TheRoleofHistoneAcetylationinRegulatingEarlyGene ... · 2007. 2. 17. · 6696 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER9•MARCH2,2007. cells. In general, locally increased

The Role of Histone Acetylation in Regulating Early GeneExpression Patterns during Early Embryonic StemCell Differentiation*□S

Received for publication, October 10, 2006, and in revised form, December 19, 2006 Published, JBC Papers in Press, January 4, 2007, DOI 10.1074/jbc.M609519200

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

We have examined the role of histone acetylation in the veryearliest steps of differentiation ofmouse embryonic stemcells inresponse to withdrawal of leukemia inhibitory factor (LIF) as adifferentiation signal. The cells undergo dramatic changes inmorphology and an ordered program of gene expressionchanges representing differentiation to all three germ layersover the first 3–5 days of LIF withdrawal. We observed a globalincrease in acetylation on histone H4 and to a lesser extent onhistone H3 over this time period. Treatment of the cells withtrichostatin A (TSA), a histone deacetylase inhibitor, inducedchanges in morphology, gene expression, and histone acetyla-tion that mimicked differentiation induced by withdrawal ofLIF. We examined localized histone acetylation in the regula-tory regions of genes that were transcriptionally either active inundifferentiated cells, induced during differentiation, or inac-tive under all treatments. There was striking concordance in thehistone acetylation patterns of specific genes induced by bothTSA and LIF withdrawal. Increased histone acetylation in localregions correlated best with induction of gene expression.Finally, TSA treatment did not support themaintenance or pro-gression of differentiation. Upon removal of TSA, the cellsreverted to the undifferentiated phenotype. We concluded thatincreased histone acetylation at specific genes played a role intheir expression, but additional events are required for mainte-nance of differentiated gene expression and loss of the pluripo-tent state.

Mouse embryonic stem (mES)2 cells are characterized bypluripotency and their capacity for self-renewal (1). Upon

exposure to a differentiation signal these cells are capable ofmulti-lineage differentiation into cells of the three germ layers:ectoderm, mesoderm, and endoderm (2). The term “commit-ment” is frequently used with regard to achieving a new differ-entiated phenotype, assayed by changes in morphology andpatterns of gene expression (3–5). Commitment to the differ-entiated phenotype is probably a different, although not com-pletely independent cellular process than loss of the undiffer-entiated state. The loss of the undifferentiated state occurs afterexposure to a differentiation signal that results in an irreversibleloss of the mES cell phenotype, characterized by loss of tightcolony growth, altered self-renewal, and a pattern of markergene expression that no longer resembles undifferentiatedmEScells.We are interested in identifying themolecular events thatregulate loss of the undifferentiated state ofmES cells and com-mitment to lineage specific differentiation.Mouse ES cells are maintained in the undifferentiated state

by adding leukemia inhibitory factor (LIF) to the culturemedia.A commonmethod of inducing differentiation is withdrawal ofLIF from the culture media, resulting in multi-lineage differen-tiation, indicated by the induced expression of lineage markergenes (6). During earlymES cell differentiation, the rate of tran-scription of large numbers of genes is substantially altered in atime-dependent fashion. Epigenetic changes are proposed toplay a role in regulating both local and global gene expressionthrough post-translational histonemodifications (7). Some his-tone modifications result in transcriptional activation, whereasothers result in transcriptional repression (8). Some of thesemodifications appear to spread over broad genomic areas,resulting in large regional effects. In developmental models,covalent modifications of the histone tails have been proposedto act as epigenetic marks that impart transient or permanent“cellular memory” of the transcriptional activation state of spe-cific genes or regions of the genome (9, 10). Chromatin domainswith both transcriptional activating and repressive histone tailmodifications are seen in undifferentiated mES cells (11). Wepropose that commitment and changes in transcription associ-ated with very early differentiation of mES cells are at least inpart regulated by local and/or global modifications of histonetails.We examined the role of acetylation of histones H3 andH4

in regulating the early events during differentiation of mES

* This work was supported in part by NIDDK, National Institutes of HealthPublic Health Service Grant RO1DK64243 (to M. K. F.), a University ofWisconsin/Howard Hughes Medical Institute Faculty Development Pro-gram award (to M. K. F.), a Society for Pediatric Pathology Young Inves-tigator research grant (to M. K. F.), a University of Wisconsin Hilldaleaward (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 thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental material.

1 To whom correspondence should be addressed: University of Wisconsin,Dept. of Pathology and Laboratory Medicine, 5250 MSC, 1300 UniversityAve, Madison, WI 53706. Tel.: 608-263-5351; Fax: 608-265-3301; E-mail:[email protected].

