The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes
- Lorena E Rosaleny†1,
- Ana B Ruiz-García†1,
- José García-Martínez2,
- José E Pérez-Ortín1 and
- Vicente Tordera1Email author
© Rosaleny et al.; licensee BioMed Central Ltd. 2007
Received: 20 November 2006
Accepted: 20 June 2007
Published: 20 June 2007
Specific histone modifications can perform several cellular functions, for example, as signals to recruit trans-acting factors and as modulators of chromatin structure. Acetylation of Lys14 of histone H3 is the main target of many histone acetyltransferases in vitro and may play a central role in the stability of the nucleosome. This study is focused on the genome-wide binding of Saccharomyces cerevisiae histone acetyltransferases that are specific for Lys14 of histone H3.
We have used a variation of the genome-wide location analysis method, based on a macroarray platform, to identify binding sites of yeast histone acetyltransferase catalytic subunits and to correlate their positions with acetylation of Lys14 of histone H3. Our results revealed that the histone acetyltransferases Sas3p and Gcn5p are recruited to a pool of intensely transcribed genes and that there is considerable overlap between the two cohorts of Sas3p and Gcn5p bound gene pools. We also demonstrate a positive correlation between binding sites of both proteins and the acetylation state of Lys14 of histone H3. Finally, a positive correlation between the decrease of H3 Lys14 acetylation in a GCN5 deleted strain and the Gcn5p genome occupancy is shown.
Our data support a model in which both Gcn5p and Sas3p act as general activators of an overlapping pool of intensely transcribed genes. Since both proteins preferentially acetylate Lys14 of histone H3, our data support the hypothesis that acetylation of this specific residue facilitates the action of the transcriptional apparatus.
Most of the protein mass in eukaryotic chromatin is composed of histones, which are crucial for the organization of the highly conserved nucleosomal structure. Histones are subjected to a wide variety of post-translational modifications, most of them reversible [1, 2], that can influence chromatin functions by different mechanisms. This epigenetic information, composed of histone modifications, has been called the histone code [3–6] and can be considered as specific binding surfaces for the recruitment of activators or repressors of biological processes. While the term code has raised some controversy, there is growing evidence suggesting that histone modifications act as recognition signals for proteins that regulate gene expression and other chromatin functions . The existence of the histone code does not exclude the hypothesis that several histone modifications may also directly affect the nucleosomal structure or the folding properties of the chromatin, facilitating the action of the protein complexes that regulate expression, repair, recombination and other essential functions of the eukaryotic genome.
Several types of enzymatic activities, generically referred to as chromatin regulators , can carry out the modifications of chromatin structure. The best-studied group is that of the histone acetyltransferases (HATs). These enzymes acetylate specific lysine residues, most of which are localized in the amino-terminal tail of the histones, and hence are responsible for many of the signals of the histone code. Nine different proteins that exhibit HAT activity in vitro have been described to date in Saccharomyces cerevisiae: Gcn5p, Hat1p, Elp3p, Hpa2p, Esa1p, Sas2p, Sas3p, Nut1p and Taf1p. Some of the properties of this set of enzymes are consistent with the histone code hypothesis. For instance, they are able to selectively acetylate particular histone residues. Moreover, most of them have physical and functional connections to transcriptional regulation and act as complexes composed of several subunits, which can modulate their activities. Nevertheless, there are also some features of these enzymes that are hard to reconcile with their proposed role in a combinatorial signaling system, which involves four different histones, each one with several lysine targets. Thus, with the data available to date, if we omit Hat1p  and Esa1p [10, 11], which selectively modify histone H4, the remainder of the HATs have a marked preference, at least in vitro, for histone H3, and particularly Lys14 (H3K14). Hence, there seems to be something special about this position because most HATs have potential H3K14 acetylation (H3K14ac) activity, exclusively or accompanied by other specificities. More specifically, it has been reported that Gcn5p [12–14], Sas3p , Hpa2p , Elp3p  and Taf1p  preferentially acetylate H3K14 in vitro. Nut1p also preferentially acetylates H3, but its specificity is unknown. Finally, the SAS complex, which is composed of the Sas2p acetylase bound to the Sas4 and Sas5 proteins, acetylates free H3 at Lys14 , although its specificity for nucleosome-assembled histones seems to be limited to H4 .
