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Originally published In Press as doi:10.1074/jbc.M609519200 on January 4, 2007

J. Biol. Chem., Vol. 282, Issue 9, 6696-6706, March 2, 2007
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The Role of Histone Acetylation in Regulating Early Gene Expression Patterns during Early Embryonic Stem Cell Differentiation*Formula

Kevin W. McCool, Xiaojie Xu, Don B. Singer, Fern E. Murdoch, and Michael K. Fritsch1

From the Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, October 10, 2006 , and in revised form, December 19, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse embryonic stem (mES)2 cells are characterized by pluripotency and their capacity for self-renewal (1). Upon exposure to a differentiation signal these cells are capable of multi-lineage differentiation into cells of the three germ layers: ectoderm, mesoderm, and endoderm (2). The term "commitment" is frequently used with regard to achieving a new differentiated phenotype, assayed by changes in morphology and patterns of gene expression (3-5). Commitment to the differentiated phenotype is probably a different, although not completely independent cellular process than loss of the undifferentiated state. The loss of the undifferentiated state occurs after exposure to a differentiation signal that results in an irreversible loss of the mES cell phenotype, characterized by loss of tight colony growth, altered self-renewal, and a pattern of marker gene expression that no longer resembles undifferentiated mES cells. We are interested in identifying the molecular events that regulate loss of the undifferentiated state of mES cells and commitment to lineage specific differentiation.

Mouse ES cells are maintained in the undifferentiated state by adding leukemia inhibitory factor (LIF) to the culture media. A common method of inducing differentiation is withdrawal of LIF from the culture media, resulting in multi-lineage differentiation, indicated by the induced expression of lineage marker genes (6). During early mES cell differentiation, the rate of transcription of large numbers of genes is substantially altered in a time-dependent fashion. Epigenetic changes are proposed to play a role in regulating both local and global gene expression through post-translational histone modifications (7). Some histone modifications result in transcriptional activation, whereas others result in transcriptional repression (8). Some of these modifications appear to spread over broad genomic areas, resulting in large regional effects. In developmental models, covalent modifications of the histone tails have been proposed to act as epigenetic marks that impart transient or permanent "cellular memory" of the transcriptional activation state of specific genes or regions of the genome (9, 10). Chromatin domains with both transcriptional activating and repressive histone tail modifications are seen in undifferentiated mES cells (11). We propose that commitment and changes in transcription associated with very early differentiation of mES cells are at least in part regulated by local and/or global modifications of histone tails.

We examined the role of acetylation of histones H3 and H4 in regulating the early events during differentiation of mES cells. In general, locally increased acetylation of histones H3 and H4 is associated with increased gene transcription. Histone acetylation may increase transcription factor access to the DNA by relieving its interaction with the histone octomer, and high levels of acetylation can also disrupt higher order chromatin folding (12-14). We observed a global increase in acetylation of histones H3 and H4 during the first 5 days of mES cell differentiation induced by LIF withdrawal. Examination of a set of marker genes showed that increased histone acetylation was not seen at all local sites. Rather, it occurred within the regulatory regions of specific genes and temporally correlated with the increase in transcription from each gene. We demonstrated that hyperacetylation of histones H3 and H4 by treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced a rapid morphologic and molecular (gene expression pattern) program that directly mimicked early differentiation induced by LIF withdrawal. Yet the TSA-treated cells were not committed to a differentiation pathway. Withdrawal of TSA resulted in a reversal to the undifferentiated cell phenotype. We conclude that global acetylation of histones H3 and H4 can induce the expression of some marker genes, but is not sufficient for early commitment to differentiation and loss of the pluripotent state.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—CCE mES cells were used for all studies and were obtained from Dr. John Gearhart (Johns Hopkins University) with permission from Dr. Gordon Keller (15, 16). Culture conditions were as previously described (6).

Preparation of Total RNA—RNA samples were isolated from cultured cells (usually one 10-cm plate/sample) using Trizol® reagent (Invitrogen) as previously described (6).

Conventional Reverse Transcription-PCR—The synthesis of cDNA and performance of conventional PCR was as previously reported (6). Additional primers used and detection are described in the supplemental data.

Histone Purification—Cells (10-20 x 106) were washed twice with cold phosphate-buffered saline with 1 mM phenylmethylsulfonyl fluoride (PMSF), scraped into PBS-PMSF, and centrifuged at 300 x g at 4 °C. Hypotonic buffer (10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 30 mM sodium butyrate, 0.5 mM dithiothreitol, 100 µM leupeptin, 10 µg/ml aprotinin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride) was added (0.5 ml), and the cells were centrifuged immediately at 300 x g. The cells were resuspended in 1 ml of hypotonic buffer, allowed to swell for 10 min at 4 °C, and then homogenized in a Dounce homogenizer with 10 strokes of a B pestle. The sample was centrifuged at 5000 rpm for 5 min at 4 °C in a microcentrifuge, and the supernatant was removed. The pellet-containing nuclei was resuspended in 0.2 M H2SO4 at about 2 mg of DNA/ml (about 400 µl), sonicated for 2-3 s at power setting 4, and allowed to stand at 4 °C overnight. This was sonicated again for 2-3 s at 4 °C and centrifuged at the maximum setting for 10 min at 4 °C in a microcentrifuge. One-third volume (133 µl) of 100% trichloroacetic acid (final, 25%) was added and mixed well, and proteins were allowed to precipitate for 30 min at 4 °C followed by centrifugation at maximum speed for 10 min at 4 °C in a microcentrifuge. The histone pellet was washed once with 1 ml of 100% acetone, 0.05 M HCl at 4 °C with vortexing and then centrifuged. The resulting pellet was washed once with 100% acetone at 4 °C and centrifuged. The pellet was allowed to air dry then resuspended in 200-800 µl of water at room temperature and stored at -80 °C. Total protein levels were determined using the BCA kit from Pierce. Histones were separated by SDS-PAGE on 12% gels and immunoblotted as described below.