2 The abbreviations used are: mES cells, mouse embryonic stem cells; LIF,leukemia inhibitory factor; TSA, trichostatin A; Fgf, fibroblast growthfactor; PR, progesterone receptor; Brachy, brachyury; Afp, �-fetopro-

tein; Ins, insulin; Prl, prolactin; ChIP, chromatin immunoprecipitation;Hoxb1, homeobox b1; PR, progesterone receptor; PBS, phosphate-buff-ered saline; RT, room temperature; AcH3, acetylated histone H3.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 9, pp. 6696 –6706, March 2, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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cells. In general, locally increased acetylation of histones H3and H4 is associated with increased gene transcription. His-tone acetylation may increase transcription factor access tothe DNA by relieving its interaction with the histoneoctomer, and high levels of acetylation can also disrupthigher order chromatin folding (12–14). We observed a glo-bal increase in acetylation of histones H3 and H4 during thefirst 5 days of mES cell differentiation induced by LIF with-drawal. Examination of a set of marker genes showed thatincreased histone acetylation was not seen at all local sites.Rather, it occurred within the regulatory regions of specificgenes and temporally correlated with the increase in transcrip-tion from each gene.We demonstrated that hyperacetylation ofhistones H3 and H4 by treatment with the histone deacetylaseinhibitor trichostatin A (TSA) induced a rapid morphologicand molecular (gene expression pattern) program that directlymimicked early differentiation induced by LIF withdrawal. Yetthe TSA-treated cells were not committed to a differentiationpathway.Withdrawal of TSA resulted in a reversal to the undif-ferentiated cell phenotype.We conclude that global acetylationof histones H3 and H4 can induce the expression of somemarker genes, but is not sufficient for early commitment todifferentiation and loss of the pluripotent state.

EXPERIMENTAL PROCEDURES

Cell Culture—CCE mES cells were used for all studies andwere obtained fromDr. John Gearhart (Johns Hopkins Univer-sity) with permission from Dr. Gordon Keller (15, 16). Cultureconditions 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 PCRwas as previouslyreported (6). Additional primers used and detection aredescribed in the supplemental data.Histone Purification—Cells (10–20 � 106) were washed

twice with cold phosphate-buffered saline with 1 mM phenyl-methylsulfonyl fluoride (PMSF), scraped into PBS-PMSF, andcentrifuged at 300� g at 4 °C.Hypotonic buffer (10mMHEPES,pH 7.4, 1.5 mMMgCl2, 10 mMKCl, 1 mM phenylmethylsulfonylfluoride, 30mM sodium butyrate, 0.5mM dithiothreitol, 100�Mleupeptin, 10�g/ml aprotinin, 1mM4-(2-aminoethyl)benzene-sulfonyl fluoride) was added (0.5 ml), and the cells were centri-fuged immediately at 300 � g. The cells were resuspended in 1ml of hypotonic buffer, allowed to swell for 10 min at 4 °C, andthen homogenized in a Dounce homogenizer with 10 strokes ofa B pestle. The sample was centrifuged at 5000 rpm for 5min at4 °C in a microcentrifuge, and the supernatant was removed.The pellet-containing nuclei was resuspended in 0.2 M H2SO4at about 2 mg of DNA/ml (about 400 �l), sonicated for 2–3 s atpower setting 4, and allowed to stand at 4 °C overnight. Thiswas sonicated again for 2–3 s at 4 °C and centrifuged at themaximum setting for 10 min at 4 °C in a microcentrifuge. One-third volume (133 �l) of 100% trichloroacetic acid (final, 25%)was added and mixed well, and proteins were allowed to pre-cipitate for 30 min at 4 °C followed by centrifugation at maxi-mum speed for 10min at 4 °C in amicrocentrifuge. The histone

pelletwaswashed oncewith 1ml of 100% acetone, 0.05MHCl at4 °C with vortexing and then centrifuged. The resulting pelletwas washed once with 100% acetone at 4 °C and centrifuged.The pellet was allowed to air dry then resuspended in 200–800�l of water at room temperature and stored at �80 °C. Totalprotein levels were determined using the BCA kit from Pierce.Histones were separated by SDS-PAGE on 12% gels and immu-noblotted as described below.Immunofluorescence—Cells were harvested as a single cell

suspension using trypsin followed by inactivation of the trypsinusing growth media-containing serum. The cells were centri-fuged onto slides using aCytoproTMcytocentrifuge (Wescor) at1400 rpm for 5 min at room temperature (RT). Cells were fixedwith 2% paraformaldehyde in PBS for 20 min at RT. Cells wererinsed twice with 0.1% Triton X-100 in PBS and blocked with3% goat serum in 0.1%Triton X-100-PBS for 1 h at RT. Primaryantibody 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 thenrinsed 3 times with blocking agent for 2 min each at RT, andsecondary antibody at 1:800 dilution was applied for 1 h at RT.The cells were counterstained with 4�,6-diamidino-2-phen-ylindole at 1:50,000 dilution, and coverslips were appliedusing Fluoromount�. Primary antibodies include Oct3/4 (BDTransduction Laboratories) and Nestin clone Rat 401 (StemCell Technologies). Secondary antibody was Alexa Fluor 488goat 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 stand-ard methods. Primary antibodies were against acetylated his-tone H3 (Upstate), acetylated histone H4 (Upstate), native his-tone H3 (Upstate), and dimethylated lysine 4 of histone H3(Upstate). Blotting for total histone H3 was used as a loadingcontrol. Appropriate conjugated secondary antibodies wereused, and binding was detected by either enhanced chemilumi-nescence or chemifluorescence (Amersham Biosciences).Chromatin Immunoprecipitation (ChIP)—ChIP was per-

