LETTER

Histone Deacetylase Activity Is Required for EmbryonicStem Cell DifferentiationJeong-Heon Lee, Suzanne R.L. Hart, and David G. Skalnik*Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology/Oncology, Department of Pediatricsand Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana

Received 12 August 2003; Accepted 23 October 2003

Summary: Mammalian development requires commit-ment of cells to restricted lineages, which requires epi-genetic regulation of chromatin structure. Epigeneticmodifications were examined during in vitro differentia-tion of murine embryonic stem (ES) cells. Global histoneacetylation, a euchromatin marker, declines dramati-cally within 1 day of differentiation induction and par-tially rebounds by day 2. Histone H3-Lys9 methylation, aheterochromatin marker, increases during in vitro differ-entiation. Conversely, the euchromatin marker H3-Lys4methylation transiently decreases, then increases to un-differentiated levels by day 4, and decreases by day 6.Global cytosine methylation, another heterochromatinmarker, increases slightly during ES cell differentiation.Chromatin structure of the Oct4 and Brachyury genepromoters is modulated in concert with their pattern ofexpression during ES cell differentiation. Importantly,prevention of global histone deacetylation by treatmentwith trichostatin A prevents ES cell differentiation.Hence, ES cells undergo functionally important globaland gene-specific remodeling of chromatin structureduring in vitro differentiation. genesis 38:32–38, 2004.© 2003 Wiley-Liss, Inc.

Key words: epigenetics; Oct4; Brachyury; cytosine methyl-ation; histone code

Epigenetic regulation of gene expression is critical forrestriction of stem cell progeny to distinct cell lineagesand for the phenomenon of cell memory (Frances andKingston, 2001; Orlando, 2003). Regulation of chroma-tin structure is the molecular mechanism underlyingepigenetics and cells contain elaborate machinery forcontrolling chromatin structure (Li, 2002; Reik et al.,2001; Surani, 2001).

Cytosine methylation and covalent modification ofhistones are two mechanisms for the epigenetic controlof chromatin structure. In mammalian cells cytosinemethylation occurs predominantly at CpG dinucleotides.Cytosine methylation is associated with transcriptionallyinactive heterochromatin and is catalyzed by mainte-nance (Dnmt1) and de novo (Dnmt3a/Dnmt3b) DNAmethyltransferases (Li et al., 1992; Okano et al., 1999).Cytosine methylation plays an important role in genomic

imprinting and X-chromosome inactivation and is essen-tial for normal development. Individual disruption of theDnmt1, Dnmt3a, or Dnmt3b genes leads to an embry-onic lethal phenotype (Li et al., 1992; Okano et al.,1999). Furthermore, perturbations of both global andgene-specific patterns of cytosine methylation are com-monly observed in cancer (Baylin and Herman, 2000).

The “histone code” hypothesis predicts that distinctcovalent histone modifications lead to functionally dis-tinct chromatin structures (Jenuwein and Allis, 2001;Strahl and Allis, 2000). More than two dozen site-specifichistone modifications have been described. For exam-ple, methylation of histone H3-Lys4 (H3-Lys4) and his-tone acetylation are associated with euchromatin. Con-versely, methylation of histone H3-Lys9 (H3-Lys9) isassociated with heterochromatin and transcriptional si-lencing (Nakayama et al., 2001; Roth et al., 2001; Santos-Rosa et al., 2002).

The mammalian epigenome undergoes global re-modeling during early development. For example, thezygotic genome undergoes both active and passiveglobal cytosine demethylation immediately followingfertilization and reaches a nadir of �20% CpG meth-ylation at the blastocyst stage (Li, 2002). This is fol-lowed by a wave of de novo methylation prior togastrulation that reprograms chromatin structure andrestores the global level of CpG methylation to �60%(Chapman et al., 1984; Santos et al., 2002). This reg-ulation of cytosine methylation is critical for mamma-lian development, as embryonic stem (ES) cells con-taining reduced or elevated levels of 5-methylcytosine

* Correspondence to: David G. Skalnik, Ph.D., Herman B Wells Center forPediatric Research, Cancer Research Building, Room 472, Indiana Univer-sity School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202.