Immunofluorescence—Cells were harvested as a single cell suspension using trypsin followed by inactivation of the trypsin using growth media-containing serum. The cells were centrifuged onto slides using a CytoproTM cytocentrifuge (Wescor) at 1400 rpm for 5 min at room temperature (RT). Cells were fixed with 2% paraformaldehyde in PBS for 20 min at RT. Cells were rinsed twice with 0.1% Triton X-100 in PBS and blocked with 3% goat serum in 0.1% Triton X-100-PBS for 1 h at RT. Primary antibody at 1:400 dilution was applied in 0.1% Triton X-100/PBS for 1 h at RT in a humidity chamber. The slides were then rinsed 3 times with blocking agent for 2 min each at RT, and secondary antibody at 1:800 dilution was applied for 1 h at RT. The cells were counterstained with 4', 6-diamidino-2-phenylindole at 1:50,000 dilution, and coverslips were applied using Fluoromount®. Primary antibodies include Oct3/4 (BD Transduction Laboratories) and Nestin clone Rat 401 (Stem Cell Technologies). Secondary antibody was Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes).

Use of TSA—TSA (Sigma) was dissolved to 1 mM in 100% ethanol and used at 100 nM in 0.1% final ethanol concentration.

Immunoblotting—Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunostained using standard methods. Primary antibodies were against acetylated histone H3 (Upstate), acetylated histone H4 (Upstate), native histone H3 (Upstate), and dimethylated lysine 4 of histone H3 (Upstate). Blotting for total histone H3 was used as a loading control. Appropriate conjugated secondary antibodies were used, and binding was detected by either enhanced chemiluminescence or chemifluorescence (Amersham Biosciences).

Chromatin Immunoprecipitation (ChIP)—ChIP was performed as previously described with minor alterations as detailed in the supplemental data (17). PCR amplification was performed on DNA recovered from the immunoprecipitation and the total chromatin input. Primers used for PCR of genomic DNA recovered by ChIP for regulatory regions are shown in supplemental Table 1. The PCR products were separated on a 1% agarose gel, stained with vistra green, and detected using the Storm 860 PhosphorImager (GE Healthcare) with quantitation analysis performed using ImageQuant 5.2 software. Background signals for IgG or no antibody controls were subtracted from the immunoprecipitation numbers and generally represented less than 10-15% of the signal with specific antibody. The ratio of the corrected immunoprecipitation value over the total input value for the +LIF (undifferentiated mES cells) sample was arbitrarily set equal to one, and all other ratios were determined relative to this as a -fold acetylation relative to +LIF. For determination of the relative amount of acetylation of histones H3 and H4 at each promoter in the +LIF cells, the highest levels were always at the Oct4 regulatory region, and therefore, this was arbitrarily set equal to 100%, and all other values were expressed relative to this. Three independent experiments were performed, and the data were plotted as the mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Global Histone Acetylation Increased during Early Differentiation of mES Cells Induced by LIF Withdrawal—Total histones were prepared from undifferentiated mES cells maintained in the presence of LIF and from cells induced to differentiate by LIF withdrawal for 1-4 days. As shown in Fig. 1A, levels of acetylated histones H3 and H4 increased over the 4 days of differentiation. The total pool of histone H4 showed an increase in global acetylation (a modification usually associated with localized transcriptional activation) beginning on day 2 after LIF withdrawal, reaching 2.5 on day 3 and persisting through day 4 (Fig. 1B). The level of acetylation of histone H3 also changed, but more slowly, and only achieved a modest 50% increase by day 4. The level of dimethylation on lysine 4 of histone H3 did not significantly change during this period.


Figure 1
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  		FIGURE 1.
Global histone acetylation during very early differentiation of mES cells induced by LIF withdrawal.A, total histones were purified from undifferentiated mES cells (day 0, (+) LIF) or from cells after LIF withdrawal for the indicated days. A representative Western blot analysis for acetylated histone H3 (AcH3), acetylated histone H4 (AcH4), and histone H3 (H3) as a loading control are shown. diMe, dimethylated. B, quantitation of immunoblotting data for AcH3 (white bars), AcH4 (gray bars), or dimethylation of lysine 4 on histone H3 (black bars) is shown. The signal intensity for the modified histone was divided by the intensity for the native histone H3 signal, and that ratio for the undifferentiated cells was arbitrarily set equal to 1. All other ratios were expressed as -fold changes relative to the undifferentiated ratio. The data represent the means ± S.E. of the mean for three independent experiments. C, the pattern of expression of a marker gene set during mES cell differentiation induced by LIF withdrawal was assayed by reverse transcription-PCR on total RNA samples. Time 0 represents undifferentiated mES cells. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a loading control.

