Acetylation of GATA-4 Is Involved in the Differentiation of Embryonic Stem Cells into Cardiac Myocytes* □ S

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin con-figuration. However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. In an ES cell model of developing embryoid bodies, an acetylated form of GATA-4 and its DNA

During embryogenesis, cell type-specific gene expression plays a pivotal role in the determination of cell fate determination, including differentiation, proliferation, and apoptosis. In contrast to other cell types, cardiac muscle cells are highly organized and their developmental processes require a number of cell type-specific transcription factors (1). Among these, a zinc finger protein, GATA-4, is expressed at the earliest stage during heart development. In addition, embryonic stem (ES) 1 cell lines overexpressing GATA-4 show enhancement of cardiac myocyte differentiation, whereas GATA-4-deficient ES cell lines are impaired in differentiation. These findings suggest that GATA-4 is one of the DNA binding transcription factors regulating cardiac muscle cell differentiation (2).
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate gene expression patterns by affecting chromatin structure. These regulators are recruited to target genes in association with specific DNA binding transcription factors. HATs facilitate chromatin opening by acetylating the N-terminal tails of nucleosomal histones, which are rich in lysine residues, thus promoting active transcription (3). Conversely, HDACs deacetylate lysine residues and induce compaction of chromatin, making the access of transcription factors to nearby promoters more difficult and thereby resulting in gene silencing (4). A transcriptional coactivator, p300, serves as one of the intrinsic HATs and plays central roles in a wide range of cellular processes, including proliferation, apoptosis, and differentiation (3,5). In addition, p300 is able to acetylate several DNA binding transcription factors and enhance their DNA binding activities (3). For example, p300 acetylates p53, increases its DNA binding activity, and activates the transcription of p53-inducible genes, contributing to cell cycle arrest or apoptosis (6,7). In cardiac myocytes, p300 acetylates GATA-4, enhances its DNA binding, and plays an important role in the transactivation of hypertrophy-responsive genes (8). However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. Specifically, we would like to know whether the acetylation of GATA-4 occurs in ES cells during their differentiation into cardiac myocytes and, if so, whether pharmacological augmentation of the acetylation could facilitate the differentiation. The present study was performed to address these questions.

MATERIALS AND METHODS
Cell Line and Cell Culture of Embryonic Stem Cells-The 129/Oladerived ES cell lines we used in the present study were ht7 and its derivative (Nkx2.5-GFP ES cells) in which green fluorescent protein (GFP) is knocked into the Nkx2.5 locus (9). These cells were maintained and differentiated as previously described (9). In brief, the cells were grown on gelatinized dishes without feeder cells by using culture me-* This work was supported in part by the Advanced and Innovational Research Program in Life Science and grants from the Ministry of Education, Science, and Culture of Japan (to T. Kita and K. Hasegawa). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. dium containing Glasgow-modified Eagle's medium, 1000 units/ml leukemia inhibitory factor (Chemicon International), 100 mg/ml hygromycin (Invitrogen), 10% heat-inactivated fetal calf serum, 1 ϫ nonessential amino acids, 1 mmol/liter sodium pyruvate, 50 units/ml penicillin, 0.05 mg/ml streptomycin, and 0.1 mmol/liter 2-mercaptoethanol. Differentiation of ES cells was induced through formation of embryoid bodies (EBs). After incubation in hanging drops for 2 days, 60 EBs were transferred into 10-cm bacterial Petri dishes together with 10 ml of differentiation medium and cultured as floating EBs until dissociation for the experiments.
Western Blotting, Immunoprecipitation, and Analysis of the Acetylation State of GATA-4 -Nuclear protein extracts from ES cells were prepared as previously described (8). Immunoprecipitation and Western blotting for p300, GATA-4, and ␤-actin were performed as previously described (8). Briefly, aliquots of the lysates containing 100 g of protein were immunoprecipitated by incubating with goat anti-GATA-4 polyclonal antibody (Santa Cruz Biotechnology) or normal goat IgG in low stringency buffer for 16 h at 4°C and then incubating with protein G beads (Amersham Biosciences) for 2 h at 4°C. The precipitate was washed four times in the same buffer and subjected to Western blotting by using rabbit polyclonal antibody against acetylated lysine (Cell Signaling) or rabbit anti-GATA-4 polyclonal antibody (Santa Cruz Biotechnology). The detection of acetylated GATA-4 was also analyzed by pulse labeling as previously described (8). Briefly, EBs were resuspended in 2.5 ml of medium containing 0.05 mCi of [1-14 C]acetic acid sodium salt/ml (200 Ci/ml; Amersham Biosciences) and incubated for 3 h. Ethanol, the solvent of [1-14 C]acetic acid sodium salt, was removed by evaporation to minimize the cytotoxic effect of the ethanol in the culture medium. Nuclear extracts from these cells were immunoprecipitated using goat anti-GATA-4 antibody (Santa Cruz Biotechnology) or normal goat IgG in low stringency buffer for 16 h at 4°C and incubated with protein G beads for 1 h at 4°C. The precipitate was washed four times in the same buffer, resuspended in 50 l of sodium dodecyl sulfate (SDS) lysis buffer, heated to 95°C for 2 min, electrophoresed in an SDS-polyacrylamide gel, fixed, and autoradiographed using a bioimaging analyzer (BAS 2000; FUJIX).
