The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2′-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1n/n mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression. The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2′-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1n/n mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression. In mammalian embryogenesis the first cellular differentiation begins at the end of the third cleavage, which leads to compaction and formation of the blastocyst. The inner cell mass (ICM) 1The abbreviations used are: ICM, inner cell mass; ChIP, chromatin immunoprecipitation; RT, reverse transcription; dpc, days postcoitum; ES cells, embryonic stem cells; TS cells, trophoblast stem cells; T-DMR, tissue-dependent, differentially methylated region; 5-aza-dC, 5-aza-2′-deoxycytidine; TSA, trichostatin A; MeCP2, methyl-CpG-binding protein 2; DE, distal enhancer; PE, proximal enhancer. of the blastocyst is known to generate all fetal somatic cells and germ cells, whereas the outer cell layer, the trophectoderm, gives rise to the trophoblastic components of the placenta. The explant culture of ICM or epiblast cells produces pluripotent embryonic stem (ES) cell lines, which contribute to all of the ICM lineages in chimeric embryos (1Martin G.R. Proc. Natl. Acad. Sci. 1981; 78: 7634-7636Crossref PubMed Scopus (4355) Google Scholar, 2Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9424-9428Google Scholar). Trophoblast stem (TS) cell lines, which have the ability to differentiate into trophoblast lineage in vitro, have also been established from blastocyst or early postimplantation trophoblastic tissue (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). TS cells do not contribute to the embryo proper in chimeras but differentiate only into the trophoblastic cells of the placenta. We previously investigated the genome-wide DNA methylation status of CpG islands by RLGS (restriction landmark genomic scanning) of mouse stem cells, i.e. ES, embryonic germ, and TS cells, before and after differentiation, as well as germ cells isolated from testis and some somatic tissues (4Shiota K. Kogo Y. Ohgane J. Imamura T. Urano A. Nishino K. Tanaka S. Hattori N. Genes Cells. 2002; 7: 961-969Crossref PubMed Scopus (180) Google Scholar). These analyses revealed that genes having tissue-dependent, differentially methylated regions (T-DMRs) were numerous. The methylation pattern of T-DMRs was specific but varied according to cell lineage and tissue type (4Shiota K. Kogo Y. Ohgane J. Imamura T. Urano A. Nishino K. Tanaka S. Hattori N. Genes Cells. 2002; 7: 961-969Crossref PubMed Scopus (180) Google Scholar, 5Imamura T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar). We have also shown that individual cloned mice have different methylation aberrations, mainly at tissue-specific methylated loci (6Ohgane J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). Considering that DNA methylation is involved in various biological phenomena, such as tissue-specific gene expression, cell differentiation, X-chromosome inactivation, genomic imprinting, changes in chromatin structure, and tumorigenesis (7Eden S. Hashimshony T. Keshet I. Cedar H. Thorne A.W. Nature. 1998; 394: 842Crossref PubMed Scopus (240) Google Scholar, 8Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2252) Google Scholar, 9Bird A. Wolffe A. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar, 10Cho J. Kimyra H. Mimami T. Ohgane J. Hattori N. Tanaka S. Shiota K. Endocrinology. 2001; 142: 3389-3396Crossref PubMed Scopus (54) Google Scholar), it is conceivable that the formation of specific DNA methylation patterns is one of the principal epigenetic events underlying mammalian development. In mice, Oct-4, a member of the POU transcription factors, is expressed in the oocyte and preimplantation embryo but is later restricted to only the ICM of the blastocyst (11Okamoto K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 12Rosner M.H. Vigano M.A. Ozato K. Timmons P.M. Poirier P.W. Rigby P.W.J. Staudt L.M. Nature. 