 
As shown in Fig. 1C, LIF withdrawal initiated a specific pattern of gene expression and order of induction and repression of marker genes over this time course. After 5 days of LIF withdrawal, this pattern of gene expression was quite dissimilar from the undifferentiated mES cells at day 0, providing a distinct fingerprint for early, multi-lineage differentiation. The gene expression pattern at 0 days of LIF withdrawal represented that of undifferentiated mES cells, with readily detectable transcripts for Oct4, Rex1, and Fgf4. This group of marker genes was termed "ON" to represent their expression in the pluripotent stem cell state. The transcript levels of all three of these genes decreased subsequent to LIF withdrawal but at different rates. The second set of marker genes, referred to here as "INDUCED," were not expressed in the undifferentiated mES cell but displayed a time-dependent, ordered increase in their expression during LIF withdrawal. Transcripts for the PR increased first, within 1 day of LIF withdrawal, whereas levels for Hoxb1 (a patterning gene), Brachy (early mesoderm marker), and Nestin (early neural ectoderm marker) rose between days 2 and 3. The last set of genes, termed "OFF," were not detectable during the early stages of differentiation examined in these studies. Afp (endoderm) and betaH1 globin (later mesoderm) did not increase significantly until after 5 days of LIF withdrawal (6). Prl and Ins are tissue-specific genes that were not expressed after LIF withdrawal in our model system even after longer induction times. Positive controls were performed on samples from cell lines expressing Prl and Ins (data not shown). Glyceraldehyde-3-phosphate dehydrogenase was expressed in undifferentiated cells, and RNA levels did not significantly change over this period. It was used as an internal control.

Localized Histone Acetylation Patterns in Gene Regulatory Regions Correlated with Transcription and Not the Global Pattern—To determine whether changes in gene expression that occurred during early differentiation coincided with localized or global histone acetylation, we examined regulatory regions of the marker gene set during early differentiation using ChIP. We chose to examine regulatory regions, including promoters and enhancers, regardless of their location in the gene since increased histone acetylation in such regions is predicted to be permissive for transcription. Known regulatory regions of the marker gene set (diagrammed in Fig. 2) were amplified after immunoprecipitation of the chromatin with either anti-acetylated histone H3 or anti-acetylated histone H4 antibodies. Figs. 3, 4, and 5 show changes in levels of histone acetylation relative to the undifferentiated (+LIF, day 0) cells for the three gene sets: ON, INDUCED, and OFF. Fig. 3, A and B, show the relative acetylation of histones H3 and H4, respectively, in regulatory regions of the ON genes, which are expressed in undifferentiated mES cells but decrease over 5 days of differentiation (Figs. 1 and 6). The regulatory region of Oct4 demonstrated a small decrease in both histones H3 and H4 acetylation within 1 day of LIF withdrawal. Rex1 and Fgf4 showed a small decrease in histone H4 acetylation only. This loss of histone acetylation, although modest, was in contrast to the global increase in histone acetylation, which represented the average of all localized changes.


Figure 2
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  		FIGURE 2.
Diagram of the locations of the PCR products within the regulatory regions of the genes used for ChIP analysis. The black rectangles represent the PCR products amplified by the primers shown in supplemental Table 1 for each gene. Genes are the marker set from Fig. 1. Regulatory regions were selected based on published work: OCT4 (40), REX1 (41), FGF4 (42), PR (6), HOXb1 (43-45), BESTUB (46), BRACHY (47), GLOBIN Bh1 (48, 49), AFP (50, 51), PRL (52, 53), and INS (54).

 


Figure 3
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  		FIGURE 3.
ChIP for acetylated histones H3 and H4 at the regulatory regions of the ON gene set as diagrammed in Fig. 2. The level of acetylated histones at times 4 h and 1, 2, 3, and 4 days of LIF withdrawal is expressed as a -fold change relative to that for undifferentiated mES cells (+LIF). Each data point represents the means ± S.E. of the mean from three independent experiments. A, -fold change in acetylated histone H during early differentiation. B, -fold change in AcH4 during early differentiation.