Electrophoretic Mobility Shift Assays (EMSAs)-EMSAs were carried out as previously described (8). The sequence of the sense strands of the double-stranded oligonucleotides containing a GATA-4 site in the endothelin-1 promoter were as follows: Wt-GATA, 5Ј-CCTCTAGAGC-CGGGTCT TATCTCCGGCTGCACGTTGC-3Ј, and Mut-GATA, 5Ј-CCTCTAGAGCCGGGTCTGCACTCCGGCTGCACGTTGC-3Ј (mutated sequence is underlined). We also used a double-stranded oligonucleotide that contained the Sp-1 binding site as a control probe (purchased from Santa Cruz Biotechnology, Inc.).
Acid Extraction of Proteins from ES Cells and Detection of Histone Acetylation-Histones were isolated by acid extraction using a commercial kit (Upstate) according to the manufacturer's recommendations. In brief, cells were washed with phosphate-buffered saline, resuspended in lysis buffer with 0.2 mol/liter hydrochloric acid, and incubated on ice for 30 min. After centrifugation at 11000 ϫ g for 10 min, the supernatant fraction was dialyzed against 0.1 mol/liter acetic acid twice and against H 2 O three times. After dialysis, the protein concentrations of extracts were measured, and equal aliquots of extracts were subjected to 18% SDS-PAGE. Western blotting for acetylated histone-3/4 and for total histone-3/4 was performed using goat anti-acetylated histone-3/4 polyclonal antibodies (Santa Cruz Biotechnology) and rabbit anti-histone-3/4 polyclonal antibodies (Santa Cruz Biotechnology), respectively.
Chromatin Immunoprecipitation Assay and Real-time PCR Analysis-Chromatin immunoprecipitation assays were performed as previously described (10) with the following modifications. In brief, after fixation of the genomic DNA and nuclear proteins with formalin, extracts were sonicated, subsequently immunoprecipitated with goat polyclonal anti-acetylated histone-4 antibody (Santa Cruz Biotechnology), goat polyclonal anti-GATA-4 antibody (Santa Cruz Biotechnology), or control goat IgG, and immunocomplexes were captured by adding protein G beads. After the precipitates were washed four times in the low stringency buffer, DNA was purified by phenol-chloroform extraction, and precipitated with ethanol. To detect the atrial natriuretic factor (ANF) promoter, which contains two GATA-4 sites (Ϫ281/ Ϫ273 and Ϫ123/Ϫ118), collected DNA was subjected to real-time quantitative PCR analysis using a thermal cycler (ABI Prism®7900HT sequence detection system) with the detection probe and specific primers for the ANF promoter (Ϫ292/Ϫ93). Sequences of the primers were as follows: FAM-AATGTGACTCTTGCAGCTGAGGGTCTGG-TAMRA (detection probe; Applied Biosystems), 5Ј-GAGCGCCCAGGAAGATAACC-3Ј(sense for the ANF promoter), and 5Ј-GCCAGGAGAAGATGCCCTTT-3Ј(antisense for the ANF promoter).

Immunocytochemistry and Western Blotting for Cardiac Myosin
Heavy Chain-After EBs were incubated with trypsin-EDTA, the cells were dissociated, resuspended in the medium, and grown in flask-style chambers with human fibronectin-coated slides (BD Biosciences). After a 24-h incubation, cells were subjected to immunocytochemistry for ␤-myosin heavy chain (MHC) as previously described (11). Briefly, after fixation the cells were incubated with anti-cardiac ␤-MHC monoclonal antibody (Novocastra Laboratories) at a dilution of 1:50, followed by incubation with anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) at a dilution of 1:200. 30 g of whole cell lysates extracted from EBs were subjected to Western blotting using anti-cardiac MHC monoclonal antibody that reacts with both ␣and ␤-cardiac MHC. The levels of signals were quantified by densitometry using NIH image1.61.