1990; 345: 686-692Crossref PubMed Scopus (771) Google Scholar, 13Schöler H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (598) Google Scholar), indicating that expression is restricted to totipotent and pluripotent cells. In Oct-4-deficient embryos, the ICM loses pluripotency and the trophoblast cells no longer proliferate to form the placenta (14Nichols J. Zevnik B. Anastassiadis K. Niwa H. Klewe-Nebenius D. Chambers I. Schöler H.R. Smith A. Cell. 1998; 95: 379-391Abstract Full Text Full Text PDF PubMed Scopus (2703) Google Scholar). Expression of the Oct-4 gene is found in ES cells but not in TS cells (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, 15Palmieri S.L. Peter W. Hess H. Schöler H.R. Dev. Biol. 1994; 166: 259-267Crossref PubMed Scopus (529) Google Scholar). Reduction in Oct-4 gene expression leads to the trans-differentiation of ES cells into TS cells under adequate culture conditions (16Niwa H. Miyazaki J. Smith A.G. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2926) Google Scholar), demonstrating that suppression of the Oct-4 gene is a critical step for the determination of the potency of these stem cells. The Oct-4 gene has a GC-rich and TATA-less minimal promoter (17Okazawa H. Okamoto K. Ishino F. Ishino-Kaneko T. Takeda S. Toyoda Y. Muramatsu M. Hamada H. EMBO J. 1991; 10: 2997-3005Crossref PubMed Scopus (156) Google Scholar), and several trans-activators and repressors have been reported as the regulators of Oct-4 expression (17Okazawa H. Okamoto K. Ishino F. Ishino-Kaneko T. Takeda S. Toyoda Y. Muramatsu M. Hamada H. EMBO J. 1991; 10: 2997-3005Crossref PubMed Scopus (156) Google Scholar, 18Pikarsky E. Sharir H. Ben-Shushan E. Bergman Y. Mol. Cell. Biol. 1994; 14: 1026-1038Crossref PubMed Scopus (105) Google Scholar, 19Schoorlemmer J. van Puijenbroek A. van Den Eijnden M. Jonk L. Pals C. Kruijer W. Mol. Cell. Biol. 1994; 14: 1122-1136Crossref PubMed Scopus (91) Google Scholar, 20Sylvester I. Schöler H.R. Nucleic Acids Res. 1994; 22: 901-911Crossref PubMed Scopus (73) Google Scholar, 21Ben-Shushan E. Sharir H. Pikarsky E. Bergman Y. Mol. Cell. Biol. 1995; 15: 1034-1048Crossref PubMed Google Scholar, 22Fuhrmann G. Sylvester I. Schöler H.R. Cell. Mol. Biol. (NoisyLe-Grand). 1999; 45: 717-724PubMed Google Scholar, 23Pesce M. Marin G.M. Philipsen S. Schöler H.R. Cell. Mol. Biol. (Noisy-Le-Grand). 1999; 45: 709-716PubMed Google Scholar, 24Barnea E. Bergman Y. J. Biol. Chem. 2000; 275: 6608-6619Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 25Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Schöler H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). However, specific regulatory mechanisms conferring the developmental stage- and cell type-specific expression of the Oct-4 gene have not been conclusively revealed to date. In this report, to determine whether DNA methylation is involved in the regulation of Oct-4 gene expression, we investigated 1) the effect of a DNA methylation inhibitor on Oct-4 non-expressing cells, 2) the methylation status of Oct-4 enhancer/promoter locus in both ES and TS cells, 3) the effect of in vitro methylation on Oct-4 enhancer/promoter activity, 4) Oct-4 expression in the DNA methyltransferase-1 (Dnmt1)-deficient conceptus, and 5) the chromatin structure of Oct-4 enhancer/promoter region in both ES and TS cells. Reagents, Cell Culture, and Tissue Preparation—All reagents were purchased from Wako Pure Chemicals (Osaka, Japan) unless stated otherwise. The ES cell line MS12, derived from C57BL/6 mice (26Kawase E. Suemori H. Takahashi N. Int. J. Dev. Biol. 1994; 38: 385-390PubMed Google Scholar), was kindly provided by Dr. H. Suemori and cultured in a standard condition (27Matise M.P. Auerbach W. Joyner A.L. Joyner A.L. Gene Targeting: A Practical Approach. Oxford University Press, New York2000: 102-132Google Scholar). The cells were kept undifferentiated by the addition of leukemia inhibitory factor (Chemicon, Temecula, CA (28Williams R.L. Hilton D.J. Pease S. Willson T.A. Stewart C.L. Gearing D.P. Wagnew E.F. Metcalf D. Nicola N.A. Gough N. Nature. 