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1 Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2 Samuel Runenfeld Research Institute, Mt. Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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Methylation of DNA is essential for normal mammalian development (Li 2002), and proper formation of cell type-specific DNA methylation profiles is fundamental to cellular differentiation (Shiota et al. 2002). ES cells showed unique DNA methylation profiles different from those of both TS cells and embryonic germ cells (Shiota et al. 2002). Methylation of DNA, which causes gene silencing and heterochromatin formation, is a chief mechanism of epigenetic regulation (Bird & Wolffe 1999; Fazzari & Greally 2004). There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the mammalian genome (Ohgane et al. 1998; Imamura et al. 2001; Shiota et al. 2002). In general, DNA methylation of T-DMRs in gene loci causes transcriptional repression (Cho et al. 2001; Imamura et al. 2001; Hattori et al. 2004,; Nishino et al. 2004; Ko et al. 2005; Tomikawa et al. 2006). In addition to DNA methylation, histone modifications play an important role in heterochromatin formation, mitosis, chromosomal territory and gene regulation (Jenuwein & Allis 2001; Li 2002). Histone methylation is associated directly and indirectly with changes in DNA methylation profile (Selker et al. 2002; Tamaru et al. 2003; Hattori et al. 2004; Ikegami et al. 2007). Acetylation of histones and methylation of histone H3 lysine 4 (H3-K4) residue affect gene activation by relaxing chromatin (Eberharter & Becker 2002; Lachner & Jenuwein 2002; Hayashi et al. 2005). In contrast, methylation of H3 lysine 9 (H3-K9) and lysine 27 (H3-K27) residues is correlated with gene silencing by condensing chromatin (Lachner & Jenuwein 2002; Tachibana et al. 2002).
We previously found that DNA methylation and histone deacetylation are responsible for Oct-4 repression in TS cells (Hattori et al. 2004). Here we provide evidence that the Nanog gene is also regulated by an epigenetic mechanism. Intriguingly, however, the combination of the epigenetic mechanisms is unique from that of the Oct-4 gene.
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Results |
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The expression of the Nanog gene was investigated in ES cells, TS cells, NIH/3T3 cells and adult liver by RT-PCR (Fig. 1A). A specific band of the Nanog transcript was detected in ES cells, but not in NIH/3T3 cells or liver, confirming previous studies (Chambers et al. 2003; Mitsui et al. 2003). Nanog gene expression was barely detectable in TS cells by RT-PCR. Thus, the expression of the Nanog gene is restricted to limited types of cells.
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Region I was totally hypomethylated and Region II was hypermethylated in both ES and TS cells. However, in Region III, six CpGs at the proximal upstream region were completely unmethylated, while the other ten CpGs in the distal upstream region showed partial methylation (29%) in ES cells. In contrast, in TS cells, the Nanog locus was hypermethylated (78%) in the proximal upstream region, whereas the distal upstream region was moderately methylated (50%). Similarly, the Nanog locus in NIH/3T3 cells was hypermethylated both in the proximal (63%) and distal (68%) upstream regions. Thus, the DNA methylation status of Region III in ES cells is different from other cell types we examined, indicating that there is a tissue-dependent and differentially methylated region (T-DMR) in the 5'-upstream region of the Nanog gene as found in the Oct-4 gene. In this study, we temporarily divided the T-DMR into distal and proximal T-DMRs (Fig. 1B). The DNA methylation status of the proximal T-DMR was clearly different between ES cells and the other cell types, whereas differences in methylation in the distal T-DMR were not as great between ES cells and TS and NIH/3T3 cells.
DNA methylation repressed the promoter activity of the Nanog gene
The effect of DNA methylation on Nanog promoter activity was investigated by luciferase assay using two reporter constructs: the –358 Luc construct corresponding to the proximal T-DMR and the –983 Luc construct covering the proximal and distal T-DMR (Fig. 2A). The –358 Luc construct and the –983 Luc construct exhibited 18.3- and 10.3-fold promoter activity, respectively, relative to that of the control vector in ES cells (Fig. 2B). In vitro DNA methylation by SssI methylase caused severe suppression of activity. The luciferase assay was further performed using differentiated TS cells. The –358 Luc and the –983 Luc showed higher activity than the empty vector (Fig. 2C). The transcriptional activities of these constructs were dramatically reduced by in vitro CpG methylation in trophoblast cell lineage as found in ES cells. Results from the luciferase assays and sodium bisulfite sequencing of TS cells provide evidence that transcription of the Nanog gene is repressed by CpG methylation in TS cells.