 
Fig. 4 shows the relative histone acetylation in the regulatory regions of genes that were not expressed in undifferentiated cells but were induced during LIF withdrawal (INDUCED). Local increases in histone acetylation over the differentiation time course were observed for all of these genes. PR is a complex gene, which we have recently shown is turned on in mES cells 12-24 h after LIF withdrawal (Fig. 1) (6). Two isoforms of PR, PRA and PRB, are encoded by a single gene with two separate promoters (18). The PRB promoter is upstream of the PRA promoter; therefore, the PRB transcript shares all the PRA sequence plus an additional 5' sequence. The PCR primers we used in Fig. 1C did not distinguish between the two PR transcripts. As shown in Fig. 4, A and B, the PRB promoter demonstrated a 2-fold increase in histone H4 acetylation and a smaller increase in histone H3 acetylation as early as 4 h after LIF withdrawal. In contrast, acetylation of histone H4 at the PRA promoter showed a transient increase at 2 days and little change in acetylation of histone H3. The increased histone acetylation of PRB preceded the increase in PR transcripts observed at 12-24 h (6). Two known regulatory regions for the Hoxb1 gene, one in the 5' promoter and the other in the 3' region of the gene, were examined (19). Acetylation of histone H4 in the 3' region increased by day 2, reaching 2-fold by day 4 of differentiation, with little change in histone H3 acetylation (Fig. 4, A and B). H4 acetylation of the 5' region rose within 4 h and persisted to day 4 (Fig. 4, C and D). Acetylation of histone H3 also rapidly increased, reaching a 3.8-fold increase by day 4. The increase in histone H3 acetylation preceded the increased expression of Hoxb1 seen at 2-3 days (Figs. 1 and 6). As shown in Fig. 4, C and D, acetylation of both histones H3 and H4 in the Nestin regulatory region increased 3 days after LIF withdrawal, coincident with increased gene expression. Acetylation of histone H3 in the Brachy regulatory region also increased 3 days after LIF withdrawal (Fig. 4C), coincident with increased accumulation of RNA (Fig. 1). Increased acetylation of histone H4 in this region was not visible until after 4 days (Fig. 4D). Overall, increased acetylation of either or both histones H3 and H4 was observed in at least one regulatory region of each INDUCED gene preceding or coincident with RNA accumulation.


Figure 4
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  		FIGURE 4.
ChIP for acetylated histones H3 and H4 at the regulatory regions of the Induced gene set as diagrammed in Fig. 2. Data are expressed as in Fig. 3. A and C, -fold change in acetylated histone H3 during early differentiation. B and D, -fold change in AcH4 during early differentiation.

 


Figure 5
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  		FIGURE 5.
ChIP for acetylated histones H3 and H4 at the regulatory regions of the OFF gene set as diagrammed in Fig. 2. Data are expressed as in Fig. 3. A and C, -fold change in acetylated histones H3 during early differentiation. B and D, -fold change in AcH4 during early differentiation.

 
Fig. 5, A and B, show the relative acetylation of histones H3 and H4, respectively, in three regulatory regions of two genes, whose transcripts accumulate only after 5 days of LIF withdrawal (6). The beta-globin gene locus is complex, containing multiple beta-globin genes and regional as well as gene-specific regulatory regions. An upstream DNase hypersensitive site (locus control region HS2) is necessary for initial de-repression of the entire beta-globin locus (20-23). The betaH1 globin (Hbb-bh1, hemoglobin z) gene is expressed early in development (24). The local promoter of this gene (bH1) acquired a small, 1.5-fold increase in histone H3 acetylation after 3 days of LIF withdrawal, but little change in the level of H4 acetylation occurred over the 4 days (Fig. 5, A and B). The HS2 locus control region showed a unique pattern, with increased acetylation of both histones H3 and H4 only 4 h after LIF withdrawal followed by a return of acetylated histone H4 to the level seen in undifferentiated cells and a fall in acetylated histone H3 to 50% that seen in undifferentiated cells. This lower level was maintained throughout the 4 days of LIF withdrawal (Fig. 5A). The Afp regulatory region demonstrated an average 1.5-fold increase in the acetylation of histone H3 over the entire 4 days of LIF withdrawal and little change at H4 (Fig. 5, A and B). We have previously reported that both betaH1 globin and Afp are expressed later in this cell model (6), and these local increases in histone acetylation may be necessary to maintain these genes in a permissive chromatin structure during this early differentiation period. Finally, levels of histone acetylation were not altered in the regulatory regions of two genes (Ins and Prl) that were not expressed even at later times in this mES cell model (Fig. 5, C and D).

Basal Acetylation of Histones H3 and H4 in Undifferentiated mES Cells Varied across the Regulatory Regions of the Marker Gene Set—We used ChIP to obtain a measure of the relative basal acetylation levels of the histone tails at the regulatory regions of the marker gene set in the undifferentiated mES cells as shown in Fig. 6. In all experiments the level of acetylation of both histones H3 and H4 was highest in the Oct4 regulatory region. We, therefore, arbitrarily set this to 100%, and levels in other regulatory regions are shown relative to the Oct4. The three genes of the ON set (Oct4, Rex1, Fgf4) showed some of the overall highest levels of acetylation of histones H3 (Fig. 6A) and H4 (Fig. 6B). The relative levels of histone acetylation in the regulatory regions of the rest of the marker gene set were quite variable. There was no apparent relationship between the level of histone acetylation in the undifferentiated state and the order of transcriptional activation that is seen in response to LIF withdrawal for the INDUCED set. This was indicated by the relatively high level of basal histone acetylation for Brachy despite having the slowest induced expression (as seen in Fig. 1). PRB, both Hoxb1 regulatory regions, and Nestin had lower basal acetylation of histones H3 and H4 than the ON set of genes yet were rapidly activated by LIF withdrawal. The basal acetylation of histones H3 and H4 for the "OFF" set of genes was also lower than for the ON set. Overall, integrating the results from Fig. 6 with Figs. 3, 4, and 5, differentiation induced the largest increases in local histone acetylation in gene regulatory regions that both had a low level of basal histone acetylation and were transcriptionally activated by differentiation.