formed as previously described with minor alterations asdetailed in the supplemental data (17). PCR amplification wasperformed on DNA recovered from the immunoprecipitationand the total chromatin input. Primers used for PCR ofgenomic DNA recovered by ChIP for regulatory regions areshown in supplemental Table 1. The PCR products were sepa-rated on a 1% agarose gel, stained with vistra green, anddetected using the Storm860PhosphorImager (GEHealthcare)with quantitation analysis performed using ImageQuant 5.2software. Background signals for IgG or no antibody controlswere subtracted from the immunoprecipitation numbers andgenerally represented less than 10–15% of the signal with spe-cific antibody. The ratio of the corrected immunoprecipitationvalue over the total input value for the �LIF (undifferentiatedmES cells) sample was arbitrarily set equal to one, and all otherratios were determined relative to this as a -fold acetylationrelative to �LIF. For determination of the relative amount ofacetylation of histonesH3 andH4 at each promoter in the�LIFcells, the highest levels were always at the Oct4 regulatoryregion, and therefore, this was arbitrarily set equal to 100%, and

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all other valueswere expressed relative to this. Three independ-ent experiments were performed, and the data were plotted asthe mean � S.E.

RESULTS

Global Histone Acetylation Increased during Early Differ-entiation of mES Cells Induced by LIF Withdrawal—Totalhistones were prepared from undifferentiated mES cellsmaintained in the presence of LIF and from cells induced todifferentiate by LIF withdrawal for 1–4 days. As shown inFig. 1A, levels of acetylated histones H3 and H4 increasedover the 4 days of differentiation. The total pool of histoneH4 showed an increase in global acetylation (a modificationusually associated with localized transcriptional activation)beginning on day 2 after LIF withdrawal, reaching 2.5 on day3 and persisting through day 4 (Fig. 1B). The level of acety-lation of histone H3 also changed, but more slowly, and onlyachieved a modest 50% increase by day 4. The level of di-methylation on lysine 4 of histone H3 did not significantlychange during this period.As shown in Fig. 1C, LIF withdrawal initiated a specific pat-

tern of gene expression and order of induction and repressionof marker genes over this time course. After 5 days of LIF with-drawal, this pattern of gene expression was quite dissimilarfrom the undifferentiated mES cells at day 0, providing a dis-tinct fingerprint for early, multi-lineage differentiation. Thegene expression pattern at 0 days of LIFwithdrawal representedthat of undifferentiated mES cells, with readily detectable tran-scripts for Oct4, Rex1, and Fgf4. This group of marker geneswas termed “ON” to represent their expression in the pluripo-tent stem cell state. The transcript levels of all three of thesegenes decreased subsequent to LIF withdrawal but at differentrates. The second set of marker genes, referred to here as“INDUCED,” were not expressed in the undifferentiated mEScell but displayed a time-dependent, ordered increase in theirexpression during LIF withdrawal. Transcripts for the PRincreased first, within 1 day of LIF withdrawal, whereas levelsfor Hoxb1 (a patterning gene), Brachy (early mesodermmarker), and Nestin (early neural ectoderm marker) rosebetween days 2 and 3. The last set of genes, termed “OFF,” werenot detectable during the early stages of differentiation exam-ined in these studies. Afp (endoderm) and �H1 globin (latermesoderm) did not increase significantly until after 5 days ofLIF withdrawal (6). Prl and Ins are tissue-specific genes thatwere not expressed after LIF withdrawal in our model systemeven after longer induction times. Positive controls were per-formed on samples from cell lines expressing Prl and Ins (datanot shown). Glyceraldehyde-3-phosphate dehydrogenase wasexpressed in undifferentiated cells, and RNA levels did not sig-nificantly change over this period. It was used as an internalcontrol.Localized Histone Acetylation Patterns in Gene Regulatory

Regions Correlated with Transcription and Not the GlobalPattern—To determine whether changes in gene expressionthat occurred during early differentiation coincided with local-ized or global histone acetylation, we examined regulatoryregions of themarker gene set during early differentiation usingChIP. We chose to examine regulatory regions, including pro-

FIGURE 1. Global histone acetylation during very early differentiation ofmES cells induced by LIF withdrawal. A, total histones were purified from undif-ferentiated mES cells (day 0, (�) LIF) or from cells after LIF withdrawal for theindicated days. A representative Western blot analysis for acetylated histone H3(AcH3), acetylated histone H4 (AcH4), and histone H3 (H3) as a loading control areshown. diMe, dimethylated. B, quantitation of immunoblotting data for AcH3(white bars), AcH4 (gray bars), or dimethylation of lysine 4 on histone H3 (blackbars) is shown. The signal intensity for the modified histone was divided by theintensity for the native histone H3 signal, and that ratio for the undifferentiatedcells was arbitrarily set equal to 1. All other ratios were expressed as -fold changesrelative to the undifferentiated ratio. The data represent the means � S.E. of themean for three independent experiments. C, the pattern of expression of amarker gene set during mES cell differentiation induced by LIF withdrawal wasassayed by reverse transcription-PCR on total RNA samples. Time 0 representsundifferentiated mES cells. The housekeeping gene glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) is used as a loading control.