Despite the important role that acetylation of H3K14 could play, the HATs responsible for this modification in vivo are not well known. There are some studies where the acetylation state of bulk H3 obtained from different HAT mutant strains has been analyzed. However, most of these studies used an antibody specific for histone H3 acetylated at both K9 and K14 and, hence, it is not possible to determine if a lower signal in histone H3 isolated from the mutant strains is the consequence of a decrease in the acetylation state of H3K9, H3K14 or both. For instance, the acetylation analysis of a gcn5Δ strain did not definitively show which position was affected [15, 21, 22]. For sas3Δ [15, 21], elp3Δ [21, 22] or hpa2Δ  strains no differences were observed when comparing the relative amounts of H3K14ac in the mutant and the wild-type strain. When site-specific antibodies were used in a gcn5Δ strain the results showed that Gcn5p likely acetylates all sites in H3 with the sole exception of H3K14 . These authors commented on the striking contradiction between this finding and the very strong preference of Gcn5p for acetylating H3K14 in vitro. On the other hand, using chromatin immunoprecipitation of histones and DNA-microarray analysis (ChIP-chip), a positive association between Gcn5p occupancy and transcriptional activity [8, 24] and between Gcn5p occupancy and acetylation of H3K14 has been found , suggesting an active role for this HAT in the in vivo acetylation of H3K14 and transcriptional activation. Moreover, it was reported that acetylation of H3K14 peaks at the start sites of active genes and drops substantially across the open reading frames (ORFs) on a genome-wide level . Similar results were obtained using a high-resolution tiling microarray with single-nucleosome resolution, covering 0.5 megabases of yeast genomic sequence . These observations suggest that this H3 modification may directly facilitate the action of the transcriptional apparatus, at least at the initiation step.
In this study, we have investigated the genome-wide binding of known Saccharomyces cerevisiae HATs that have shown specificity for H3K14. With this aim, we have developed a ChIP-chip method based on the use of macroarrays. Using this technique, we have shown that Sas3p, like Gcn5p, is generally recruited to a similar pool of actively transcribed protein-coding genes. We also have shown that Gcn5p and Sas3p are involved in the acetylation state of H3K14 in vivo. Additionally, we found a relationship between Sas2p and double-strand break repair and ubiquitin-specific protease activity.
Results and discussion
Testing the protocol
Since the development of ChIP-chip technology, this method has been extensively used to analyze the histone modification patterns of chromatin and the binding sites of histone-modifying enzymes in yeast. These studies employ microarrays to hybridize the PCR-amplified, immunoprecipitated DNA. We have developed a method of ChIP-chip (see Materials and methods) based on the utilization of membrane-based DNA macroarrays. Macroarrays, while less widespread than microarrays, are also used by many laboratories and offer an interesting alternative. Macroarrays can be hybridized many times, up to ten times, without a measurable reduction in their performance , thus greatly reducing the cost of the experiments. The nature of DNA present in chips utilized in chromatin modification studies is variable. Most of the procedures employ microarrays containing ORFs, intergenic regions (IGRs), or both, from the yeast genome. There are also DNA microarrays that use oligonucleotide probes, which cover most of the yeast genome , and high-density oligonucleotide microarrays with approximately 20 base-pair (bp) resolution, which cover a small percentage of the yeast genome .