E-mail: dskalnik@iupui.eduContract grant sponsor: National Institutes of Health, Contract grant

number: HL69974 (to D.G.S.), Contract grant sponsor: Riley Children’sFoundation.

DOI: 10.1002/gene.10250

© 2003 Wiley-Liss, Inc. genesis 38:32–38 (2004)

are viable but unable to differentiate (Biniszkiewicz etal., 2002; Lei et al., 1996).

In contrast to cytosine methylation, little is known re-garding histone modifications during the early stages ofmammalian development. Murine ES cells were used tostudy modulation of epigenetic markers during stem celldifferentiation. ES cells exhibit dramatic and complex alter-ations to both global and gene-specific chromatin structurethat are necessary for in vitro differentiation.

RESULTS AND DISCUSSION

Chromatin-bound histones were isolated at various timesfollowing the initiation of in vitro ES cell differentiation andglobal levels of specific covalent histone modificationswere assessed by Western analysis (Fig. 1a). Both histoneH3 and H4 are dramatically deacetylated within 1 day of LIFremoval, followed by partial reacetylation by day 2. HistoneH3-Lys4 methylation also decreases at day 1, reboundsbetween days 2–4 of differentiation, and then decreasesagain on days 5 and 6. These results suggest an erasure ofeuchromatin epigenetic marks upon initiation of in vitro EScell differentiation, followed immediately by reprogram-ming of the epigenome. Conversely, histone H3-Lys9 di-and tri-methylation are low in undifferentiated ES cells andprogressively increase during the 6-day time course of invitro differentiation. Histone H3-Lys9 mono-methylationwas nearly undetectable at all time points examined. Thesedata indicate a global increase in heterochromatin levelsduring in vitro differentiation of ES cells. This is consistentwith the model that stem cell differentiation is accompa-nied by a restriction of the set of genes that can be ex-pressed (Surani, 2001).

Global levels of cytosine methylation were comparedbetween undifferentiated and differentiated ES cells (Fig.1b). ES cell genomic DNA exhibits a small but statisti-cally significant increase in global cytosine methylationlevels following in vitro differentiation. The lack of signalfor 5-methylcytosine following digestion with the methyl-sensitive enzyme HpaII serves as a control for the specific-ity of this assay. Although ES cells are derived from 3.5 dpc(days postcoitus) blastocysts, a time of development thatcorresponds to a minimal level of cytosine methylation (Li,2002), undifferentiated ES cells are thought to reflect the4.5–5.5 dpc stage of development (Weng et al., 1995).Undifferentiated ES cells exhibit slightly less than 50% CpGmethylation, and have hence already acquired the majorityof global cytosine methylation. These findings indicate thatglobal cytosine methylation is largely established prior toerasure and reprogramming of the histone code that occursupon in vitro differentiation of ES cells. However, theseresults do not exclude the possibility that dynamic changesin cytosine methylation patterns might occur followinginduction of ES cell differentiation.

Epigenetic modifications of developmentally regulatedgenes were analyzed during in vitro ES cell differentiation(Fig. 2a). As previously documented (Okamoto et al., 1990;Rosner et al., 1990), the expression level of the Oct4 genedecreases with ES cell differentiation (Fig. 2b). ChIP analy-

sis of the Oct4 promoter region shows a progressive de-crease of H3-Lys4 methylation and H3-Lys9 acetylation, anda concomitant increase of H3-Lys9 methylation during dif-ferentiation (Fig. 2c). Furthermore, analysis of a CpG motifat –288 bp of the Oct4 gene promoter revealed a progres-sive acquisition of DNA methylation, as previously reported(Gidekel and Bergman, 2002), and was completely meth-ylated at day 10 of differentiation (Fig. 2d). A CpG motif at–22 bp was not analyzed in these studies, as it resides nearthe end of the NcoI restriction fragment under analysis.Hence, these results indicate a shift from euchromatin toheterochromatin at the Oct4 gene coincident with extinc-tion of Oct4 gene expression during in vitro ES cell differ-entiation.