1988; 336: 684-687Crossref PubMed Scopus (1543) Google Scholar)). TS cells were derived from C57BL/6NCrj mice according to the methods described previously (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). To maintain the undifferentiated state of these TS cells, they were cultured on feeder cells in the presence of FGF-4 and heparin (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). TS cells from ICR mice were maintained in the undifferentiated state by culturing in 70% embryonic fibroblast-conditioned medium supplemented with FGF-4 and heparin on gelatin-coated culture dishes. NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. Prior to treatment with 5-aza-2′-deoxycytidine (5-aza-dC) (DNA methylation inhibitor, Sigma), TS cells were pre-cultured for 48 h and then cultured for 2 days in medium containing 0, 1, or 5 μm 5-aza-2′-dC with or without 200 nm trichostatin A (TSA; inhibitor of histone deacetylase 1, Wako). NIH/3T3 cells were similarly cultured in medium supplemented with 0, 1, or 5 μm 5-aza-dC for 3 days following preculture. For treatment with TSA, NIH/3T3 cells were incubated for 24 h and then exposed to 200 or 400 nm TSA with or without 5 μm 5-aza-2′-dC for 72 h. Dnmt1 heterozygote mice (Dnmt1n/+) were purchased from The Jackson Laboratory. They were maintained on a 14-h light/10-h dark schedule and allowed free access to food and water. Noon of the day on which a vaginal plug was found was designated as 0.5 days postcoitum (dpc). Dnmt1n/n embryos and placentas were obtained from intercrosses between Dnmt1n/+ mice and dissected at 10.5 dpc, frozen in liquid nitrogen, and stored at –80 °C until used. Determination of the GC Content and CpG Frequency—We analyzed the GC content and CpG frequency of the genomic DNA fragments with a computer program (CpG view version 1.2.2) provided by the National Institute of Infectious Diseases (www.nih.go.jp/yoken/genebank/). Sodium Bisulfite Genomic Sequencing—Genomic DNA from cultured cells and tissues of C57BL/6NCrj mice and Dnmt1n/n placentas was extracted as described previously (6Ohgane J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). Briefly, each sample was treated with proteinase K (Merck, Darmstadt, Germany) following phenol/chloroform/isoamyl alcohol (50:49:1) extraction and dissolved in 200 μm TE buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8.0). Genomic DNA, digested with EcoRI, was denatured by adding 0.3 m NaOH and incubating for 15 min at 37 °C. After the incubation, sodium metabisulfite, pH 5.0, and hydroquinone were added to final concentrations of 2.0 m and 0.5 mm, respectively, and the mixture was further incubated at 55 °C for 14 h in dark. The modified DNA was purified through the Wizard DNA Clean-Up system (Promega, Madison, WI), and the bisulfite reaction was terminated with NaOH (0.3 m, final) at 37 °C for 15 min. The solution was then neutralized by adding NH4Oac, pH 7.0, to a final concentration of 3 m. The ethanol-precipitated DNA was resuspended in water and then amplified by PCR using primers designed as follows: PRO F, 5′-TGGGTTGAAATATTGGGTTTATTT-3′; PRO R, 5′-CTAAAACCAAATATCCAACCATA-3′; DE2 F, 5′-GTGTGT TTATGT AATTGT AGATGATT-3′; DE2 R, 5′-TCTACCCCCTTTAAAAATCACTTTAA-3′; DE4 F, 5′-TAAAGGGAGTAGGTATATTT-3′; DE4 R, 5′-CAAATAACCACACTTACTAAA-3′; DE5 F, 5′-GTGATATATTTTAGTAGAAGGTTAGGTT-3′; DE5 R, 5′-TTACAAAATATACCTACTCCCTTT A-3′. PCR fragments were digested with HpyCH4IV (New England Biolabs) or TaqI (Roche Applied Science) to evaluate the methylation status of Oct-4 upstream region. Because only unmethylated cytosine residues are changed to thymines by the sodium bisulfite reaction, PCR fragments from nonmethylated genomic DNA are resistant to HpyCH4IV or TaqI, and those from methylated DNA are digested by the enzymes. The resultant products of restriction mapping were assessed by agarose gel electrophoresis. The images were recorded and quantified using NIH Image 1.61 software provided by the National Institutes of Health (rsbweb.nih.gov/pub/nih-image/nih-image/nihimage161_fat.hqx). The electrophoresed PCR fragments were cloned into pGEM T-Easy vector (Promega), and 10 clones were sequenced for each sample. Analysis of Oct-4 Expression by RT-PCR—Total RNA was prepared with TRIzol reagent (Invitrogen). Before synthesis of first strand cDNA, total RNA was treated with RNase-free DNase I (Invitrogen) to eliminate any residual genomic DNA. The mixture of total RNA was converted into first strand cDNA synthesis with random hexamer and SuperScript II™ First Strand Synthesis System for RT-PCR (Invitrogen). PCR was performed with forward (5′-GGCGTTCGCTTTGGAAAGGTGTTC-3′) and reverse (5′-CTCGAACCACATCCTTCTCT-3′) primers to detect Oct-4 cDNA, whereas control detection of β-actin was performed with forward (5′-GTGGGCCGCTCTAGGCACCAA-3′) and reverse (5′-CTCTTTGATGTCACGCACGATTTC-3′) primers. In the case of Oct-4, PCR reactions were performed under the following conditions: 95 °C, 30 s; 35 cycles of 94 °C, 30 s; 62 °C, 1 min; 72 °C, 1 min; final extension 72 °C, 5 min. For β-actin, PCR reactions were performed under the following condition: 95 °C, 30 s; 35 cycles of 94 °C, 30 s; 65 °C, 1 min; 72 °C, 1 min; final extension 72 °C, 5 min. Luciferase Reporter Assays—The 5′-flanking region of the Oct-4 gene including the proximal enhancer/promoter (–1524 to –30) was amplified by PCR from the genomic DNA of C57BL/6NCrj background, and the fragment was cloned into pGL3-Basic vector (Promega). Amplification of the reporter construct was carried out using the dam–,dcm– bacterial strain, SCS110 (Stratagene, La Jolla, CA), which lacks two methylase found in Escherichia coli. To obtain methylated reporter, the construct was incubated with 3 units/μg SssI methylase (New England Biolabs (29Renbaum P. Abrahamove D. Fainsod A. Wilson G.G. Rottem S. Razin A. Nucleic Acids Res. 1990; 18: 1145-1152Crossref PubMed Scopus (166) Google Scholar)) in the presence of 160 μm S-adenosylmethionine at 37 °C for 3 h. Completion of the methylation was confirmed by resistance to HpaII digestion. NIH/3T3 cells plated at 2.5 × 104/well in a 24-well dish were incubated for 48 h and then were transfected transiently with 0.38 μgofthe luciferase reporter construct using LipofectAMINE reagent (Invitrogen). To normalize the luciferase activity, 0.02 μg of a control plasmid having a Renilla luciferase sequence was co-transfected into the cells. In a parallel study, a mixture of 0.2 μg of the luciferase reporter construct and 0.2 μg of MeCP2 expression vectors was co-transfected into NIH/3T3 cells. The activities of both luciferases were determined using a Dual-Luciferase Reporter System (Promega) according to the manufacturer's instructions. Assays were performed three times each in triplicate. Chromatin Immunoprecipitation Assays—A chromatin immunoprecipitation assay was performed using anti-acetylated histone H3 and H4 antibodies (Upstate Biotechnology, Lake Placid, NY (30Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar)). In brief, cells were rinsed twice with phosphate-buffered saline and treated with 1% formaldehyde for 20 min at room temperature to form DNA-protein cross-links. Each sample was then sonicated on ice and incubated with the antibodies at 4 °C overnight. PCR was performed with aliquots of the extracted DNA from the immunocomplexes for 28 cycles. The amount of each product was evaluated with an ethidium bromide-stained gel image using NIH Image 1.61 software. The following primers were used to amplify the Oct-4 proximal enhancer (Primer A), promoter (Primer B), and mouse β-actin: Primer A forward, 5′-GGAGGTGCAATGGCTGTCTTGTCC-3′, and reverse, 5′-CTGCCTTGGGTCACCTTACACCTCAC-3′; Primer B forward, 5′-GTGAGGTGTCGGTGACCCAAGGCAG-3′, and reverse, 5′-GGCGAGCGCTATCTGCCTGTGTC-3′; β-actin forward, 5′-AAATGCTGCACTGTGCGGCG-3′, and reverse, 5′-AGGCAACTTTCGGAACGGCG-3′, at an annealing temperature of 63, 66, and 63 °C, respectively. Breakdown of Oct-4 Gene Silencing by Treatments with Reagents Affecting Epigenetic Status—The Oct-4 gene is expressed only in restricted types of cells. The expression of Oct-4 was not detected in TS cells or in NIH/3T3 cells, even by RT-PCR analyses (Fig. 1). Similarly, the Oct-4 gene was silenced in other somatic tissues and cells (data not shown). Oct-4 mRNA became detectable in TS cells following treatment with 5-aza-dC, an inhibitor of DNA methylation, in a concentration-dependent manner. In addition, the Oct-4 expression was also observed in TS cells treated with an inhibitor of histone deacetylase 1, TSA. This suggests that Oct-4 gene activity is kept in the suppressed state in TS cells by DNA methylation and chromatin remodeling (Fig. 1A). The expression of Oct-4 in NIH/3T3 cells, however, was not attained by treatment with either 5-aza-dC or TSA alone. Interestingly, the