Figure 6
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  		FIGURE 6.
Basal levels of acetylation of histones H3 and H4 in undifferentiated mES cells vary substantially between gene regulatory regions. Histone acetylation in the regulatory regions shown in Fig. 2 was assayed by ChIP on undifferentiated mES cells. The level of histone acetylation at the Oct4 promoter was arbitrarily set equal to 100%, and the levels of acetylation of histone H3 (panel A) or histone H4 (panel B) are expressed as a percentage relative to the level of acetylation of the Oct4 promoter. The data represent the mean ± S.E. from three independent experiments.

 
TSA Treatment Induced an Early Differentiation Phenotype That Mimics LIF Withdrawal—Based on the observation that global histone acetylation increased during differentiation induced by LIF withdrawal (Fig. 1), we tested whether induction of global histone acetylation alone was sufficient for early differentiation of mES cells. TSA is a potent inhibitor of class I and II histone deacetylases. Inhibition of these histone deacetylases results in increased global acetylation of histones in most cell types including mES cells (Ref. 25 and supplemental Fig. 1). As shown in Fig. 7A, TSA treatment induced dramatic changes to cell morphology in mES cells maintained in LIF as seen by comparing panels 1 and 2. The morphology of TSA-treated cells closely resembled that of LIF withdrawn cells (panel 4), wherein the tight colonies were disrupted, cellular extensions were easily seen, and individual cell borders were clear. TSA treatment of cells grown in the absence of LIF resulted in a similar differentiated morphology (panel 5). The morphological changes induced by TSA were reversible upon its removal from LIF-containing media as seen in the third column of Fig. 7A, panels 3 and 6. Colonies were scored for phenotype, and quantitative data are presented in supplemental Fig. 2. Approximately 90% of control colonies (no TSA, grown in LIF) showed an undifferentiated morphology. Nearly 100% of colonies showed a differentiated morphology after TSA treatment in the presence of LIF. More than 65% of the colonies regained an undifferentiated morphology after withdrawal of TSA for 2 days (TSA WD2d).

As shown in Fig. 7B, TSA treatment of mES cells in the presence of LIF induced an ordered change in gene expression that mimicked LIF withdrawal, although it was markedly accelerated. Oct4, a gene from the ON set, was expressed in undifferentiated (time 0) mES cells and decreased after 10 h of TSA treatment. TSA treatment caused a time-dependent, ordered induction of the genes in the INDUCED set; PR, Hoxb1, and Nestin followed by Brachy. The OFF genes Prl and betaH1 globin were not induced by TSA treatment; however, a small amount of Afp was detectable in some replicates at 23 h. The pattern of gene expression observed after 23 h of TSA in the presence of LIF matched that induced by LIF withdrawal for 5 days (compare Figs. 7B with 1C). These results support a model in which the hyperacetylation induced by TSA is permissive for the execution of a highly regulated gene expression program.

As shown in Fig. 7C, the effects of TSA were reversible at the level of gene expression. The expression pattern for genes altered by TSA in Fig. 7B was determined over a time course after removal of TSA. Cells were treated with TSA for 20 h (0 h recovery time after TSA), then TSA was removed, and cells were cultured for the indicated recovery times in +LIF media. As shown in Fig. 7C, the gene expression pattern reverted to that of the undifferentiated cells as early as 24 h after TSA withdrawal and was maintained to 48 h.


Figure 7
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  		FIGURE 7.
TSA induces cell morphology and gene expression changes that mimic differentiation induced by LIF withdrawal, but the TSA effects are reversible. A, changes in colony morphology induced by various treatments. In the far left panels cells were grown without TSA and in the presence (panel 1) or absence (panel 4) of LIF for 3 days. In the middle panels the cells were treated with TSA for the last 20 h of culture in the presence (panel 2) or absence (panel 5) of LIF for 3 days. In the far right panels the cells were grown in LIF containing media and treated with TSA for 20 h, then TSA was withdrawn for 1 (panel 3) or 2 (panel 6) days. All photos taken at 100x magnification. B, mES cells were grown for 71 h in media containing LIF the entire time with either 0.1% ethanol (time 0) or 100 nM TSA added for the last 2, 5, 10, or 23 h of culture. RNA was prepared, and reverse transcription-PCR was performed for the expression levels of the marker gene set. C, cells were treated with 100 nM TSA for 20 h in the presence of LIF, and then TSA was withdrawn (time 0), and cells were maintained in LIF-containing media for the indicated times. RNA was prepared, and reverse transcription-PCR was performed for the expression levels of the marker gene set.