The Brachyury gene, a marker of mesoderm differenti-ation, is expressed at a low level in undifferentiated EScells, is induced at day 5 of differentiation, and is silencedby day 10 (Fig. 2b) (Keller et al., 1993). ChIP analysis of theBrachyury gene promoter reveals significant mono- anddi-methylation of histone H3-Lys4 at both days 0 and 5 ofdifferentiation, but a specific induction of H3-Lys4 tri-meth-ylation at day 5, coinciding with the time of highestBrachyury gene expression (Fig. 2c). All forms of H3-Lys4methylation are absent at day 10 of differentiation, corre-lating with the loss of Brachyury gene expression. Con-versely, methylation of H3-Lys9 is absent at day 5, but di-and tri-methyl Lys9 are present at day 10, coinciding withextinction of Brachyury expression. Mono-methylation ofboth H3-Lys4 and H3-Lys9 are present at day 0, indicatingthat this modification is not a useful marker of chromatinstructure in this context. Surprisingly, acetylation of Lys9within the Brachyury gene promoter does not correlatewith gene expression, as this modification is downregu-lated upon induction of ES cell differentiation. Similar tothe Oct4 gene promoter, cytosine methylation of theBrachyury gene promoter correlates well with the level ofgene expression. The –250 bp Brachyury gene promoterregion is partially methylated in ES cells that express theBrachyury gene, but becomes hypermethylated by day 10coinciding with loss of Brachyury gene expression (Fig.2d). No intermediate-sized bands were detected by South-ern analysis, suggesting that several CpG motifs upstreamof –250 bp are not methylated in the absence of cytosinemethylation at –250 bp.

These studies demonstrate that both H3-Lys9 di- andtri-methylation correlate with DNA methylation and generepression during in vitro ES cell differentiation. Interest-ingly, H3-Lys4 tri-methylation is associated with theBrachyury gene only at the time of highest gene expres-sion stage (day 5), suggesting that H3-Lys4 tri-methylation isthe most informative epigenetic marker of transcriptionallyactive chromatin at this locus. These data are in agreementwith a previous report that active genes are associated withH3-Lys4 tri-methylation in yeast (Santos-Rosa et al., 2002).

Additional studies were performed to assess the func-tional significance of global histone deacetylation thatoccurs upon induction of ES cell differentiation. ES cellswere treated with the histone deacetylase inhibitor tri-chostatin A (TSA) for 6 days following the removal of LIF.

33EPIGENETICS OF ES CELL DIFFERENTIATION

Treatment with 10 nM TSA effectively blocks the initialwave of histone deacetylation normally observed uponinduction of ES cell differentiation (Fig. 3a). This treat-ment also drastically inhibits ES cell differentiation, asrevealed by inhibition of embryoid body formation (Fig.

3b). Also, cells treated with TSA fail to downregulatealkaline phosphatase activity (Fig. 3c), which normallyoccurs upon ES cell differentiation (Wobus et al., 1997).A more modest effect on ES cell differentiation is ob-served following treatment with 5 nM TSA (Fig. 3b,c).

FIG. 1. Modulation of global his-tone modifications and DNAmethylation during ES cell differ-entiation. a: Global histone modi-fications during in vitro ES cell dif-ferentiation. Murine ES cells weredifferentiated as described in Ma-terials and Methods, and histoneswere isolated and analyzed byWestern blotting using the indi-cated modification-specific anti-bodies. Total histone levels weredetermined by Coomassie stain-ing (bottom panel). The experi-ment was performed three timesand representative data areshown. b: Global cytosine meth-ylation during in vitro ES cell dif-ferentiation. The global level of5-methylcytosine within the se-quence context of CCGG wasquantitated by thin layer chroma-tography prior to differentiation(day 0) and following in vitro dif-ferentiation (day 5), as describedin Materials and Methods. A rep-resentative experiment is pre-sented and a summary of compiledresults from three experiments isindicated below. The error bars in-dicate the mean SD and the Pvalue was determined by a stan-dard t-test. 5-mCMP, 5-methyl cy-tosine monophosphate; CMP, cy-tosine monophosphate.