combined treatment with 5-aza-dC and TSA effectively activated the Oct-4 gene in NIH/3T3 cells (Fig. 1B). Thus, DNA demethylation or histone acetylation alone was not sufficient to evoke Oct-4 transcription in NIH/3T3 cells. Nonetheless, the data suggests that the repression mechanism of the Oct-4 gene in Oct-4 non-expressing cells involves DNA methylation. If this was the case, then the 5′-flanking region of the Oct-4 gene is likely to be hypermethylated in cells or tissues in which the Oct-4 gene is silenced. DNA Methylation Status of the 5′-flanking Region of the Oct-4 Gene—The Oct-4 gene has two distinct enhancers, a distal enhancer (DE) and proximal enhancer (PE), which alternately drive the cell type-specific expression of the Oct-4 gene (31Yeom Y.I. Fuhrmann G. Ovitt C.E. Brehm A. Ohbo K. Gross M. Hubner K. Schöler H.R. Development. 1996; 122: 881-894Crossref PubMed Google Scholar, 32Yoshimizu T. Sugiyama N. De Felice M. Yeom Y.I. Ohbo K. Masuko K. Obinata M. Abe K. Schöler H.R. Matsui Y. Dev. Growth Differ. 1999; 41: 675-684Crossref PubMed Google Scholar). The DE is implicated in Oct-4 expression in preimplantation embryos, germ cells, ES and embryonic germ cells (31Yeom Y.I. Fuhrmann G. Ovitt C.E. Brehm A. Ohbo K. Gross M. Hubner K. Schöler H.R. Development. 1996; 122: 881-894Crossref PubMed Google Scholar, 32Yoshimizu T. Sugiyama N. De Felice M. Yeom Y.I. Ohbo K. Masuko K. Obinata M. Abe K. Schöler H.R. Matsui Y. Dev. Growth Differ. 1999; 41: 675-684Crossref PubMed Google Scholar). There is no CpG island at the 5′-region of the Oct-4 gene, although the promoter region is relatively rich in CpG dinucleotide sequences (Fig. 2A). There is one HpyCH4IV recognition site (at –4401 (Site 1)) and three TaqI sites (at –4028 (Site 2), –3030 (Site 3), and –2823 (Site 4)) in the DE, whereas PE has a site each for HpyCH4IV and TaqI at –638 (Site 5) and –314 (Site 6), respectively. In the promoter region, there is one HpyCH4IV site at –202 (Site 7) (Fig. 2A). We analyzed the DNA methylation status of the Oct-4 regulatory region by restriction mapping analysis, focusing on these seven recognition sites in ES cells, TS cells, and adult liver. In ES cells, PCR products generated for Site 1 were not digested with HpyCH4IV, indicating that the genomic DNA of ES cells was unmethylated at Site 1 (Fig. 2B). Similarly, PCR products including Site 2, Site 6, and Site 7 remained intact after restriction enzyme treatment in ES cells. Thus, it is clear that the Oct-4 locus was hypomethylated over a wide range of regulatory regions in ES cells (Fig. 2C). In contrast, in TS cells all PCR products were sensitive to enzymatic digestion. The overall percentage of CpG methylation was 37–91% (Fig. 2B). This clearly shows that the Oct-4 upstream region of TS cells was more highly methylated than that of ES cells (Fig. 2C). Likewise, in the liver, in which the Oct-4 gene is also silenced (data not shown), all recognition sites of restriction enzymes were digested, indicating that the Oct-4 is highly methylated over the regulatory regions in this tissue (54–94%) (Fig. 2, B and C). Thus, there is a T-DMR covering the promoter, proximal, and distal enhancer regions of the Oct-4 gene (Fig. 2C). Importantly, the T-DMR was hypomethylated in ES cells that express the Oct-4 gene, whereas TS cells and the liver, which do not express Oct-4, have methylated T-DMR. DNA Methylation Profile of the Oct-4 Promoter Region—We further investigated the methylation status of each CpG immediately upstream of the Oct-4 gene containing the promoter region up to –470 bp from the translation start site (designated as +1) by sodium bisulfite genomic sequencing (Fig. 3A). There are 16 CpGs in this region, and the CpG density is higher than in other regions upstream of the Oct-4 gene (see diagram in Fig. 2A). In ES cells, the overall percentage of CpG methylation was only 5%, and the region is almost unmethylated. In contrast, the region was heavily methylated in TS cells, exhibiting 74% methylation. Similarly, the liver showed 74% methylation of CpGs. Thus, we confirmed that the promoter region of Oct-4 is hypomethylated in ES cells but hypermethylated in TS cells and liver. Taking these results together with those of the 5-aza-dC treatment, it is highly probable that Oct-4 gene expression is regulated through DNA methylation-mediated gene silencing. Repression of Oct-4 Regulatory Region Activity by DNA Methylation—The promoter activity of Oct-4 without the enhancer region was low (31Yeom Y.I. Fuhrmann G. Ovitt C.E. Brehm A. Ohbo K. Gross M. Hubner K. Schöler H.R. Development. 