 
TSA Treatment Did Not Support Commitment to Differentiation in Individual Cells—TSA treatment of mES cells mimicked differentiation induced by LIF withdrawal. The results from Fig. 7 represented a population of cells. This population of TSA-treated cells did not commit to a differentiation pathway as defined by an irreversible change in gene expression and morphology. There are two possible explanations for these results. First, this could be due to selection, where a small number of cells remained undifferentiated during TSA treatment, and those cells expanded after TSA withdrawal, whereas cells displaying the differentiated phenotype died or became quiescent. Alternatively, TSA may have induced a phenotypic change in all the cells that was truly reversible. This was the hypothesis we favored due to prior observations of the cultures that indicated morphological changes to virtually all colonies on the plate, limited cell death, and consistent population doubling times. Over 4 days the population doubling time of cultures grown in the presence or absence of LIF without LIF were the same (data not shown).

To quantitatively test if the molecular changes induced by TSA were reversible at the level of individual cells, we performed immunofluorescence analysis for Oct4 (a marker of undifferentiated cells) and Nestin (a marker of differentiated cells) protein expression levels. As seen in Fig. 8A, high levels of Oct4 protein (top left panel) were seen in almost every undifferentiated cell. TSA treatment in the presence of LIF resulted in a dramatic decrease in Oct4 protein in almost every cell (Fig. 8A, top right panel). LIF withdrawal for 3 days also resulted in a decrease in cells expressing Oct4 protein. Within 24 h after TSA withdrawal, Oct4 protein was re-expressed in almost 75% of cells, and this was maintained at 2 days of TSA withdrawal (bottom right panel of Fig. 8A). Nestin staining of cells from the same set of treatments is shown in Fig. 8B. Virtually no Nestin protein was seen in any undifferentiated cell (top left, Fig. 8B). TSA treatment in the presence of LIF resulted in a small amount of perinuclear Nestin protein in about 50% of cells (top right, 8B). Withdrawal of LIF for 3 days also induced Nestin protein in more than 30% of cells (lower left panel, Fig. 8B). By 2 days after TSA withdrawal less than 25% of cells contained Nestin protein, and the intensity of staining was substantially decreased (bottom right, Fig. 8B). Quantitation of three independent experiments is shown in Fig. 8C. Clearly, the differentiated phenotype induced by TSA treatment was reversible at the individual cell level.

We considered the possibility that a small population of undifferentiated mES cells in the culture were unaffected by TSA, and these cells simply repopulated the culture as undifferentiated cells upon withdrawal of TSA. To test the possibility of this cell selection model, we performed cell counts using trypan blue exclusion during a time course of TSA treatment and withdrawal. As can be seen in supplemental Fig. 3, during the 18 h of TSA treatment there was no effect on the cell growth rate (doubling time of about 12 h). A 2-fold decrease in cell growth was observed for the 24 h after TSA withdrawal (doubling time about 24 h), and then the doubling time recovered to the control rate of about 12 h. Changes in cell growth rate are an average of cell proliferation and cell death. These data suggested that potentially there was a small component of selection imposed by TSA treatment. However, the small decease in doubling time for 1 day could not totally account for the percentage of cells with reversed phenotypes seen in Fig. 8, A-C. There were at least 3-4-fold more Oct4-positive cells in the TSA withdrawn cultures than would be expected if 100% of the positive cells resulted only from selection and not from a reversible phenotype.

Increased Acetylation of Histones H3 and H4 after TSA Treatment Did Not Occur at the Regulatory Regions of All Genes—We used ChIP to obtain a measure of the relative basal acetylation levels of the histone tails at the regulatory regions of the marker gene set in the undifferentiated mES cells (Fig. 6) and after TSA treatment. Fig. 9 shows the -fold increase in histone acetylation for each gene regulatory region induced by TSA treatment relative to undifferentiated cells. These data are also plotted as % acetylation and directly compared with the basal levels of histone acetylation from Fig. 6 (see supplemental Fig. 4). The three genes of the ON group showed no increased acetylation of histones H3 (Fig. 9A) and H4 (Fig. 9B) in response to TSA. These gene regions had high levels of basal histone acetylation (as shown in Fig. 6) and were actively transcribed in the undifferentiated cells (Fig. 1C). The ability of TSA to increase the histone acetylation levels of regulatory regions of the INDUCED genes was related to their basal acetylation state (seen in Fig. 6). The regions with the lowest levels of basal acetylation, such as PRB, Hoxb1 3' and 5', and Nestin, showed the greatest increases induced by TSA. Perhaps most interesting was the behavior of the OFF set of genes. TSA induced a significant increase in histone acetylation in the regulatory regions of the two genes that were expressed at later times in our culture system, betaH1 globin and Afp. The levels of acetylation did not change for the two genes that were completely transcriptionally silent in this model system, Prl and Ins. Overall, the effects of TSA on localized histone acetylation were very similar to the effects of LIF withdrawal. We concluded that increased histone acetylation was an active event in triggering the expression of early differentiation genes but was not sufficient for activation of very late genes, maintenance of the differentiated phenotype, or irreversible loss of the undifferentiated state.