Percentage of tRNAs detected over a range of distances from an adjacent ORF
Total no. of tRNAs
Percentage of detected tRNAs
Accumulated percentage of detected tRNAs
Sas3p and Gcn5p are recruited to a similar set of active genes
Genes bound by Gcn5p and Sas3p
No. of genes in category
No. of genes bound by Gcn5p (P value)
No. of genes bound by Sas3p (P value)
No. of genes bound by both (P value)
43 (3.6 × 10-26)
54 (3.2 × 10-29)
17 (9.4 × 10-16)
51 (1.1 × 10-23)
67 (8.6 × 10-28)
21 (6.8 × 10-16)
There is no correlation between Sas2p, Hpa2p and Nut1p binding and transcription rate
Genes bound by Sas2p
No. of genes in category
No. of genes bound by Sas2p (P value)
7 (2.5 x 10-5)
RAD50, YKU70, SIR3, SIN3, LIF1, POL4, RAD1
Ubiquitin-specific protease activity
6 (2.8 x 10-5)
UBP2, UBP3, UBP6, UBP7, UBP10, UBP12
In vivoH3K14 acetylation by Gcn5p and Sas3p HATs
Sas3p is the catalytic subunit of NuA3, a complex identified on the basis of its ability to preferentially acetylate Lys14 and, to a lesser extent, Lys23 of nucleosome-bound histone H3 in vitro [39, 42]. Moreover, it has recently been reported that Yng1, a component of NuA3, interacts directly with K4 trimethylated H3 amino-terminal tail (H3K14me3) and that this interaction enhances in vitro H3K14-specific NuA3 HAT activity . Furthermore, mutation of H3K14 to arginine phenocopies the sas3Δ phenotype . All these findings strongly suggest that Sas3p acetylates H3K14 in vivo. On the other hand, whereas the preference of Gcn5p for H3K14 in vitro is well-documented [12–14], the role of this HAT in the acetylation of this position in vivo is not well known. It is surprising that ten years after the identification of Gcn5p as a HAT, its role on the in vivo acetylation of H3K14 remains unclear. Thus, it has been reported that Gcn5p acetylates H3 lysines 9, 18, 23 and 27 but not Lys14 in vivo [22, 23]. In contrast, a genome-wide positive association between Gcn5p occupancy, acetylation of H3K14 and transcriptional activity has also been demonstrated .
Genes that showed a significantly decreased H3K14 acetylation level in the sas3Δ mutant
No. of genes in category
No. of genes
Cell cycle/cell-division cycle
2.8 × 10-7
Mitotic cell cycle
5.8 × 10-7
2.2 × 10-6
3.7 × 10-6
9.5 × 10-6
Plasma membrane/bacterial inner membrane/cell membrane/cytoplasmic membrane/juxtamembrane/Plasmalemma
1.1 × 10-6
Cell organization and biogenesis
1.6 × 10-5
Cytoskeleton organization and biogenesis
1.6 × 10-5
We have developed a new ChIP-chip method based on the use of ORF macroarrays that can be reutilized several times and, therefore, applied to several samples. The methodology of ChIP-chip described allows for the rapid scanning of HAT localization of chromatin binding proteins in yeast and could represent a low-cost alternative to microarrays. Nevertheless, the methodology described here has several limitations. For instance, with this macroarray, which contains entire ORF sequences as probes, it is clearly impossible to distinguish between IG binding sites and those within an ORF. Binding sites within large IGRs will also be undetectable. On the other hand, promoter binding detected by this method will be biased towards sites that are closest to an ORF and we cannot discard the possibility that this represents a functionally distinct subset of promoters. Finally, probes prepared from very long ORFs (longer than 3 kb) will work poorly for detection of promoter regions and detection of protein binding will weaken as ORF length increases . However, these probes represent only 7% of the total. Despite these limitations, in this report we have demonstrated that macroarrays can reproduce the same results previously obtained by other researchers using microarrays.