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moters and enhancers, regardless of their location in the genesince increased histone acetylation in such regions is predictedto be permissive for transcription. Known regulatory regions ofthemarker gene set (diagrammed in Fig. 2) were amplified afterimmunoprecipitation of the chromatin with either anti-acety-lated histone H3 or anti-acetylated histone H4 antibodies. Figs.3–5 show changes in levels of histone acetylation relative to theundifferentiated (�LIF, day 0) cells for the three gene sets: ON,INDUCED, and OFF. Fig. 3, A and B, show the relative acetyla-tion of histones H3 and H4, respectively, in regulatory regionsof the ON genes, which are expressed in undifferentiated mEScells but decrease over 5 days of differentiation (Figs. 1 and 6).The regulatory region ofOct4 demonstrated a small decrease inboth histones H3 and H4 acetylation within 1 day of LIF with-drawal. Rex1 and Fgf4 showed a small decrease in histone H4acetylation only. This loss of histone acetylation, althoughmodest, was in contrast to the global increase in histone acety-lation, which represented the average of all localized changes.Fig. 4 shows the relative histone acetylation in the regulatory

regions of genes that were not expressed in undifferentiatedcells but were induced during LIF withdrawal (INDUCED).Local increases in histone acetylation over the differentiationtime coursewere observed for all of these genes. PR is a complexgene, which we have recently shown is turned on in mES cells12–24 h after LIF withdrawal (Fig. 1) (6). Two isoforms of PR,PRA and PRB, are encoded by a single gene with two separatepromoters (18). The PRB promoter is upstream of the PRA

promoter; therefore, the PRB transcript shares all the PRAsequence plus an additional 5� sequence. The PCR primers weused in Fig. 1C did not distinguish between the two PR tran-scripts. As shown in Fig. 4, A and B, the PRB promoter demon-strated a 2-fold increase in histoneH4 acetylation and a smallerincrease in histone H3 acetylation as early as 4 h after LIF with-drawal. In contrast, acetylation of histone H4 at the PRA pro-moter showed a transient increase at 2 days and little change inacetylation of histone H3. The increased histone acetylation ofPRB preceded the increase in PR transcripts observed at 12–24h (6). Two known regulatory regions for theHoxb1 gene, one inthe 5� promoter and the other in the 3� region of the gene, wereexamined (19). Acetylation of histone H4 in the 3� regionincreased by day 2, reaching 2-fold by day 4 of differentiation,with little change in histoneH3 acetylation (Fig. 4,A and B). H4acetylation of the 5� region rose within 4 h and persisted to day4 (Fig. 4, C and D). Acetylation of histone H3 also rapidlyincreased, reaching a 3.8-fold increase by day 4. The increase inhistone H3 acetylation preceded the increased expression of

FIGURE 2. Diagram of the locations of the PCR products within the regu-latory regions of the genes used for ChIP analysis. The black rectanglesrepresent the PCR products amplified by the primers shown in supplementalTable 1 for each gene. Genes are the marker set from Fig. 1. Regulatoryregions 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. ChIP for acetylated histones H3 and H4 at the regulatoryregions of the ON gene set as diagrammed in Fig. 2. The level of acetylatedhistones 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 datapoint represents the means � S.E. of the mean from three independentexperiments. A, -fold change in acetylated histone H during early differentia-tion. B, -fold change in AcH4 during early differentiation.

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Hoxb1 seen at 2–3 days (Figs. 1 and 6). As shown in Fig. 4,C andD, acetylation of both histones H3 and H4 in the Nestin regu-latory region increased 3 days after LIF withdrawal, coincidentwith increased gene expression. Acetylation of histone H3 inthe Brachy regulatory region also increased 3 days after LIFwithdrawal (Fig. 4C), coincident with increased accumulation

of RNA (Fig. 1). Increased acetyla-tion of histone H4 in this region wasnot visible until after 4 days (Fig.4D). Overall, increased acetylationof either or both histonesH3 andH4was observed in at least one regula-tory region of each INDUCED genepreceding or coincident with RNAaccumulation.Fig. 5, A and B, show the relative

acetylation of histones H3 and H4,respectively, in three regulatoryregions of two genes, whose tran-scripts accumulate only after 5 daysof LIF withdrawal (6). The �-globingene locus is complex, containingmultiple �-globin genes andregional as well as gene-specific reg-ulatory regions. An upstreamDNase hypersensitive site (locuscontrol region HS2) is necessary forinitial de-repression of the entire�-globin locus (20–23). The �H1globin (Hbb-bh1, hemoglobin z)gene is expressed early in develop-ment (24). The local promoter ofthis gene (bH1) acquired a small,1.5-fold increase in histone H3acetylation after 3 days of LIF with-drawal, but little change in the levelof H4 acetylation occurred over the4 days (Fig. 5, A and B). The HS2locus control region showed aunique pattern, with increasedacetylation of both histones H3 andH4 only 4 h after LIFwithdrawal fol-lowed by a return of acetylated his-toneH4 to the level seen in undiffer-entiated cells and a fall in acetylatedhistone H3 to 50% that seen inundifferentiated cells. This lowerlevel was maintained throughoutthe 4 days of LIF withdrawal (Fig.5A). The Afp regulatory regiondemonstrated an average 1.5-foldincrease in the acetylation of his-toneH3 over the entire 4 days of LIFwithdrawal and little change at H4(Fig. 5,A andB).Wehave previouslyreported that both �H1 globin andAfp are expressed later in this cellmodel (6), and these local increases

in histone acetylationmay be necessary tomaintain these genesin a permissive chromatin structure during this early differen-tiation period. Finally, levels of histone acetylation were notaltered in the regulatory regions of two genes (Ins and Prl) thatwere not expressed even at later times in this mES cell model(Fig. 5, C and D).