Figure 8
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  		FIGURE 8.
TSA induces changes in the protein levels of Oct4 and Nestin within individual cells that are reversible upon removal of TSA. Cytospins of cells were prepared, and immunofluorescence for Oct4 (A) and Nestin (B) protein was performed. The upper left panel in both A and B shows undifferentiated mES cells (Control, +LIF). The upper right panel in both A and B shows cells harvested immediately after 20 h of TSA treatment while in LIF-containing media. The lower left panel in both A and B shows cells cultured without LIF for 3 days (-LIF). The lower right panel in both A and B shows cells culture in LIF-containing media for 2 days after withdrawal of TSA treatment. All images are at 200x. C, the immunofluorescence data were quantitated and plotted as the percentage of positively staining cells for Oct4 (black bars) and Nestin (gray bars). The data represent the mean ± S.E. for three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Major reorganization of the chromatin structure including changes in histone modifications and DNA methylation are features of early embryonic development as well as of ES cell loss of pluripotency and commitment to differentiation (26-30). Considerable work has been done on chromatin changes during late, lineage-specific differentiation, X chromosome inactivation, and in germ cells where an increase in heterochromatin is a major feature, whereas relatively little investigation has centered on the very early time frame (0-96 h) after an mES cell is exposed to a differentiation signal. Loss of compact colony morphology and dramatic changes in the expression of a set of marker genes characterize the phenotype associated with this very early differentiation of mES cells in response to LIF withdrawal. We have focused on this early time frame because of the rapid changes in gene expression and loss of pluripotency that we expect are accompanied and indeed facilitated by epigenetic changes in the chromatin. We expect some epigenetic changes are part of the mechanism of immediate gene expression changes, but others are important to mark genes for later changes in expression and for prevention of phenotypic reversion. Understanding the program of epigenetic marks that drive the cell into a differentiation pathway will contribute to efforts to derive specific cell lineages for therapeutic purposes.

The experiments presented here were designed to examine the role of histone acetylation, generally associated with transcriptional activation, in the very earliest steps of the differentiation pathway induced by LIF withdrawal. During this early time frame the expression of markers of all three germ cell lineages is seen, but the cells are not yet committed to specific lineages (3, 6). We observed a global increase in the acetylation of both histone H3 and H4 by either LIF withdrawal or TSA treatment. Global induction of histone acetylation by either treatment leads to the immediate gene expression changes characteristic of the very early differentiation program. It is also potentially important for marking genes that will be expressed later in differentiation such as Afp and betaH1 globin. However, histone acetylation alone is not sufficient for irreversible exit from the stem cell state and progression to a lineage specific differentiation program as demonstrated by the TSA withdrawal results (Figs. 7 and 8).


Figure 9
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  		FIGURE 9.
The effect of TSA on histone H3 and H4 acetylation levels varies substantially between genes. Histone acetylation in the regulatory regions shown in Fig. 2 was assayed by ChIP after treatment for 20 h with or without TSA in LIF-containing media. The acetylation level in the presence of TSA was divided by that in the absence of TSA to calculate the -fold effect of TSA. The -fold changes in acetylation of histone H3 (panel A) or histone H4 (panel B) in response to TSA are shown. The data represent the mean ± S.E. from three independent experiments.

 
Role of Histone Acetylation in Gene Expression during Early mES Cell Differentiation—The global change in acetylation of the total histone pool we observed in Fig. 1 is an average of the differential response to LIF withdrawal across regions of heterochromatin and euchromatin. The few studies that have quantitated changes in global histone acetylation in mES cells during differentiation have focused on later, more differentiated cells or on heterochromatin and X inactivation. A recent study on epigenetic changes between mES cells and neural progenitor cells showed a robust increase in the distribution and amount of methylation of lysine 9 on histone H3 and a decrease in acetylation on both histones H3 and H4 on day 7 of their differentiation protocol (31). The only other time point examined was 24 h of LIF withdrawal at which a slight increase in H4 acetylation was observed similar to our own result. A series of studies have shown that a protocol of embryoid body differentiation in female mES cells leads to inactivation of one of the X chromosomes and increases overall heterochromatin formation over 1-3 weeks (32). One study focused only on sites of heterochromatin formation where a decrease in histone acetylation is observed at those sites (33). Another study examined global histone acetylation during embryoid body differentiation of mES cells and saw an initial decrease and then recovery of histone acetylation levels (34). The differentiation protocol of embryoid body formation used in that report is significantly different from our own. This overview of the published data and our own results suggest that there is a very early increase in global histone acetylation, whereas epigenetic events to restrict active chromatin including histone deacetylation, histone methylation, and DNA methylation dominate later time points. Histone acetylation is a dynamic process resulting from the balance between acetylase and deacetylase enzymes (35). We expect that the activity and targeting of these enzymes to specific loci will be affected by differentiation. We postulate several roles for this very early increase in global histone acetylation during mES cell differentiation. The first is immediate changes in transcription at specific genes. The second is as a permissive mark for genes that will be expressed later and need to be protected during subsequent events to restrict chromatin. The third is as part of a mechanism to propel the cell irreversibly out of the pluripotent state.