Most of the HATs present in yeast exhibit HAT activity in vitro towards histone H3 and, especially, to H3K14. In this study, we have used our methodology to investigate the genome-wide association of known S. cerevisiae HATs that have shown specificity for this residue. Considering that there are at least 12 lysines that could act as possible acetylation sites in the yeast inner histones, H3, H4, H2A and H2B, we think that this preferential specificity for H3K14 may be of particular importance. For instance, this position may have a central role in the stability of the nucleosome. Thus, this specific modification of this H3 residue may directly facilitate the action of the transcriptional apparatus and other chromatin functions. Among the HATs analyzed, genome-wide occupancy of Gcn5p and Sas3p correlates with the transcriptional rate of the genes, whereas in the case of Elp3p, Sas2p, Hpa2p and Nut1p, no significant correlation was observed. These results demonstrate that Sas3p, a protein initially described as implicated in transcriptional silencing, is recruited to intensely transcribed genes, similar to what has been observed for Gcn5p. In this sense, it has recently been proposed that NuA3 is targeted to and/or retained at sites of H3K4me3 through interaction with Yng1, promoting H3K14ac, positively regulating downstream transcription events . The observation that Sas3p and Gcn5p HATs are recruited to similar active genes also supports the hypothesis  that Gcn5p and Sas3p have redundant functions. Moreover, we obtained a positive correlation between Sas3p (and Gcn5p) binding and the acetylation state of H3K14. The observation that Sas3p and Gcn5p are related to processes such as protein biosynthesis or cell division, in which an intense transcriptional activity is required, supports the hypothesis that H3K14ac plays a role in facilitating transcription. These findings strongly suggest that Gcn5p and Sas3p acetylate H3K14 in vivo. However, analysis of the genome-wide effect of the loss of GCN5 or SAS3 on the H3K14ac state may be subject to different interpretations. Thus, the negative correlation found between H3K14ac in the gcn5Δ strain and the Gcn5p occupancy using an epitope-tagged strain provides good evidence that Gcn5p acetylates this residue in vivo. On the contrary, the positive correlation found between H3K14ac in the sas3Δ strain and the Sas3p occupancy in the epitope-tagged strain suggests that Sas3p occupancy has a negative influence on the H3K14ac state. This striking result can be interpreted by considering that there is another HAT in vivo with H3K14 specificity that acetylates intensely this position in vivo in the absence of Sas3p. Based on the results shown here, we suggest that this HAT is Gcn5p. In any case, these results show implicitly that, in vivo, different HATs with overlapping specificities compete for chromatin binding sites and that this competition can play an important role in the regulation of the establishment of the histone code signals. Thus, one possible role of Sas3p could be to displace Gcn5p from certain key sites.
Materials and methods
Yeast strains and genetic methods
Strains used in this study
MATa, ura3-52, lys2-801, ade2-101,trp1-Δ1,his3-Δ200, leu2Δ1
MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63,his3-Δ200, leu2Δ1, rpc160Δ1::HIS3, pC160-240 (TRP1 CEN4 HA3-RPC160)
MATα, ura3Δ0, lys2Δ0, his3Δ1, leu2Δ0
The targets of catalytic HAT subunits and the Rpc160 subunit of RNA Pol III from yeast were identified by combining chromatin immunoprecipitation and ORF macroarray hybridization. We designed a specific protocol for ORF macroarrays based on a previously described method originally devised for intergenic microarrays .
Three independent cultures from tagged and control strains were grown. A volume of 40 ml YPD (1% yeast extract, 2% peptone, 2% glucose) culture from each one (OD 600 ≈ 0.5-0.8) was set aside, and proteins were cross-linked to their target sites in vivo by adding formaldehyde to a final concentration of 1%. Cells were incubated for 30 minutes at room temperature with occasional mixing, and cross-linking was quenched by adding glycine to a final concentration of 125 mM. Cells were washed three times with 30 ml of ice-cold phosphate-buffered saline buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Pelleted cells were frozen after this step. Cells were thawed on ice and resuspended in 300 μl lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 1 mM benzamidine and 1 pill of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) dissolved in every 25 ml of buffer). The equivalent of 0.2 ml of frozen glass beads (425-600 mm; Sigma-Aldrich, St Louis, MO, USA) was added to the cellular suspension and cells were lysed at 4°C by 25 minutes of vortexing in a Genie 2 vortex with Turbo mix at maximum power. Lysis buffer (400 μl) was added and the extract was transferred to a new tube. Sonication of DNA was performed in order to obtain DNA fragments between 400 and 3,000 bp, with an average size of 800 bp. Chromatin was sonicated on ice, 3 pulses of 10 s at 38% amplitude in a Vibracell VCX500 (Sonics&Materials, INC., Newtown, CT, USA). Cell debris was removed by centrifugation at 13,000 rpm at 4°C for 10 minutes. An aliquot of 50 μl of this whole cell extract (WCE) was kept to check the quality and size of the isolated chromatin.