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

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

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Basal Acetylation of Histones H3 and H4 in UndifferentiatedmES Cells Varied across the Regulatory Regions of the MarkerGene Set—We used ChIP to obtain a measure of the relativebasal acetylation levels of the histone tails at the regulatoryregions of themarker gene set in the undifferentiatedmES cellsas shown in Fig. 6. In all experiments the level of acetylation ofboth histones H3 and H4 was highest in the Oct4 regulatoryregion. We, therefore, arbitrarily set this to 100%, and levels inother regulatory regions are shown relative to the Oct4. Thethree genes of theONset (Oct4, Rex1, Fgf4) showed someof theoverall highest levels of acetylation of histones H3 (Fig. 6A) andH4 (Fig. 6B). The relative levels of histone acetylation in theregulatory regions of the rest of the marker gene set werequite variable. There was no apparent relationship betweenthe level of histone acetylation in the undifferentiated state

and the order of transcriptional activation that is seen inresponse to LIF withdrawal for the INDUCED set. This wasindicated by the relatively high level of basal histone acety-lation for Brachy despite having the slowest induced expres-sion (as seen in Fig. 1). PRB, both Hoxb1 regulatory regions,and Nestin had lower basal acetylation of histones H3 andH4 than the ON set of genes yet were rapidly activated by LIFwithdrawal. The basal acetylation of histones H3 and H4 forthe “OFF” set of genes was also lower than for the ON set.Overall, integrating the results from Fig. 6 with Figs. 3–5,differentiation induced the largest increases in local histoneacetylation in gene regulatory regions that both had a lowlevel of basal histone acetylation and were transcriptionallyactivated by differentiation.TSA Treatment Induced an Early Differentiation Phenotype

That Mimics LIF Withdrawal—Based on the observation thatglobal histone acetylation increased during differentiationinduced byLIFwithdrawal (Fig. 1), we testedwhether inductionof global histone acetylation alone was sufficient for early dif-ferentiation ofmES cells. TSA is a potent inhibitor of class I andII histone deacetylases. Inhibition of these histone deacetylasesresults in increased global acetylation of histones in most celltypes including mES cells (Ref. 25 and supplemental Fig. 1). Asshown in Fig. 7A, TSA treatment induced dramatic changes tocell morphology inmES cellsmaintained in LIF as seen by com-paring panels 1 and 2. The morphology of TSA-treated cellsclosely resembled that of LIFwithdrawn cells (panel 4), whereinthe tight colonies were disrupted, cellular extensions were eas-ily seen, and individual cell borders were clear. TSA treatmentof cells grown in the absence of LIF resulted in a similar differ-entiated morphology (panel 5). The morphological changesinduced by TSA were reversible upon its removal from LIF-containing media as seen in the third column of Fig. 7A, panels3 and 6. Colonies were scored for phenotype, and quantitativedata are presented in supplemental Fig. 2. Approximately 90%of control colonies (no TSA, grown in LIF) showed an undiffer-entiated morphology. Nearly 100% of colonies showed a differ-entiated morphology after TSA treatment in the presence ofLIF. More than 65% of the colonies regained an undifferenti-ated morphology after withdrawal of TSA for 2 days (TSAWD2d).As shown in Fig. 7B, TSA treatment of mES cells in the pres-

ence of LIF induced an ordered change in gene expression thatmimicked LIF withdrawal, although it was markedly acceler-ated. Oct4, a gene from the ON set, was expressed in undiffer-entiated (time 0) mES cells and decreased after 10 h of TSAtreatment. TSA treatment caused a time-dependent, orderedinduction of the genes in the INDUCED set; PR, Hoxb1, andNestin followed by Brachy. The OFF genes Prl and �H1 globinwere not induced by TSA treatment; however, a small amountof Afp was detectable in some replicates at 23 h. The pattern ofgene expression observed after 23 h of TSA in the presence ofLIF matched that induced by LIF withdrawal for 5 days (com-pare Figs. 7B with 1C). These results support a model in whichthe hyperacetylation induced by TSA is permissive for the exe-cution 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

FIGURE 6. Basal levels of acetylation of histones H3 and H4 in undifferen-tiated mES cells vary substantially between gene regulatory regions. His-tone acetylation in the regulatory regions shown in Fig. 2 was assayed by ChIPon undifferentiated mES cells. The level of histone acetylation at the Oct4promoter was arbitrarily set equal to 100%, and the levels of acetylation ofhistone H3 (panel A) or histone H4 (panel B) are expressed as a percentagerelative to the level of acetylation of the Oct4 promoter. The data representthe mean � S.E. from three independent experiments.