34 LEE ET AL.

FIG. 2. Epigenetic modifications of developmentally regulated genes during ES cell differentiation. a: Schematic representations of theOct4 and Brachyury gene promoters analyzed by ChIP assays (c) and Southern blotting analysis (d). For the Oct4 gene, the proximalpromoter is boxed. The –569 to –293 bp region was used as a Southern probe and the –465 to –250 bp region was analyzed by ChIP (�1indicates the site of translation initiation). For the Brachyury gene, the –239 to –62 bp region was used as a Southern probe and the –364to –62 bp region was analyzed by ChIP (�1 indicates the transcriptional start site [arrow]). N, NcoI; S, SacI; triangles, HpaII CpG sites. b:The expression levels of the Oct4, Brachyury, and HPRT genes were analyzed by semiquantitative RT-PCR during in vitro ES celldifferentiation as described in Materials and Methods. The experiment was performed three times and representative data are shown. c:ChIP analysis of the Oct4 and Brachyury gene promoters during in vitro ES cell differentiation. ChIP assays were performed as describedin Materials and Methods using modification-specific antibodies. The experiment was performed three times and representative data areshown. d: DNA methylation analysis of the Oct4 and Brachyury gene promoters during in vitro ES cell differentiation. Genomic DNA sampleswere digested with NcoI/HpaII (Oct4) or SacI/HpaII/NcoI (Brachyury) and hybridized with radioactive probes as described above. Theexperiment was performed three times.

FIG. 3. (Continued)

Withdrawal of TSA after 3 days of treatment leads to theinitiation of histone deacetylation (compare day 4 of “-TSADay4” with day 3 of “10 nM TSA”), followed by a reboundat day 5 (Fig. 3a). This resembles the wave of histonedeacetylation and re-acetylation observed in untreated EScells following removal of LIF (Fig. 1a). Interestingly, em-bryoid body formation occurred more rapidly followingremoval of TSA compared to untreated cells following theremoval of LIF (Fig. 3b). Mature embryoid bodies appear incontrol cultures 5–6 days following removal of LIF. How-ever, similar embryoid bodies are apparent 3 days follow-ing removal of TSA. This suggests that cells treated withTSA respond to the withdrawal of LIF, although they do notacquire differentiated characteristics, and rapidly completethe differentiation program upon subsequent removal ofTSA. Removal of TSA also led to a 3.7-fold increase in theextinction of alkaline phosphatase activity (a measure of EScell differentiation) compared to cells treated continuouslywith TSA (Fig. 3c).

Thus, global deacetylation of histones is necessary for invitro differentiation of ES cells. Similarly, Marin-Husstege etal. (2002) reported an erasure of histone acetylation and arequirement for histone deacetylase activity during oligo-dendrocyte lineage progression. Furthermore, Kim et al.(2003) showed that histone deacetylation is an importantfeature of nuclear reprogramming in oocytes during meio-sis. We conclude that histone deacetylases and acetylasestransmit differentiation signals to initiate appropriate epi-genetic modifications, such as erasure of preexisting chro-matin structure and establishment of new histone modifi-cation patterns during in vitro differentiation of ES cells.