The antibodies used were 2 μg of rat anti-HA 3F10 (Roche), 20 μg of mouse anti-MYC 9E11 (Roche), 15 μl of rabbit α-K14acH3 chip grade (Upstate, Lake Placid, NY, USA) and 20 μg of rabbit α-H3Ct (Abcam Inc., Cambridge, UK). Rat and mouse antibody complexes were collected using 100 μl of a 50% (v/v) suspension of Protein G Sepharose 4FF (Amersham Biosciences, Piscataway, NJ, USA) equilibrated in 5 mg/ml bovine serum albumin (BSA) lysis buffer. Rabbit antibody complexes were collected using 50 μl of a suspension of sheep α-rabbit IgG M-280 dynabeads (Dynal, Invitrogen Corp., Carlsbad, CA, USA) in a final volume of 120 μl. Suspensions were incubated overnight at 4°C. DNA fragments specifically cross-linked to the epitope-tagged protein were purified by immunoprecipitation with the indicated monoclonal antibodies coupled to Protein G Sepharose for 4 h at 4°C. We included control immunoprecipitations of lysates from wild-type strains with α-HA, α-MYC or α-H3Ct antibodies. Beads were washed twice with lysis buffer, twice with 500 mM NaCl lysis buffer, twice with wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA pH 8.0, 1 mM PMSF, 1 mM benzamidine and 1 pill of protease inhibitor cocktail/25 mL (Roche)), once with TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 1 mM PMSF, and finally collected. Two successive elutions were performed with 100 and 150 μl of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubating for 10 minutes at 65°C each time. The eluted fraction of protein cross-linked to DNA was treated overnight at 65°C to reverse the cross-linking. For WCE, 200 μl of elution buffer were added before reversal of cross-linking (from 1 h to overnight).
DNA purification and annealed linker ligation
Proteins were degraded by adding 50 μg of proteinase K and SDS to 2.5% final concentration, and incubation at 37°C for 1 h. DNA was purified by phenol/chloroform/isoamylic alcohol extraction. For WCE, the aqueous phase was directly ethanol precipitated, resuspended in 15 μl of milliQ water, and treated for 30 minutes with 10 μg of RNAse A. The WCE chromatin was visualized by agarose electrophoresis to check the quantity and size of the fragments. For immunoprecipitated DNA samples, the aqueous phase was subsequently purified with Montage PCR columns (Millipore Corp., Billerica, MA, USA). The total eluted volume was treated with 10 μg of RNAse A for 30 minutes at 37°C. Ethanol precipitation in the presence of 20 μg of glycogen as a carrier was performed. The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μl BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche). The reaction was allowed to proceed for 20 minutes at 12°C. After phenol/chloroform/isoamylic alcohol extraction, DNA was ethanol-precipitated in the presence of 11 μg of glycogen and was ligated in a final volume of 50 μl with annealed linkers oJW102 and oJW103 (1.5 μM of each primer) . The reaction was carried out overnight at 16°C and ligated DNA was precipitated and resuspended in 25 μl milliQ water.
DNA amplification and macroarray hybridization
Ligation Mediated PCR  was used for DNA amplification. Briefly, ligated DNA was dissolved in final volume of 40 μl (1× Biotaq buffer (from Bioline, London, UK), 2 mM MgCl2, 0.25 mM dNTPs, 1.25 μM oligonucleotide oJW102). The reaction was started by incubating for 2 minutes at 55°C and paused to add 10 μl of reaction mix (1× Biotaq buffer, 2 mM MgCl2 and 5 U BioTaq from Bioline). The program was resumed with 5 minutes at 72°C, 2 minutes at 95°C and 33 cycles of 30 s at 95°C, 30 s at 55°C and 2 minutes at 72°C. A 5 μl DNA aliquot of the LM-PCR was analyzed on a 1.2% agarose gel (to check for a smear from 200-2,500 bp and an average size of 600 bp), and the rest was purified with Montage PCR columns. DNA was precipitated overnight and resuspended in 25 μl of milliQ water. The concentration of nucleic acids was determined using a spectrophotometer.