Flow Cytometry-For the quantitative analysis of the number of GFP-positive cells in Nkx2.5-GFP ES cells, EBs were dissociated into single cells and analyzed by flow cytometry (BD Biosciences) as previously described (9).
Gene Expression Analysis by Real-time RT-PCR-Total RNA was isolated from EBs by using TRIzol® reagent (Invitrogen). RNA samples were treated with DNaseI (Invitrogen) to eliminate genomic DNA contamination, and cDNA was synthesized by using SuperScriptTMII reverse transcriptase (Invitrogen). For real-time PCR, the reaction was performed with a SYBR® Green PCR master mix (Applied Biosystems),

FIG. 1. Acetylated form of GATA-4 and GATA-4/DNA binding increases in concert with expression of p300 during the differentiation of ES cells.
A, protein extracts from embryoid bodies (EBs days 3, 5, 7, respectively) were subjected to Western blotting for p300, GATA-4, and ␤-actin. B, the extracts (200 g of protein) were immunoprecipitated with anti-GATA-4 antibody or IgG, followed by sequential Western blotting with anti-acetylated lysine antibody and anti-GATA-4 antibody. C, the extracts from EBs (day 7) were probed with a radiolabeled double-stranded oligonucleotide containing the GATA site. Unlabeled competitor DNAs were present at a 100-fold molar excess where indicated. Lane 2, a wild-type GATA oligonucleotide (Wt-GATA); lane 3, a mutant GATA oligonucleotide (Mut-GATA). Supershift assays were performed in the presence of 4 g of either anti-GATA-4 antibody or IgG as indicated (lanes 4 and 5). D and E, the amounts of GATA-4/DNA binding (D) and Sp-1/DNA binding (E) were compared among the same extracts used for panels A and B. and the products were analyzed with a thermal cycler (ABI Prism®7900HT sequence detection system). Levels of GAPDH transcript were used to normalize cDNA levels. Gene-specific primers were used as previously described (9).
Statistical Analysis-Data are presented as means Ϯ S.E. Statistical comparisons were performed using unpaired two-tailed Student's t tests or analysis of variance with Scheffe's test where appropriate, with a probability value Ͻ0.05 taken to indicate significance.

Expression of p300 and the Acetylated Form of GATA-4 Concomitantly Increases during the Differentiation of ES Cells into
Cardiac Myocytes-We examined the time dependence of the expression of p300 and GATA-4 proteins during the differentiation of ES cells into cardiac myocytes. The day on which EBs were transferred into Petri dishes was set as day 0. The expression level of GATA-4 was almost constant on days 3, 5, and 7, though the level slightly increased from day 3 to day 5 (Fig.  1A, middle panel). In contrast, p300 expression progressively increased from day 3 to day 7 (Fig. 1A, upper panel). The expression level of ␤-actin was almost constant. As p300 is able to acetylate GATA-4, we examined whether the acetylated form of GATA-4 also increased in a time-dependent manner during the ES cell differentiation. Protein extracts from EBs at days 3, 5, and 7 were subjected to immunoprecipitation with an anti-GATA-4 antibody, followed by Western blotting using antiacetylated lysine antibody. As shown in Fig. 1B, upper panel, the acetylated form of GATA-4 progressively increased from day 3 to day 7, consistent with the expression pattern of p300. The anti-acetylated lysine antibody was stripped, and then the membrane was reprobed with the anti-GATA-4 antibody. GATA-4 was similarly immunoprecipitated with anti-GATA-4 antibody in these three groups (Fig. 1B, lower panel). No protein was immunoprecipitated with control IgG (lane four).
GATA-4/DNA Binding Increases Concomitantly with GATA-4 Acetylation during ES Cell Differentiation-Next, to examine the changes in the DNA binding activity of GATA-4 during ES cell differentiation, EMSAs were performed. The same extracts from EBs used to detect acetylation were probed with a radiolabeled double-stranded oligonucleotide containing a GATA site. As shown in Fig. 1C, competition EMSAs demonstrated that the retarded band represented specific binding, as evidenced by the fact that it was competed out by an excess of unlabeled GATA oligonucleotide (lane 2) but not by the same amount of an oligonucleotide containing a GATA site with a mutation (lane 3). Furthermore, the retarded band was supershifted by anti-GATA-4 antibody (lane 5) but not by control goat IgG (lane 4). These data confirm that the retarded band represents an interaction of the probe with GATA-4. The amount of GATA-4/DNA binding (Fig. 1D) was markedly increased from day 3 to day 7, whereas Sp-1/DNA binding (Fig.  1E) was not altered. This was consistent with the increase in the amounts of p300 and acetylated GATA-4.