MATERIALS AND METHODS

Cell CultureMale murine ES (CCE) cells (Evans and Kaufman, 1981;

Keller et al., 1993) were cultured in Dulbecco’s modified

Eagle’s medium (Life Technologies, Carlsbad CA) supple-mented with 25 mM glucose, 150 IU/ml of penicillin G, 150�g/ml of streptomycin sulfate, 0.007 �g/ml of �-mercapto-ethanol, 1000 IU/ml mouse leukemia inhibitory factor(LIF), and 10% fetal bovine serum (Sigma Chemical, St.Louis, MO). Cells were grown on gelatinized tissue culturedishes at 37°C and 5% CO2. To initiate differentiation,1–10 � 105 ES cells were seeded into bacterial culturedishes in medium lacking LIF, containing 5 or 10 nM tri-chostatin A (TSA) (Calbiochem, San Diego, CA) whereindicated, and medium was changed every 24 h. Alterna-tively, 20 �l of medium (200–300 cells) were placed on thelids of bacterial culture dishes containing PBS. The embry-onic bodies that form in these hanging drops were trans-ferred to new plates after 2 days of culture. Alkaline phos-phatase activity, which is downregulated upon ES celldifferentiation (Wobus et al., 1997), was histochemicallydetected using an alkaline phosphatase assay kit (Sigma).

Histone Preparation and Western Analysis

Histones were isolated by acid extraction, essentially asdescribed by Panyim et al. (1971). Protein concentrationswere determined by the Bradford (1976) method and Coo-massie R-250 staining and core histones were solubilized inLaemmli sample buffer. Following electrophoresis on a12% SDS polyacrylamide gel, proteins were transferredonto polyvinylidene difluoride membrane (AmershamPharmacia Biotech, Piscataway, NJ). The membrane wasincubated with one of the following modification-specificantisera: antihistone H3-Lys4 mono-methyl (Abcam, Cam-bridge, UK), anti-histone H3-Lys4 di-methyl (Upstate Bio-tech., Lake Placid, NY), anti-histone H3-Lys4 tri-methyl (Ab-cam), anti-histone H3-Lys9 mono-methyl (Abcam), anti-histone H3-Lys9 di-methyl (Upstate, Lake Placid, NY), anti-histone H3-Lys9 tri-methyl (Abcam), anti-acetylated histoneH3 antibody (Lys9, Lys14) (Upstate), or anti-acetylated his-tone-4 (Lys5, Lys8, Lys12, Lys16) (Upstate). Membraneswere then incubated with horseradish peroxidase-labeledsecondary antibody and detected by an ECL detection kit(Amersham, Arlington Heights, IL) according to the manu-facturer’s instructions.

Analysis of DNA Methylation

Analysis of 5-methylcytosine in the context of thesequence CCGG was performed as described by Li et al.(1992). Briefly, genomic DNA was digested with therestriction enzyme MspI or the methyl-sensitive isoschi-zomer HpaII, labeled with T4 polynucleotide kinase and[�-32P]ATP, and digested with nuclease P1. 5-Methylcy-tosine monophosphate and cytosine monophosphatewere separated by thin-layer chromatography and visu-alized by autoradiography.

Chromatin Immunoprecipitation Assay

ES cells were treated with 1% formaldehyde for 10 min at37°C to cross-link DNA and associated proteins, and chro-matin immunoprecipitation (ChIP) was performed using aChIP assay kit as described by the manufacturer (Upstate).ChIP products were analyzed using polymerase chain re-

FIG. 3. Histone deacetylation is required for ES cell differentiation.a: TSA treatment blocks histone deacetylation. ES cells were dif-ferentiated in vitro for the indicated number of days following re-moval of LIF. Following removal of LIF, ES cells were grown in thepresence of 10 nM TSA for 6 days. Alternatively, TSA was removedfrom the medium following 3 days of treatment and ES cells weregrown in the absence of TSA for another 3 days (-TSA Day4).Histones were isolated as described in Materials and Methods.Histone modifications were analyzed by Western blotting usingmodification-specific antibodies. The experiment was performedthree times and representative data are shown. b: TSA treatmentprevents embryoid body formation. ES cells were examined mor-phologically for embryoid body formation as a measure of differen-tiation. ES cells were grown as described above. The experimentwas performed three times and representative data are shown. c:ES cells were examined for alkaline phosphatase activity as a mea-sure of differentiation, as described in Materials and Methods. Al-kaline phosphatase activity is downregulated upon ES cell differen-tiation (Wobus et al., 1997) and the percentage of ES cells thatexhibited downregulated alkaline phosphatase activity is indicated.The data represents a summary of three independent experiments.Error bars indicate the mean SD and P value was determined by astandard t-test.

