yeast cells (overall) is essential for viability and provide evidence that the YEATS domain itself is important for function, as mutations in conserved residues in the domain confer significant phenotypes. At present, we are not able to determine whether these substitutions eliminate YEATS domain function entirely or just impair it significantly; therefore, it remains possible that the YEATS domain is required for all functions of Yaf9 and Taf14. As a YEATS family member is present in all three currently identified chromatin remodeling and modifying complexes involved in telomere-proximal gene expression (SAS/NuA4/SWR1), identification of the binding partner for the YEATS domain may reveal a unifying role.
Recent work strongly suggests that the human TIP60 complex is the human counterpart of the yeast NuA4 complex, as they contain 11 paralogous subunits (5, 12, 13). The TIP60 complex contains the YEATS protein Gas41, and the yeast protein most similar to Gas41 is Yaf9 (Fig. (Fig.1)1) (32). Our data show further that the Yaf9 C terminus is required for the assembly of Yaf9 into NuA4. This result is interesting in light of the critical role of the C terminus in MLL fusions involving YEATS proteins; the C terminus is a constant feature of all leukemogenic fusions, whereas the YEATS domain can be absent. Current models suggest that the MLL amino terminus present in fusion proteins retains the domains important for targeting to HOX genes. We suggest that the C terminus of the YEATS partner may represent a domain for an HAT-remodeler interaction that enables the misregulation of HOX genes by improper HAT-remodeler recruitment.
Recent data from several laboratories indicates the coordinated efforts of HATs, remodelers, and Htz1 in opposing the spreading of Sir proteins at yeast telomeres (see the introduction). For the acetylation component, H4 acetylation appears to be key, although there is also evidence that H3 acetylation plays a role at certain telomeres (28). Clearly, the factor with the most significant impact at telomeres is the SAS complex (26, 49), based on the large spectrum of telomeres involved (see Table S1 in the supplemental data). The SAS complex (Sas2/4/5) consists of the MYST family HAT Sas2p, Sas4, and the YEATS protein Sas5 (33, 40). Sas2 has a clear preference for H4K16 in vitro, and cells lacking Sas2 show a significant reduction in H4K16 acetylation (33, 49). Earlier studies established the concept that H4K16 acetylation by Sas2 at telomeres is important for limiting Sir spreading (26, 49) and focused on the role of Sas2 and its impact at chromosome 6R. Here, our transcription profiling of sas5Δ strains revealed a significant impact on silencing at the vast majority of yeast telomeres (see Table S1 in the supplemental data). This result is consistent with the observations that the SAS complex lacking Sas5 shows reduced HAT activity in vitro and that sas5 mutations affect silencing at HMR (51, 53). Taken together, these studies establish an important role for the YEATS protein Sas5 in assisting the SAS complex at yeast telomeres.
A yaf9Δ mutation has a clear effect on telomere-proximal gene expression. However, as Yaf9 is present in both SWR1 and NuA4, it is difficult to attribute Yaf9 functions to a particular complex. To help clarify its role in NuA4, we examined the role of histone acetylation by Esa1. A role for Esa1 in promoting gene expression at genes near telomere 6R had been demonstrated previously (49), whereas we assessed the impact of an esa1 mutation genome-wide and observed downregulation at several telomeres, revealing a fairly broad role for Esa1. Importantly, we found a significant overlap in the genes affected near telomeres in esa1-L254P and yaf9Δ strains (Fig. (Fig.5D).5D). However, as the esa1-L254P allele is still partially functional, the full impact of Esa1 on telomere-proximal gene expression (and its overlap in function with Yaf9) may be significantly greater than was observed with esa1-L254P. Furthermore, we show that H4 acetylation is reduced at affected telomeres in yaf9Δ strains. Although this result is consistent with Yaf9 assisting NuA4 in acetylation at these loci, reductions at these loci are also observed in strains lacking Htz1 (34). Thus, the reductions in H4 acetylation observed in yaf9Δ strains may be due to a combination of reduced NuA4 function, increased Sir2/3/4 (histone deacetylase) spreading, and the lack of Htz1 at these loci.