We observe a striking coincidence in the pattern of local histone acetylation changes when cells are treated with either LIF withdrawal or TSA. The global increases in histone acetylation are not evenly distributed across regulatory regions of our marker genes (Figs. 3, 4, and 5 and 9) but correlate best with the transcriptional activity of that marker gene. All of the genes in the INDUCED set show an increase in either or both histone H3 and H4 acetylation in at least one regulatory region coincident with RNA accumulation. However, genes that are already transcriptionally active (ON) or inactive in our model (Ins and Prl) show little or no increase in histone acetylation and even some decrease for the ON set. These results are consistent with the first postulated role for the global increase in histone acetylation; that is, immediate changes in the transcriptional activation of specific genes.

We observed an increase in acetylated histone H3 in the regulatory region of Afp and the transcriptional start site of betaH1 globin by 2-3 days of LIF withdrawal or TSA treatment despite the lack of expression of these genes during this time course. However, expression of Afp and betaH1 globin is seen within 4-5 days of LIF withdrawal in embryoid bodies (6). This distinguishes the expression pattern of these two genes from Ins and Prl for which no expression is seen even with embryoid body formation for 16 days (data not shown). These two genes are an example of the second postulated role of early increased histone acetylation; the genes are marked for future expression, but increased histone acetylation alone is not sufficient to induce transcription. It is possible a repressive histone modification must be removed.

Increased Histone Acetylation Is Not Sufficient for mES Cells to Commit to Differentiation—Treatment of mES cells with TSA induces changes that mimic the withdrawal of LIF for 3-5 days. Cells that have been cultured in the absence of LIF for 3 days are committed to differentiation.3 Specifically, returning cells differentiated by 3 days of LIF withdrawal to culture in media with LIF does not restore the tight colony morphology or gene expression pattern seen in the pluripotent mES cell. TSA treatment results in a reversible phenotypic change, and the cells can revert to the pluripotent mES cell phenotype upon removal of TSA. We conclude from these data that the increase in histone acetylation observed upon LIF withdrawal and induced by histone deacetylase inhibition with TSA is not sufficient for maintenance or progression of differentiation. Evidently, additional events are required that occur with LIF withdrawal but that are not induced by TSA.

We hypothesize that a second, essential signal is missing when the cells receive TSA treatment alone. This signal may be a change in the activity of a specific transcription factor (activator or repressor) or other specific epigenetic changes different from the histone acetylation studied here. Possible epigenetic changes would include other histone tail modifications, substitutions of histone variants into nucleosomes, and DNA methylation at either a local or global level (8, 36, 37-39). Recent work has identified the presence of a chromatin domain with bivalent epigenetic marks in the regulatory regions of a large set of developmental genes in mES cells (11). The marks are methylation on lysine 4 of histone H3 and methylation on lysine 27 of histone H3. Lys-4 methylation is generally associated with active chromatin, whereas Lys-27 methylation is a repressive modification. These authors propose that the presence of both marks keeps genes silent but poised for activation in mES cells. In retinoic acid-differentiated cells, the two marks tend to resolve with a gene having only one mark remaining that is correlated with its transcriptional status. This type of opposed and balanced epigenetic marks would be consistent with our own observations. Increased acetylation alone induced by TSA can support the expression of a set of genes poised for transcription. However, the removal of repressive marks may be required to maintain the expression of those poised genes. Removal of activating or repressive marks on additional genes may be required to commit the cell to a differentiation pathway. Our data suggest that regulation of gene transcription during mES cell differentiation may best be described by a multivalent model of histone tail modifications including removal of repressive marks such as Lys-27 methylation (11) and the maintenance or addition of activating marks such as Lys-4 methylation (11) and acetylation (this report).

This report adds to our understanding of the role for epigenetic marks in mES cell differentiation. The great promise of ES cells is the hope of producing highly pure and functional progenitor cells for treatment of disease. Elucidation of the program of epigenetic marks required to achieve a specific cell type will contribute to realizing this promise.


   FOOTNOTES
 
* This work was supported in part by NIDDK, National Institutes of Health Public Health Service Grant RO1DK64243 (to M. K. F.), a University of Wisconsin/Howard Hughes Medical Institute Faculty Development Program award (to M. K. F.), a Society for Pediatric Pathology Young Investigator research grant (to M. K. F.), a University of Wisconsin Hilldale award (to K. W. M.), and a Pfizer, Inc. research fellowship (to K. W. M.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental material. Back

1 To whom correspondence should be addressed: University of Wisconsin, Dept. of Pathology and Laboratory Medicine, 5250 MSC, 1300 University Ave, Madison, WI 53706. Tel.: 608-263-5351; Fax: 608-265-3301; E-mail: mkfritsch{at}wisc.edu.

2 The abbreviations used are: mES cells, mouse embryonic stem cells; LIF, leukemia inhibitory factor; TSA, trichostatin A; Fgf, fibroblast growth factor; PR, progesterone receptor; Brachy, brachyury; Afp, {alpha}-fetoprotein; Ins, insulin; Prl, prolactin; ChIP, chromatin immunoprecipitation; Hoxb1, homeobox b1; PR, progesterone receptor; PBS, phosphate-buffered saline; RT, room temperature; AcH3, acetylated histone H3. Back

3 M. K. Fritsch, D. B. Singer, and F. E. Murdoch, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Carley N. Sauter, Rebecca L. McDermid, and Jeremy M. Roberts for technical assistance.



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