An HDAC Inhibitor, TSA, Induces Acetylation of Histone Tails and Increases the Accessibility of GATA-4 to the ANF Promoter-To examine the effect of TSA, a specific HDAC inhibitor, on the differentiation of ES cells, we first determined whether TSA treatment induces the acetylation of histone tails in these cells. Seven days after the transfer of EBs onto Petri dishes, ES cells were stimulated with TSA for 24 h. Protein extracts from these cells were then subjected to immunoblotting for the acetylated states of histone-3 and histone-4. As shown in Fig. 2A, TSA treatment of ES cells enhanced the acetylation of both histone-3 and histone-4. GATA-4 binds two GATA elements (Ϫ281/Ϫ273 and Ϫ123/118) within the ANF promoter and activates this promoter in a sequence-specific manner (12). As GATA-4 is a substrate of an intrinsic HAT, p300, using chromatin immunoprecipitation assays we examined whether histones near GATA-4 sites within the ANF promoter are acetylated as a result of TSA treatment and, if so, whether the treatment increases the accessibility of GATA-4 to the ANF promoter. Before immunoprecipitation, input genomic DNA was measured using GAPDH primers (Applied Biosystems) by comparison with the standard curve shown in supplemental Fig. S1A, left panels. Extracts from non-treated and TSA-treated ES cells containing the same amounts of GAPDH

FIG. 2. Analysis of histone acetylation and GATA-4/DNA binding induced by trichostatin A (TSA).
A, EBs (day 7) were stimulated with TSA (10 ng/ ml) for 24 h. Proteins from the EBs were then isolated by acid extraction and subjected to Western blotting for acetylated histone-3/4 and histone-3/4 as indicated. B, EBs stimulated with TSA (10 ng/ml) for 24 h (days 7-8) were subjected to chromatin immunoprecipitation followed by quantification by real-time PCR. After fixation by formalin, chromatin from EBs was immunoprecipitated with anti-acetylated histone-4 antibody. ANF promoter sequences (Ϫ292/Ϫ93) including two GATA elements (Ϫ281/Ϫ273 and Ϫ123/ Ϫ118) were detected by real-time PCR analysis. A graphic presentation of results obtained by real-time PCR is shown. The amount of PCR product amplified from untreated EBs was set at 1.0 in each experiment. Data are presented as the means Ϯ S.E. of three independent experiments.
were immunoprecipitated with anti-acetylated histone-4 antibody, anti-GATA-4 antibody, or with control goat IgG as a negative control. DNA was then purified from these precipitates and subjected to real-time PCR to quantify the amount of GATA-4 site-containing DNA sequences in the ANF promoter. We quantified the amounts of DNA by comparison with the standard curve shown in supplemental Fig. S1A, right panels. As shown in Fig. 2B, in extracts precipitated with anti-acetylated histone-4 antibody and those precipitated with anti-GATA-4 antibody, the amount of the ANF promoter containing two GATA-4 sites was much higher in TSA-treated ES cells than in non-treated ES cells. However, this promoter was not detectable in the extracts precipitated with IgG. These findings demonstrate that treatment of ES cells with TSA induces acetylation of histones near GATA-4 sites within the ANF promoter and increases the accessibility of GATA-4 to these sites.