Studies of Esa1 specificity in vitro and in vivo strongly suggest that Esa1 (also a MYST family HAT) contributes the majority of acetylation at H4K5/8/12 and a portion of the acetylation at H4K16 (2, 46, 49). Thus, together, Sas2 and Esa1 constitute the majority of H4 acetyltransferase activity in yeast cells, and it seems reasonable to propose that these two HATs work together to regulate H4 acetylation at telomeres. Furthermore, their different specificities raise the possibility that acetylation by one HAT may improve acetylation by the other HAT. Support for these proposals was revealed in the acetylation profile of sas2 esa1 double mutants at 6R, which showed greater-than-additive reductions in H4K16 acetylation near telomeres than either single mutant (49). As both the SAS complex and the NuA4 complex contain YEATS proteins, it is tempting to speculate that YEATS proteins may assist in the targeting of these complexes to telomeres through the recognition of an aspect of telomeric chromatin, such as Htz1.
Important roles for Htz1 in boundary formation and SWR1 in Htz1 replacement were recently established, but much about the regulation of this process is not understood (see Introduction). Here we provide several lines of evidence that Yaf9 contributes to the Htz1 deposition process. First, yaf9Δ strains and htz1Δ strains show significant overlap in phenotypes. However, their phenotypes are not identical (yaf9Δ strains are more sensitive to MMS, for example), suggesting that Yaf9 has roles beyond those attributed to SWR1. These roles likely involve the functions of Yaf9 in NuA4, as it is a stable member of NuA4 and as NuA4 mutants are likewise very sensitive to DNA-damaging agents such as MMS. Second, yaf9Δ strains and htz1Δ strains show significant overlap in both affected genes and affected telomeres. Third, yaf9Δ strains show dramatic reductions in Htz1 deposition at telomeres. Taken together, these data indicate that Yaf9 plays a functional role in the SWR1 complex in Htz1 deposition. Several important questions remain, including how the SWR1 complex is targeted to particular loci, how the process of replacement is regulated by complex members, and how Htz1 replacement and H4 acetylation are linked.
Emerging evidence strongly suggests that antisilencing at telomeres requires the coordinated functions of HATs, remodelers, and a histone variant. However, several central issues remain. For example, it is not clear why spreading is observed at certain telomeres and not others as a consequence of the loss of certain HAT or SWR1 components. One might expect that the strength of the barrier at a particular telomere would correlate with the rates of transcription of resident genes. Indeed, a strongly transcribed ribosomal protein gene or the isolated promoters of highly transcribed genes can limit Sir propagation in certain contexts (4, 54). Also, tRNAs can serve as effective barriers to propagation (10, 11), although they are not uniformly present near telomere ends and the generality of their use as barriers has not been established. However, transcription itself is not required for Reb1 and Tbf1 to serve as barriers at subtelomeric X elements (15), and an examination of native boundaries and transcriptional activity has not been thoroughly performed. Thus, the relationship between transcription and barrier formation at native telomeres is still emerging and requires further work. Another interesting question is the order in which the processes occur, e.g., whether histone acetylation serves as a mark for Htz1 replacement or whether nucleosomes that bear Htz1 are themselves preferred substrates for SAS or NuA4 complexes. Either scenario would provide an effective combination of these processes to establish and or maintain boundaries.
The phenomenon of gene silencing near telomeres has revealed many interesting silencing proteins as well as important concepts in epigenetics. However, the context in which a yeast cell may utilize a position effect to increase fitness has only recently come to light. Studies by Ai and colleagues recently showed that stress conditions lead to the activation of Mpk1/Slt2 kinase and the phosphorylation of Sir3, which leads to a reduction in Sir3 occupancy of genes near telomeres (1). Remarkably, many genes that participate in the response to stress involving the cell wall are telomere proximal, leading to their expression under cell wall stress conditions. Therefore, in this context, the telomere may consist of two distinct regions: (i) subtelomeric regions with few or no genes, as transcription may interfere with DNA end protection and replication processes, and (ii) telomere-proximal regions bearing polymerase II genes that utilize the dynamic properties of the spreading process to control their expression programs via position effects. Clearly, understanding how HATs and SWR1/Htz1 function to control spreading dynamics will be central to understanding the regulation of these gene programs.
We thank Brian Dalley and Adrienne Tew from the HCI microarray core facility for assistance. We thank Bob Schackmann for oligonucleotide synthesis and Mitch Smith for strain HA-HTZ1.
This work was supported by National Institutes of Health grants 5-T32DK07115-29 (to J. Kushner, for support of D. N. Roberts and D. O. Richardson), GM60415 (to B. R. Cairns, for support of H. Zhang), and CA24014 (for core facilities). J.C. is an investigator with the Canadian Institutes of Health Research. This work was also supported by the Howard Hughes Medical Institute (HHMI). B.R.C. is an investigator with the Huntsman Cancer Institute and an assistant investigator with HHMI.
†Supplemental material for this article may be found at http://mcb.asm.org/.