37EPIGENETICS OF ES CELL DIFFERENTIATION

action (PCR) (21–23 cycles) with [�-32P]dCTP. PCR prim-ers were as follows. Oct4-F; 5�-tgggtaagcaagaactgaggagtg-3�,Oct4-R; 5�-ttcaaggtcctctcacccctgcct-3� which amplify a 216bp fragment of the Oct4 gene promoter (positions –465 to–250 bp) (Accession number: S58422), Brachyury-F; 5�-ccacttgaactcccgcaaggcgcg-3�, Brachyury-R; 5�-ccgctttgatg-gaggtgcaaac-3� which amplify a 303 bp fragment of theBrachyury gene promoter (positions –364 to –62 bp)(Accession number: AF466883). PCR products were re-solved by 6% polyacrylamide gel electrophoresis and ex-posed to X-ray film. Dose–response experiments were per-formed to confirm that each PCR reaction was in the linearrange (data not shown).

Southern Blot Analysis

Genomic DNA samples were digested with NcoI/HpaII (Oct4 analysis) or SacI/HpaII/NcoI (Brachyuryanalysis) and loaded onto 1.2% agarose gels. DNA wastransferred to nylon membrane and hybridized with ra-dioactive probes. Probes for Southern analyses weresynthesized by PCR and 50 ng was radioactively labeledby random priming. Primers used for PCR were as fol-lows. Oct4 (277 bp): (For) 5�-gagcctctaaactctggaggactg-3�, (Rev) 5�-cacctcacaaaccagttgctcgg-3� (positions –569to –293 bp); Brachyury (178 bp): (For) 5�-ccagtctga-cacggccgcgcac-3�, (Rev) 5�-ccacttgaactcccgcaaggcgcg-3�(positions –239 to –62 bp).

Semiquantitative RT-PCR

Total cellular RNA was isolated using the TRIzol re-agent (Life Technologies, Gaithersburg, MD). Total RNA(1 �g) was reverse-transcribed using avian myeloblasto-sis virus reverse transcriptase and random hexamers(Roche, Indianapolis, IN) at 42°C for 60 min. Single-stranded cDNA (0.1 �g) was amplified in a 25 �l reactionmixture that included 10 nM of dNTPs, 50 pmol of senseand antisense primers, and 1 U of Taq DNA polymerase(Roche) in buffer supplied by the manufacturer. Sampleswere heat-denatured at 94°C for 2 min, followed by28–30 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for30 s, and finally 10 min at 72°C. PCR products (10 �l)were subjected to electrophoresis on 1.5% agarose gels.Dose–response experiments were performed to confirmthat the assay was in the linear response range.

ACKNOWLEDGMENT

We thank Paula Ladd and Erika Dobrota for technicalsupport and helpful advice.

LITERATURE CITED

Baylin SB, Herman JG. 2000. DNA hypermethylation in tumorigenesis.Trends Genet 16:168–173.

Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humph-erys D, Mastrangelo M-A, Jun Z, Walter J, Jaenisch R. 2002. Dnmt1overexpression causes genomic hypermethylation, loss of imprint-ing, and embryonic lethality. Mol Cell Biol 22:2124–2135.

Bradford MM. 1976. A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of pro-tein-dye binding. Anal Biochem 72:248–254.

Chapman V, Forrester L, Sanford J, Hastie N, Rossant J. 1984. Celllineage-specific undermethylation of mouse repetitive DNA. Na-ture 307:284–286.