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One may predict that epigenetic status of the Nanog gene is similar to that of the Oct-4 gene (Hattori et al. 2004). TS cells were treated with 5-aza-dC, an inhibitor of DNA methylation, and/or TSA, an inhibitor of histone deacetylase. Expression of Nanog and Oct-4 genes was barely detectable in TS cells before treatment (Fig. 3A). Importantly, Nanog expression did not become detectable following treatment with 5-aza-dC or TSA. Furthermore, 5-aza-dC and TSA together could not induce Nanog gene expression (Fig. 3A). Under identical experimental conditions, the transcript of Oct-4 gene became detectable, in agreement with our previous results that the Oct-4 gene is repressed via DNA methylation and histone deacetylation (Fig. 3A; Hattori et al. 2004). Based on these results, the epigenetic status of the Nanog gene may be different from that of the Oct-4 gene.
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To clarify the distinct epigenetic status between the Nanog and Oct-4 loci, we compared the status of histone H3 and H4 acetylation of the Nanog and Oct-4 upstream regions in ES and TS cells by ChIP assay (Fig. 3B–D). In ES cells, histones H3 and H4 in the proximal T-DMR of the Nanog gene were hyperacetylated, whereas acetylation of H3 and H4 in TS cells was minimal. Histones H3 and H4 in the distal T-DMR of the Nanog gene were also hyperacetylated in ES cells. Similarly, histones H3 and H4 of the Oct-4 up-stream region were hyperacetylated in ES cells and hypoacetylated in TS cells, confirming previous findings (Hattori et al. 2004). These data clearly demonstrated that histone H3 and H4 acetylation occurs at the Nanog gene locus similar to the Oct-4 gene locus.
H3-K4 methylation status of the Nanog gene locus and the Oct-4 gene locus
Next, we investigated H3-K4 tri-, di-methylation status (Fig. 4). H3-K4 trimethylation was high at the Nanog proximal T-DMR in ES cells, but was low in TS cells. Hyper-trimethylation of H3-K4 was observed in the Nanog distal T-DMR in ES cells, whereas the trimethylation level was minimal in TS cells (Fig. 4A). Similarly, in ES cells, the level of H3-K4 dimethylation was high at both proximal and distal T-DMRs (Fig. 4A). In the Oct-4 T-DMR, H3-K4 tri- and di-methylation was higher in ES cells than in TS cells, respectively (Fig. 4B), indicating that H3-K4 methylation also occurs at the Oct-4 gene locus in addition to DNA methylation and histone acetylation (Hattori et al. 2004). Thus, histone modifications such as histone acetylation and H3-K4 methylation, which facilitate chromatin relaxation, were similar between the Nanog and Oct-4 gene loci.
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We further analyzed H3-K9 and -K27 methylation status (Fig. 4A,B). The level of H3-K9 methylation of the Nanog proximal T-DMR was relatively high in TS cells compared to ES cells, although the level was low in both cell types. In contrast, in the distal T-DMR, H3-K9 methylation was much higher in TS cells than in ES cells. The level of H3-K27 methylation was higher in TS cells than in ES cells at both proximal and distal T-DMRs. In contrast to the Nanog gene locus, the signal of H3-K9 methylation at the Oct-4 gene T-DMR was quite low in both TS and ES cells. H3-K27 methylation was detectable in TS cells but the level was low compared to the signal at the Nanog gene locus, indicating that the involvement of H3-K9 and -K27 methylation is trivial in Oct-4 gene repression. Thus, the combination of histone modifications associated with the Nanog gene is distinct from that of the Oct-4 gene.