The PCR product was used as a template for the radioactive labeling reaction. This reaction consisted of a single amplification step where Taq polymerase was incubated with a mix of 33P-dCTP, cold dCTP and the rest of dNTPs. The choice of the ratio between hot and cold dCTP is essential to produce long-sized DNA samples. Both Klenow and Taq polymerase were assayed in the presence of different radioactive/cold ratios. The labeling yield was much higher for Taq polymerase at any of the cold dCTP concentrations assayed (results not shown). Increases in the concentration of cold dCTP produced longer sizes of labeled DNA, although the incorporation of 33P-dCTP was reduced. We chose a ratio that produced DNA products with an average size of 600 bp and an acceptable threshold of radioactive labeling. The reaction mixture contained 1.5 μg of amplified DNA in 50 μl (1× Biotaq buffer, 2 mM MgCl2, 0.2 mM dATP, dTTP and dGTP, 25 μM dCTP, 1 μM oJW102, 0.8 μCi α-33P-dCTP and 5 U Biotaq). The mix was denatured for 5 minutes at 95°C, annealed 5 minutes at 50°C and was amplified during 35 minutes at 72°C. The reaction product was purified through a ProbeQuant G-50 column (Amersham Biosciences) to remove unincorporated 33P-dCTP and oligonucleotides.
The macroarrays used contained PCR products representing full-length ORFs for 6,049 genes of S. cerevisiae  (Servicio de Chips de DNA, Universitat de València). After pre-hybridizing macroarrays for 1 h in hybridization solution (5× SSC, 5× Denhart's, 0.5% SDS) at 65°C in a rotator oven, hybridizations were performed using the same solution containing labeled denatured DNA (3 × 106 dpm/ml) for 16-18 h. After hybridization, the membranes were washed once in 2× SSC, 0.1% SDS for 20 minutes, and twice in 0.2× SSC, 0.1% SDS for 30 minutes. They were exposed to an imaging plate (BAS-MP, Fujifilm) for 48-72 h and read in a PhosphorImager (FLA-3000, Fujifilm) at 50 μm resolution. After analysis of macroarray filters, they were stripped as described previously  and hybridized with an appropriate sample under the same conditions.
Image analysis and data processing
Images were quantified by using ArrayVision 7.0 software (Imaging Research, Inc., GE Healthcare Europe, Barcelona, Spain). The signal intensity for each spot was the background subtracted ARM Density (artifact removed median). Only values 1.5 times over the corresponding background were taken as valid measurements. Reproducibility of the replicates was checked using ArrayStat software (Imaging Research, Inc.). We considered data as independent and used a minimum number of two valid replicates in order to calculate the mean and standard deviation values for every gene. Normalization between conditions was done using the global median method. In order to detect the statistically significant, immunoprecipitated genes for condition, a z-test for independent data was applied and, consequently, a z-score for every gene was obtained. A P value of 0.05 and the false discovery rate method were used for controlling the false positive overall error rate. The ratio between the two conditions considered in each experiment, after normalization, was taken as the binding ratio.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a table listing the binding ratio of Rpc-160-HA, a subunit of RNA polymerase III from S. cerevisiae, in a ChIP-chip experiment using anti-HA antibodies (see Materials and methods). The binding ratio is calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies. Additional data files 2 to 7 are tables listing the binding ratio of Sas3-HA, Gcn5-MYC, Elp3-HA, Nut1-HA, Sas2-HA and Hpa2-HA, respectively, in ChIP-chip experiments using anti-HA or anti-MYC antibodies. The binding ratio was calculated as described in Additional data file 1. Additional data file 8 is a table listing the acetylated H3K14 state of the yeast genes. Acetylation at H3K14 was obtained by two ChIP-chip experiments. The first was performed by immunoprecipitating WCE with an antibody that recognizes the carboxyl termini of histone H3 (α-H3Ct) and the other one by immunoprecipitating with α-K14acH3. Ratios from the α-K14acH3 ChIP-chip experiment and the control from the α-H3Ct ChIP-chip experiment were calculated and are listed. Additional data files 9 and 10 are tables listing the acetylated H3K14 state of the yeast genes in gcn5Δ and sas3Δ strains, respectively. Acetylation at H3K14 was obtained as described in Additional data file 8.
The authors are indebted to Mercé Pamblanco for helpful discussions and critical reading of the manuscript. The authors thank Paula M Alepuz and Olivier Lefebvre for kindly providing plasmids and strains. This work was supported by research grants BFU2005-02603 to VT and BMC2003-07072-C03-02 to JEP-O from Ministerio de Educación y Ciencia, Spain, and by grant Ayudas Complementarias GVACOMP2006-159 to VT from Generalitat Valenciana. LER was supported by FP99-73562511 predoctoral grant from Ministerio de Educación y Ciencia.
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