TSA Induces Acetylation of GATA-4 and Enhances GATA-4/DNA Binding in ES Cells-We next examined whether TSA also induces acetylation of the DNA binding transcription factor GATA-4 in ES cells. On day 7, ES cells were stimulated with TSA for 24 h, pulse-labeled with [ 14 C]acetic acid sodium salt, and subjected to immunoprecipitation with antiserum against GATA-4 or with control goat IgG as a negative control. As shown in Fig. 3A, upper panel, incorporation of sodium [ 14 C]acetate into GATA-4 protein was increased by TSA treatment. TSA-induced acetylation of GATA-4 was also confirmed by immunoprecipitation with anti-GATA-4 antibody followed by Western blotting with anti-acetylated-lysine antibody (Fig. 3A,  middle panel). After the anti-acetylated lysine antibody was stripped, the membrane was reprobed with the anti-GATA-4 antibody. GATA-4 was similarly immunoprecipitated with anti-GATA-4 antibody in non-treated and TSA-treated cell extracts (Fig. 3A, lower panels). No protein was immunoprecipitated with control IgG (lane 3). These results indicate that TSA augments the acetylation of GATA-4 in ES cells. As shown in Fig. 3B, the expression of p300 was also increased by TSA stimulation, whereas the levels of GATA-4 and ␤-actin expression were almost constant. In addition, we performed EMSAs using protein extracts from ES cells to determine whether TSA treatment increased the DNA binding of GATA-4. We confirmed by competition and supershift assays that the retarded band indicated by the arrow in Fig. 3C represents specific binding of the probe with GATA-4, similar with the findings shown in Fig. 1C. The GATA-4/DNA binding was increased by TSA treatment in ES cells (Fig. 3C), whereas Sp-1/DNA binding was not altered by TSA treatment (Fig. 3D).
TSA Promotes the Differentiation of ES Cells into Cardiac Myocytes-Finally, to determine whether TSA promotes the differentiation of ES cells into cardiac myocytes, we utilized an ES cell line (Nkx2.5-GFP ES cells) that expresses GFP under the transcriptional control of the cardiac-specific Nkx2.5 promoter. The cardiac specificity of GFP-positive cells in this cell line was shown in a previous study (9). In agreement with a previous study, we confirmed by cell sorting and immunocytochemistry for ␤-MHC that more than 90% of GFP-positive cells were positive for ␤-MHC and that none of the GFP-negative cells was ␤-MHC-positive (data are not shown). EBs derived from Nkx2.5-GFP ES cells (day 7) were stimulated with TSA (10 ng/ml) for 24 h. As shown in Fig. 4A, TSA-stimulated EBs showed distinct fluorescence, whereas non-stimulated EBs showed only vague fluorescence. Then we dissociated EBs, cultured the cells on the fibronectin-coated slides for 24 h, and stained them with an antibody against cardiac ␤-MHC. As shown in Fig. 4B, cells with brown signals indicating the presence of ␤-MHC were observed predominantly in TSA-stimu-lated ES cells. For quantification, the cells dissociated from EBs were also subjected to flow cytometric analysis. In Fig. 4C, the X-and Y-axes in the dot blot represent GFP and propidium iodide (PI) signals, respectively. The percentage of GFP-positive ES cells was significantly (p Ͻ0.05) increased by TSA treatment. TSA did not affect the number of propidium iodidepositive cells, excluding the possibility of its nonspecific toxic effects on ES cells.
To confirm that TSA directs differentiation of ES cells into cardiac myocytes, we performed quantitative RT-PCR analysis using specific primers for the detection of mRNAs encoding Nkx2.5 and ANF. After total RNA was extracted from EBs (days 7-8) incubated with or without TSA for 24 h, cDNA was synthesized and subjected to real-time PCR. The amounts of cDNA for the Nkx2.5, ANF, and GAPDH genes were measured by comparison with the respective standard curves (supplemental Fig. S1B). Consistent with the data of flow cytometry, the mRNA levels of Nkx2.5 and ANF were increased by TSA treatment, as shown in Fig. 5A. Furthermore, whole cell lysates extracted from EBs treated with or without TSA for 48 h (days were immunoprecipitated with anti-GATA-4 antibody, followed by sequential Western blotting with anti-acetylated lysine antibody (middle panel) and with anti-GATA-4 antibody (lower panel). B, the extracts used for panel A before immunoprecipitation were subjected to Western blotting using the anti-p300 antibody (upper panel), anti-GATA-4 antibody (middle panel), and anti-␤-actin antibody (lower panel). C and D, the same nuclear extracts were probed with a radiolabeled doublestranded oligonucleotide containing the GATA-4 site (C) and with one containing the Sp-1 site (D). 7-9) were subjected to Western blotting for cardiac MHC. TSA increased protein expression of cardiac MHC (Fig. 5B). These findings suggest that TSA treatment promotes the differentiation of ES cells into cardiac myocytes. DISCUSSION Differentiation of ES cells into cardiac myocytes requires activation of a cardiac-specific gene program. HATs and HDACs are profoundly involved in gene expression by modulating acetylation of specific transcription factors as well as of histones. The present study was performed to examine the roles of nuclear acetylation during the ES cell differentiation into cardiac myocytes.