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Discussion |
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Nanog, Oct-4 and Sox2 are important factors for normal early mouse development (Nichols et al. 1998; Avilion et al. 2003; Chambers et al. 2003; Mitsui et al. 2003). Interestingly, Nanog transcription is controlled by a complex of Oct-4, Sox2 and/or Sox element-binding factor (Kuroda et al. 2005; Rodda et al. 2005). Furthermore, the Sox2 gene may also be under epigenetic control, because Sox2 expression was induced by 5-aza-dC treatment in embryoid bodies (Tsuji-Takayama et al. 2004). Therefore, epigenetic regulation seems to be a key mechanism that the early embryo utilizes to prevent a return to the stem condition following differentiation. In addition, ectopic expression of Oct-4 and Nanog genes has been used to characterize cancer stem cells. Since DNA hypermethylation of gene loci has been documented in several cancers (Robertson 2005; Ushijima 2005), various inhibitors of DNA methylation and histone deacetylation have been tested for use as cancer chemotherapeutic agents (Kim et al. 2003; Cheng et al. 2004). Such treatments may, however, reactivate unwanted genes such as Oct-4 and Sox2 in patients with particular types of cancer. Although Nanog was not reactivated by 5-aza-dC and TSA treatments in TS cells, careful monitoring of the reactivation of stem cell related genes would be important during epigenetic chemotherapy in cancer patients.
Although DNA methylation status of the upstream region is roughly associated with the status of histone modifications, the epigenetic component of the Nanog gene locus is complex (Fig. 5B). The Nanog gene locus has a relatively broad T-DMR compared to the Oct-4 locus. There is a p53 binding site in the distal T-DMR but not in the proximal T-DMR of the Nanog gene. Lin et al. (2005) reported that p53 induced H3 deacetylation at the Nanog gene locus through a direct interaction with a co-repressor, mSin3a. Indeed, the distal T-DMR showed hyper-acetylation in ES cells but not in TS cells. The difference in acetylation status between ES and TS cells was less in the proximal T-DMR than in the distal T-DMR. Quite interestingly, the levels of other histone modifications, including H3-K4, -K9 and -K27 methylation, were also less significant in the proximal T-DMR than the distal T-DMR. In contrast, the distal T-DMR exhibited a moderate difference in DNA methylation status between ES and TS cells, while the proximal T-DMR was heavily methylated in TS cells but not in ES cells. Thus, we conclude that histone modification is dominant in the distal T-DMR, while DNA methylation is dominant in the proximal T-DMR (Fig. 5B).
Recently, epigenetic marks, such as histone acetylation, H3-K4 trimethylation and H3-K9 dimethylation, of the Nanog gene in the ICM and the TE have been reported (O’Neill et al. 2006). These epigenetic marks of the ICM and the TE are similar to those in ES and TS cells, respectively, revealed from this study. In present study, we further found the difference in DNA methylation, H3-K4 dimethylation and H3-K27 dimethylation at the Nanog locus between ES and TS cells. Taken together, we predicted that levels of DNA methylation and H3-K27 dimethylation are lower in the ICM relative to the TE, while the level of H3-K4 dimethylation is higher in the ICM than in the TE, although further study will be needed.
Methylation of DNA occurs in the upstream regions of both Nanog and Oct-4 genes, while differences exist in the histone modifications in the T-DMRs of two gene loci. The expressions of Nanog and Oct-4 genes are crucial for normal development, and our results provide evidence that epigenetic silencing of these developmental master genes is required for normal development. Furthermore, epigenetic abnormality is one of the major causes of tumorigenesis (Robertson 2005; Ushijima 2005), and especially interesting is that cancer stem cells have been characterized by the expressions of Oct-4 and Nanog (Monk & Holding 2001; Ezeh et al. 2005; Tai et al. 2005). Thus, the mechanisms underlying the maintenance of ES cell pluripotency and normal development are composed of the distinct epigenetic stratum involved in the regulation of master genes. In conclusion, the Nanog gene is under the control of epigenetic mechanisms involving DNA methylation and histone modifications.
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Experimental procedures |
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All reagents were purchased from Wako Pure Chemicals (Osaka, Japan) unless stated otherwise.
The MS12 ES cell line derived from C57BL/6 mice was kindly provided by Dr H. Suemori (Kyoto University, Japan) (Kawase et al. 1994) and cultured in standard conditions (Matise et al. 2000). Cells were maintained in an undifferentiated state by the addition of leukemia inhibitory factor (Chemicon, Temecula, CA) (Williams et al. 1988). A TS cell line was derived from C57BL/6NCrj mouse blastocyst according to the methods previously described (Tanaka et al. 1998). The TS cells were cultured on feeder cells in the presence of FGF-4 and heparin to maintain their undifferentiated state (Tanaka et al. 1998). The NIH/3T3 cells were cultured in DMEM (Gibco BRL, Rockville, MD) supplemented with 10% FBS.