Evans MJ, Kaufman MH. 1981. Establishment in culture of pluripoten-tial cells from mouse embryos. Nature 292:154–156.

Frances NJ, Kingston RE. 2001. Mechanisms of transcriptional mem-ory. Nat Rev Mol Cell Biol 2:409–421.

Gidekel S, Bergman Y. 2002. A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification ele-ment. J Biol Chem 277:34521–34530.

Jenuwein T, Allis CD. 2001. Translating the histone code. Science293:1074–1080.

Keller G, Kennedy M, Papayannopoulou T, Wiles M. 1993. Hematopoi-etic commitment during embrynic stem cell differentiation inculture. Mol Cell Biol 13:473–486.

Kim JM, Liu H, Tazaki M, Nagata M, Aoki F. 2003. Changes in histoneacetylation during mouse oocyte meiosis. J Cell Biol 162:37–46.

Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R, Li E. 1996.De novo DNA cytosine methyltransferase activities in mouse em-bryonic stem cells. Development 122:3195–3205.

Li E. 2002. Chromatin modification and epigenetic reprogramming inmammalian development. Nat Rev Genet 3:662–673.

Li E, Bestor TH, Jaenisch R. 1992. Targeted mutation of the DNAmethyltransferase gene results in embryonic lethality. Cell 69:915–926.

Marin-Husstege M, Muggironi KM, Liu A, Casaccia-Bonnefil P. 2002.Histone deacetylase activity is necessary for oligodendocyte lin-eage progression. J Neurosci 22:10333–10345.

Nakayama J, Rice JC, Strahl BD, Allis CD, Grewal SI. 2001. Role ofhistone H3 lysine 9 methylation in epigenetic control of hetero-chromatin assembly. Science 292:110–113.

Okamoto K, Okazawa H, Okuda A, Sakai M, Murmatsu M, Hamada H.1990. A novel octamer binding transcription factor is differentiallyexpressed in mouse embryonic cells. Cell 60:461–472.

Okano M, Bell DW, Haber DA, Li E. 1999. DNA methyltransferasesDnmt3a and Dnmt3b are essential for de novo methylation andmammalian development. Cell 99:247–257.

Orlando V. 2003. Polycomb, epigenomes, and control of cell identity.Cell 112:599–606.

Panyim S, Bilek D, Chalkley R. 1971. An electrophoretic comparison ofvertebrate histones. J Biol Chem 246:4206–4215.

Reik W, Dean W, Walter J. 2001. Epigenetic reprogramming in mam-malian development. Science 293:1089–1093.

Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW,Staudt LM. 1990. A POU-domain transcription factor in early stemcells and germ cells of the mammalian embryo. Nature 345:686–692.

Roth SY, Denu JM, Allis CD. 2001. Histone acetyltransferases. Annu RevBiochem 70:81–120.

Santos F, Hendrich B, Reik W, Dean W. 2002. Dynamic reprogrammingof DNA methylation in the early mouse embryo. Dev Biol 241:172–182.

Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J, Bernstein BE, EmreNC, Schreiber SL, Mellor J, Kouzarides T. 2002. Active genes aretri-methylated at K4 of histone H3. Nature 419:407–411.

Strahl BD, Allis CD. 2000. The language of covalent histone modifica-tions. Nature 403:41–45.

Surani MA. 2001. Reprogramming of genome function through epige-netic inheritance. Nature 414:122–128.

Weng A, Magnuson T, Storb U. 1995. Strain-specific transgene methyl-ation occurs early in mouse development and can be recapitulatedin embryonic stem cells. Development 121:2853–2859.

Wobus A, Kaomei G, Shan J, Wellner M-C, Rohwedel J, Guanju J,Fleischmann B, Katus HA, Hescheler J, Franz W-M. 1997. Retinoicacid accelerates embryonic stem cell-derived cardiac differentia-tion and enhances development of ventricular cardiomyocytes. JMol Cell Cardiol 29:1525–1539.

38 LEE ET AL.