Several lines of evidence suggest that p300 is critical for the development of the embryonic heart. Expression of p300 is relatively abundant in the heart, begins to be detected as early as embryo day 7.5, and increases during the subsequent developmental stages (13,14). Homozygous p300 knock-out mice die between 9 and 11.5 days of gestation, exhibiting defects in proper heart development (13). p300 acts as a transcriptional coactivator of GATA-4, one of the earliest markers of cardiac myocyte differentiation, and is required for cardiac-specific gene expression (12,15). In addition, p300 is able to acetylate GATA-4 and enhance its DNA binding activity (8). Homozygous mice in which HAT mutant p300 is knocked in exhibit developmental defects similar to those in p300 knock-out mice (16). These findings demonstrate that the HAT activity of p300 is required for proper heart development. The present study demonstrated that the acetylated form of GATA-4 and GATA-4/ DNA binding increases in concert with the expression level of p300 during the differentiation of ES cells into cardiac myocytes. These findings suggest that p300 is involved in increasing the acetylation and DNA binding of GATA-4 during myocardial cell differentiation, while the precise roles of p300 HAT activity should be clarified by further studies.
Acetylation of nuclear proteins is also regulated by HDACs, whose functions are opposed by HATs (4,17). HDACs repress gene expression not only by affecting chromatin structure but also by directly acting on transcriptional activators, co-repressors, and DNA binding factors. HDACs directly interact with DNA binding transcription factors such as p53, MyoD, and myocyte enhancing factor 2, deacetylate these factors, and repress their transcriptional activity (18 -20). HDACs also perturb the interaction between HATs (p300/cAMP-response element-binding protein-binding protein) and their target transcription factors. Furthermore, HDACs are able to recruit other transcriptional co-repressors to the target genes (17,21). In agreement with previous reports, the present study demonstrated that inhibition of HDACs by TSA induced acetylation of histone tails in ES cells (22,23). These tails included GATA-4 site-containing DNA sequences within the ANF promoter. In addition, TSA enhanced the acetylation of GATA-4 during the differentiation of ES cells into cardiac myocytes. These findings suggest that post-translational GATA-4 modification as well as epigenetic modification are involved in the increase in GATA-4/DNA binding. The results also suggest the hypothesis that TSA treatment releases HDACs from GATA-4 and promotes interaction between p300 and GATA-4. However, TSA also increased the amount of p300 protein in ES cells. Further studies are needed to clarify the precise mechanisms by which TSA induces acetylation and DNA binding of GATA-4.
The present study demonstrated that TSA promotes the differentiation of ES cells into cardiac myocytes. In contrast, it has been reported that TSA inhibits the differentiation of adi-pocytes (22) or oligodendrocytes (23). In the skeletal muscle cell lineage, TSA either augments or inhibits the differentiation depending on the developmental stage (24). These findings suggest that TSA has differential effects in distinct cell types or developmental stages. In addition, individual HDACs play differential roles in cardiac myocytes. For example, one of the class II HDACs, HDAC9, represses the pro-hypertrophic gene program, whereas class I HDAC seems to suppress anti-hypertrophic gene expression (25)(26)(27). Therefore, further studies are needed on the role of each HDAC in different stages of ES cell differentiation into cardiac myocytes.
We have shown here that acetylation of GATA-4 as well as of histones occurs during the differentiation of ES cells into cardiac myocytes and that further augmentation of the acetylation by TSA promotes the differentiation. It is unclear at present whether acetylation of GATA-4 in whole EBs represents acetylation of GATA-4 in cardiac myocytes or their precursors. In mouse embryogenesis, however, GATA-4 is expressed in cardiac tissue during formation and bending of the heart tube at day 8 postcoitum (28). In contrast, GATA-4 is expressed later in the gut epithelium and in primitive gonads (at around day 11.5 postcoitum) (29,30). These findings suggest that GATA-4 expression is restricted to cardiac tissue during early stages of heart development. Therefore, acetylation of GATA-4 detected in the entire EBs might represent acetylation of cardiac GATA-4, which possibly contributes to the differentiation of ES cells into cardiac myocytes. These findings emphasize the important roles of nuclear acetylation in myocardial cell differentiation. Our findings might be applicable to the differentiation of endogenous stem cells into cardiac myocytes in vivo. At present, the differentiation efficiency is too low to be used for myocardial regeneration therapy for patients with end-stage heart failure. Therefore, it would be interesting to test the usefulness of TSA for this therapy in vivo.