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altered by TSA in Fig. 7B was determined over a time courseafter removal of TSA. Cells were treated with TSA for 20 h (0 hrecovery time after TSA), then TSA was removed, and cellswere cultured for the indicated recovery times in �LIF media.As shown in Fig. 7C, the gene expression pattern reverted tothat of the undifferentiated cells as early as 24 h after TSAwith-drawal and was maintained to 48 h.TSA Treatment Did Not Support Commitment to Differenti-

ation in Individual Cells—TSA treatment of mES cells mim-icked differentiation induced by LIF withdrawal. The resultsfromFig. 7 represented a population of cells. This population ofTSA-treated cells did not commit to a differentiation pathwayas defined by an irreversible change in gene expression andmorphology. There are two possible explanations for theseresults. First, this could be due to selection, where a small num-

ber of cells remained undifferentiated during TSA treatment,and those cells expanded after TSA withdrawal, whereas cellsdisplaying the differentiated phenotype died or became quies-cent. Alternatively, TSA may have induced a phenotypicchange in all the cells that was truly reversible. This was thehypothesis we favored due to prior observations of the culturesthat indicated morphological changes to virtually all colonieson the plate, limited cell death, and consistent population dou-bling times. Over 4 days the population doubling time of cul-tures grown in the presence or absence of LIF without LIF werethe same (data not shown).To quantitatively test if the molecular changes induced by

TSA were reversible at the level of individual cells, we per-formed immunofluorescence analysis for Oct4 (a marker ofundifferentiated cells) and Nestin (a marker of differentiatedcells) protein expression levels. As seen in Fig. 8A, high levels ofOct4 protein (top left panel) were seen in almost every undif-ferentiated cell. TSA treatment in the presence of LIF resultedin a dramatic decrease in Oct4 protein in almost every cell (Fig.8A, top right panel). LIF withdrawal for 3 days also resulted in adecrease in cells expressing Oct4 protein. Within 24 h afterTSA 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 thesame set of treatments is shown in Fig. 8B. Virtually no Nestinprotein was seen in any undifferentiated cell (top left, Fig. 8B).TSA treatment in the presence of LIF resulted in a smallamount of perinuclear Nestin protein in about 50% of cells (topright, 8B). Withdrawal of LIF for 3 days also induced Nestinprotein inmore than 30% of cells (lower left panel, Fig. 8B). By 2days after TSAwithdrawal less than 25% of cells containedNes-tin protein, and the intensity of staining was substantiallydecreased (bottom right, Fig. 8B). Quantitation of three inde-pendent experiments is shown in Fig. 8C. Clearly, the differen-tiated phenotype induced by TSA treatment was reversible atthe individual cell level.We considered the possibility that a small population of

undifferentiated mES cells in the culture were unaffected byTSA, and these cells simply repopulated the culture as undif-ferentiated cells uponwithdrawal of TSA. To test the possibilityof this cell selection model, we performed cell counts usingtrypan blue exclusion during a time course of TSA treatmentand withdrawal. As can be seen in supplemental Fig. 3, duringthe 18 h of TSA treatment therewas no effect on the cell growthrate (doubling time of about 12 h). A 2-fold decrease in cellgrowth was observed for the 24 h after TSA withdrawal (dou-bling time about 24 h), and then the doubling time recovered tothe control rate of about 12 h. Changes in cell growth rate are anaverage of cell proliferation and cell death. These data sug-gested that potentially therewas a small component of selectionimposed byTSA treatment. However, the small decease in dou-bling time for 1 day could not totally account for the percentageof cells with reversed phenotypes seen in Fig. 8, A–C. Therewere at least 3–4-fold more Oct4-positive cells in the TSAwithdrawn cultures than would be expected if 100% of the pos-itive cells resulted only from selection and not from a reversiblephenotype.

FIGURE 7. TSA induces cell morphology and gene expression changesthat mimic differentiation induced by LIF withdrawal, but the TSAeffects are reversible. A, changes in colony morphology induced by varioustreatments. In the far left panels cells were grown without TSA and in thepresence (panel 1) or absence (panel 4) of LIF for 3 days. In the middle panelsthe 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 cellswere grown in LIF containing media and treated with TSA for 20 h, then TSAwas withdrawn for 1 (panel 3) or 2 (panel 6) days. All photos taken at 100�magnification. B, mES cells were grown for 71 h in media containing LIF theentire time with either 0.1% ethanol (time 0) or 100 nM TSA added for the last2, 5, 10, or 23 h of culture. RNA was prepared, and reverse transcription-PCRwas performed for the expression levels of the marker gene set. C, cells weretreated with 100 nM TSA for 20 h in the presence of LIF, and then TSA waswithdrawn (time 0), and cells were maintained in LIF-containing media for theindicated times. RNA was prepared, and reverse transcription-PCR was per-formed for the expression levels of the marker gene set.

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IncreasedAcetylation ofHistonesH3 andH4after TSATreat-mentDidNotOccur at the Regulatory Regions of AllGenes—Weused ChIP to obtain a measure of the relative basal acetylationlevels of the histone tails at the regulatory regions of themarkergene set in the undifferentiatedmES cells (Fig. 6) and after TSAtreatment. Fig. 9 shows the -fold increase in histone acetylationfor each gene regulatory region induced by TSA treatment rel-ative to undifferentiated cells. These data are also plotted as %acetylation and directly compared with the basal levels of his-tone acetylation fromFig. 6 (see supplemental Fig. 4). The threegenes of the ON group showed no increased acetylation of his-tones H3 (Fig. 9A) and H4 (Fig. 9B) in response to TSA. Thesegene regions had high levels of basal histone acetylation (asshown in Fig. 6) and were actively transcribed in the undiffer-entiated cells (Fig. 1C). The ability of TSA to increase the his-tone acetylation levels of regulatory regions of the INDUCEDgenes was related to their basal acetylation state (seen in Fig. 6).The regions with the lowest levels of basal acetylation, such asPRB, Hoxb1 3� and 5�, and Nestin, showed the greatestincreases induced by TSA. Perhaps most interesting was thebehavior of the OFF set of genes. TSA induced a significantincrease in histone acetylation in the regulatory regions of thetwo genes that were expressed at later times in our culture sys-tem, �H1 globin and Afp. The levels of acetylation did notchange for the two genes that were completely transcriptionallysilent in this model system, Prl and Ins. Overall, the effects of