1996; 122: 881-894Crossref PubMed Google Scholar). We constructed the reporter plasmid with the 5′-upstream region of Oct-4 containing the PE and promoter regions (–1527 to –30, Fig. 3B). The Oct-4 reporter construct showed 4.3-fold activity relative to that of the empty vector. The transcriptional activity of Oct-4 was dramatically reduced by in vitro DNA methylation. Thus, DNA methylation at the Oct-4 regulatory region elicited strong repression in Oct-4 transcription. Co-transfection of the reporter plasmid with MeCP2 expression vector also caused severe suppression. These data clearly demonstrated that the transcription of the Oct-4 gene was regulated through CpG methylation. Expression of Oct-4 in Dnmt1-deficient Mouse Placenta— Genome-wide demethylation has been observed in Dnmt1n/n mice, which exhibit ∼30% Dnmt1 activity compared with wild type (33Li E. Bestor T.H. Janisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar). We assumed that the Oct-4 gene might be activated in Dnmt1-deficient mice if the Oct-4 promoter region is also demethylated in these mutants. We therefore analyzed the methylation status of the Oct-4 promoter region and the expression of Oct-4 in the placenta of Dnmt1n/n mice at 10.5 dpc (33Li E. Bestor T.H. Janisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar). As expected, methylated CpGs in the Oct-4 promoter region comprise only 5% of the total CpGs in the placenta of Dnmt1n/n mice, whereas methylated CpGs comprise 43% in the placenta of wild type littermates (Fig. 4A). Importantly, the expression of Oct-4 mRNA was detected in the Dnmt1n/n placenta but not in wild type placenta (Fig. 4B). Expression of the Oct-4 gene was detectable in wild type embryos due to the presence of primordial germ cells that express the Oct-4 gene. Greatly reduced expression of Oct-4 in the mutant embryo may be due to retarded growth, particularly in the caudal region. Thus, aberrant DNA demethylation results in the ectopic expression of Oct-4 gene. Chromatin Structure of the Oct-4 Gene Enhancer/Promoter Region in ES and TS Cells—Methylation of CpG dinucleotide interferes with the binding of transcription factors (34Bird A. Cell. 1992; 70: 5-8Abstract Full Text PDF PubMed Scopus (871) Google Scholar) and binding of MeCP2 and other methyl CpG-binding domain proteins physically obstructs the basal transcription machinery (35Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). Alternatively, DNA methylation induces chromatin condensation following deacetylation by histone deacetylases associated with MeCP2 and methyl CpG-binding domain proteins (36Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar). Therefore, we examined the histone acetylation status in ES cells and TS cells at the PE and promoter region of the Oct-4 gene by chromatin immunoprecipitation (ChIP) assay using antibodies that recognize acetylated histones H3 and H4 (Fig. 5). We designed two sets of primers from the Oct-4 PE (Primer A) and promoter (Primer B) regions (Fig. 5A). By ChIP assay using Primer A, enrichment of histone H3 acetylation in ES cells was more than 2-fold that of TS cells (Fig. 5, B and C), revealing that the acetylation status of histone H3 in the Oct-4 PE region was higher in ES cells than in TS cells. Furthermore, using Primer B, the acetylation status of histone H3 in ES cells was revealed to be more than three times that of TS cells, indicating that the Oct-4 promoter region was highly acetylated in ES cells compared with TS cells (Fig. 5, B and C). Similarly, the extent of histone H4 acetylation at both the Oct-4 PE and promoter was higher in ES cells than in TS cells. Thus, DNA methylation status is linked to the chromatin structure of the Oct-4 upstream region. We confirmed and expanded on the previous reports (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, 11Okamoto K. Okazawa H. Okuda A. Sakai M. M