TSA on localized histone acetyla-tion were very similar to the effectsof LIF withdrawal. We concludedthat increased histone acetylationwas an active event in triggering theexpression of early differentiationgenes but was not sufficient for acti-vation of very late genes, mainte-nance of the differentiated pheno-type, or irreversible loss of theundifferentiated state.

DISCUSSION

Major reorganization of the chro-matin structure including changesin histone modifications and DNAmethylation are features of earlyembryonic development as well asof ES cell loss of pluripotency andcommitment to differentiation(26–30). Considerable work hasbeen done on chromatin changesduring late, lineage-specific differ-entiation, X chromosome inactiva-tion, and in germ cells where anincrease in heterochromatin is amajor feature, whereas relatively lit-tle investigation has centered on thevery early time frame (0–96 h) afteran mES cell is exposed to a differen-tiation signal. Loss of compact col-ony morphology and dramatic

changes in the expression of a set of marker genes characterizethe phenotype associated with this very early differentiation ofmES cells in response to LIF withdrawal. We have focused onthis early time frame because of the rapid changes in geneexpression and loss of pluripotency that we expect are accom-panied and indeed facilitated by epigenetic changes in the chro-matin. We expect some epigenetic changes are part of themechanism of immediate gene expression changes, but othersare important tomark genes for later changes in expression andfor prevention of phenotypic reversion.Understanding the pro-gram of epigenetic marks that drive the cell into a differentia-tion pathway will contribute to efforts to derive specific celllineages for therapeutic purposes.The experiments presented here were designed to examine

the role of histone acetylation, generally associated with tran-scriptional activation, in the very earliest steps of the differen-tiation pathway induced by LIF withdrawal. During this earlytime frame the expression of markers of all three germ celllineages is seen, but the cells are not yet committed to specificlineages (3, 6). We observed a global increase in the acetylationof both histone H3 and H4 by either LIF withdrawal or TSAtreatment. Global induction of histone acetylation by eithertreatment leads to the immediate gene expression changescharacteristic of the very early differentiation program. It is alsopotentially important for marking genes that will be expressedlater in differentiation such as Afp and �H1 globin. However,

FIGURE 8. TSA induces changes in the protein levels of Oct4 and Nestin within individual cells that arereversible upon removal of TSA. Cytospins of cells were prepared, and immunofluorescence for Oct4 (A) andNestin (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 TSAtreatment while in LIF-containing media. The lower left panel in both A and B shows cells cultured without LIFfor 3 days (�LIF). The lower right panel in both A and B shows cells culture in LIF-containing media for 2 daysafter withdrawal of TSA treatment. All images are at 200�. C, the immunofluorescence data were quantitatedand plotted as the percentage of positively staining cells for Oct4 (black bars) and Nestin (gray bars). The datarepresent the mean � S.E. for three independent experiments.

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histone acetylation alone is not sufficient for irreversible exitfrom the stem cell state and progression to a lineage specificdifferentiation program as demonstrated by the TSA with-drawal results (Figs. 7 and 8).Role of Histone Acetylation in Gene Expression during Early

mES Cell Differentiation—The global change in acetylation ofthe total histone pool we observed in Fig. 1 is an average of thedifferential response to LIF withdrawal across regions of het-erochromatin and euchromatin. The few studies that havequantitated changes in global histone acetylation in mES cellsduring differentiation have focused on later, more differenti-ated cells or on heterochromatin and X inactivation. A recentstudy on epigenetic changes betweenmES cells and neural pro-

genitor cells showed a robust increase in the distribution andamount ofmethylation of lysine 9 on histoneH3 and a decreasein acetylation on both histones H3 and H4 on day 7 of theirdifferentiation protocol (31). The only other time point exam-ined was 24 h of LIF withdrawal at which a slight increase in H4acetylation was observed similar to our own result. A series ofstudies have shown that a protocol of embryoid body differen-tiation in female mES cells leads to inactivation of one of the Xchromosomes and increases overall heterochromatin forma-tion over 1–3 weeks (32). One study focused only on sites ofheterochromatin formation where a decrease in histone acety-lation is observed at those sites (33). Another study examinedglobal histone acetylation during embryoid body differentia-tion of mES cells and saw an initial decrease and then recoveryof histone acetylation levels (34). The differentiation protocolof embryoid body formation used in that report is significantlydifferent fromour own.This overviewof the published data andour own results suggest that there is a very early increase inglobal histone acetylation, whereas epigenetic events to restrictactive chromatin including histone deacetylation, histonemethylation, andDNAmethylation dominate later time points.Histone acetylation is a dynamic process resulting from thebalance between acetylase and deacetylase enzymes (35). Weexpect that the activity and targeting of these enzymes to spe-cific loci will be affected by differentiation.Wepostulate severalroles for this very early increase in global histone acetylationduring mES cell differentiation. The first is immediate changesin transcription at specific genes. The second is as a permissivemark for genes that will be expressed later and need to be pro-tected during subsequent events to restrict chromatin. Thethird is as part of amechanism to propel the cell irreversibly outof the pluripotent state.We observe a striking coincidence in the pattern of local

histone acetylation changes when cells are treated with eitherLIF withdrawal or TSA. The global increases in histone acety-lation are not evenly distributed across regulatory regions ofour marker genes (Figs. 3–5 and 9) but correlate best with thetranscriptional activity of that marker gene. All of the genes inthe INDUCEDset showan increase in either or both histoneH3and H4 acetylation in at least one regulatory region coincidentwith RNA accumulation. However, genes that are already tran-scriptionally active (ON) or inactive in our model (Ins and Prl)show little or no increase in histone acetylation and even somedecrease for the ON set. These results are consistent with thefirst postulated role for the global increase in histone acetyla-tion; that is, immediate changes in the transcriptional activa-tion of specific genes.We observed an increase in acetylated histone H3 in the reg-

ulatory region of Afp and the transcriptional start site of �H1globin by 2–3 days of LIF withdrawal or TSA treatment despitethe lack of expression of these genes during this time course.However, expression of Afp and �H1 globin is seen within 4–5days of LIF withdrawal in embryoid bodies (6). This distin-guishes the expression pattern of these two genes from Ins andPrl for which no expression is seen even with embryoid bodyformation for 16 days (data not shown). These two genes are anexample of the secondpostulated role of early increased histoneacetylation; the genes are marked for future expression, but

FIGURE 9. The effect of TSA on histone H3 and H4 acetylation levels variessubstantially between genes. Histone acetylation in the regulatory regionsshown in Fig. 2 was assayed by ChIP after treatment for 20 h with or withoutTSA in LIF-containing media. The acetylation level in the presence of TSA wasdivided 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) inresponse to TSA are shown. The data represent the mean � S.E. from threeindependent experiments.

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increased histone acetylation alone is not sufficient to inducetranscription. It is possible a repressive histone modificationmust be removed.Increased Histone Acetylation Is Not Sufficient for mES Cells

to Commit to Differentiation—Treatment of mES cells withTSA induces changes that mimic the withdrawal of LIF for 3–5days. Cells that have been cultured in the absence of LIF for 3days are committed to differentiation.3 Specifically, returningcells differentiated by 3 days of LIF withdrawal to culture inmediawith LIF does not restore the tight colonymorphology orgene expression pattern seen in the pluripotent mES cell. TSAtreatment results in a reversible phenotypic change, and thecells can revert to the pluripotent mES cell phenotype uponremoval of TSA.We conclude from these data that the increasein histone acetylation observed upon LIF withdrawal andinduced by histone deacetylase inhibition with TSA is not suf-ficient for maintenance or progression of differentiation. Evi-dently, additional events are required that occur with LIF with-drawal 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 bea change in the activity of a specific transcription factor (acti-vator or repressor) or other specific epigenetic changes differ-ent from the histone acetylation studied here. Possible epige-netic changes would include other histone tail modifications,substitutions of histone variants into nucleosomes, and DNAmethylation at either a local or global level (8, 36, 37–39).Recent work has identified the presence of a chromatin domainwith bivalent epigenetic marks in the regulatory regions of alarge set of developmental genes in mES cells (11). The marksare methylation on lysine 4 of histone H3 and methylation onlysine 27 of histone H3. Lys-4 methylation is generally associ-ated with active chromatin, whereas Lys-27 methylation is arepressive modification. These authors propose that the pres-ence of both marks keeps genes silent but poised for activationinmES cells. In retinoic acid-differentiated cells, the twomarkstend to resolve with a gene having only one mark remainingthat is correlated with its transcriptional status. This type ofopposed and balanced epigenetic marks would be consistentwith our ownobservations. Increased acetylation alone inducedby TSA can support the expression of a set of genes poised fortranscription. However, the removal of repressive marks maybe required to maintain the expression of those poised genes.Removal of activating or repressive marks on additional genesmay be required to commit the cell to a differentiation pathway.Our data suggest that regulation of gene transcription duringmES cell differentiation may best be described by a multivalentmodel of histone tail modifications including removal ofrepressivemarks such as Lys-27methylation (11) and themain-tenance or addition of activating marks such as Lys-4 methyla-tion (11) and acetylation (this report).This report adds to our understanding of the role for epige-

neticmarks inmES cell differentiation. The great promise of EScells is the hope of producing highly pure and functional pro-genitor cells for treatment of disease. Elucidation of the pro-

gramof epigeneticmarks required to achieve a specific cell typewill contribute to realizing this promise.

Acknowledgments—We thank Carley N. Sauter, Rebecca L. Mc-Dermid, and Jeremy M. Roberts for technical assistance.

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Kevin W. McCool, Xiaojie Xu, Don B. Singer, Fern E. Murdoch and Michael K. Fritschduring Early Embryonic Stem Cell Differentiation

The Role of Histone Acetylation in Regulating Early Gene Expression Patterns

doi: 10.1074/jbc.M609519200 originally published online January 4, 20072007, 282:6696-6706.J. Biol. Chem. 

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