For treatment with 5-aza-2'-deoxycytidine (5-aza-2'-dC; Sigma-Aldrich, St. Louis, MO) and trichostatin A (TSA; Sigma-Aldrich), TS cells were precultured for 2 days with FGF-4, heparin and 80% mouse embryonic fibroblast cell-conditioned medium and then cultured for additional 2 days with 5-aza-dC (1 or 5 µM) and/or TSA (200 nM).
The liver was collected from adult C57BL/6Ncrj mice. Adult C57BL/6Ncrj mice were purchased from Charles River Japan Inc. (Yokohama, Japan) and maintained on illumination cycles (14 h light and 10 h dark) with ad lib access to food and water.
Sodium bisulfite genomic sequencing
Genomic DNA from cultured cells and tissues of C57BL/6NCrj mice was extracted as previously described (Ohgane et al. 1998). Briefly, each sample was treated with proteinase K (Merck, Darmstadt, Germany) followed by phenol/chloroform/isoamylalcohol (50 : 49 : 1) extraction and ethanol precipitation, and dissolved in 200 µL of 10 mM Tris–HCl (pH 8.0) containing 1 mM EDTA.
Genomic DNA was digested with Xba I (Takara, Kyoto, Japan) and denatured in 0.3 M NaOH for 15 min at 37 °C. After 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 incubated for 14 h at 55 °C in dark. The modified DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI), and the bisulfite-treated DNA was desulfonated with NaOH (0.3 M, final) for 15 min at 37 °C. The solution was then neutralized by adding NH4OAc (pH 7.0) to a final concentration of 3 M, and the DNA was precipitated with ethanol, resuspended in water and amplified by PCR with five sets of primers to cover the Nanog up-stream region: Bisul-1 forward, 5'-TAGAGATTTTGGTAGTAAGGTTTGAT-3'; Bisul-1 reverse, 5'-CCAACCAAATCAACCTATCTAAAAA-3'; Bisul-2 forward, 5'-GTTGGGTTAGAGTGTTTTTATTTAT-3'; Bisul-2 reverse, 5'-ATCAAACCTTACTACCAAAATCTCTA-3'; Bisul-3 forward, 5'-AGGTAGATTTTTGAGTTTAAGGTT-3'; Bisul-3 reverse, 5'-ATTACCACTAAAACCTACAATA-3'; Bisul-4 forward, 5'-GAGTGTGGTAAAAGTATGTAAT-3'; Bisul-4 reverse, 5'-CCTAAAACTCACTATATAAACAAAA-3'; Bisul-5 forward, 5'-ATAAGGAATGTAGTAAGTTTGTT-3'; Bisul-5 reverse, 5'-AATTACATACTTTTACCACACTCAAA-3'. The electrophoresed PCR fragments were cloned into pGEM T-easy vector (Promega), and ten clones of each sample were sequenced.
Analysis of mRNA expression by RT-PCR
Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA). Before synthesis of first strand cDNA, total RNA was treated with RNase-free DNase I (Invitrogen) to eliminate any residual genomic DNA. The DNase I-treated total RNA was converted into first strand cDNA with random hexamer and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). The PCR amplification was performed using r-Taq Polymerase (Toyobo, Tokyo, Japan). Each PCR was performed under the following conditions: for Nanog mRNA, 95 °C for 1 min, 30 cycles of 94 °C for 30 s, 65 °C for 30 s and 72 °C for 1 min, final extension 72 °C for 5 min; for Oct-4 mRNA, 95 °C for 1 min, 30 cycles of 94 °C for 30 s, 62 °C for 30 s and 72 °C for 1 min, final extension 72 °C for 5 min; for ß-actin mRNA, 95 °C for 1 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, final extension 72 °C for 5 min. Nanog, Oct-4 and ß-actin primer sets were designed as follows: Nanog forward, 5'-AGGGTCTGCTACTGAGATGCTCTG-3'; Nanog reverse, 5'-CGGTGGGAAAAACCAGTGGTTG-3'; Oct-4 forward, 5'-GGCGTTCGCTTTGGAAAGGTGTTC-3'; Oct-4 reverse, 5'-CTCGAACCACATCCTTCTCT-3'; ß-actin forward, 5'-GACAACGGCTCCGGCATGTGCAAAG-3'; ß-actin reverse, 5'-TTCACGGTTGGCCTTAGGGTTCAG-3'.
Luciferase reporter assays
Two Nanog upstream regions, –358 to +43 and –983 to +43 relative to transcription start site, were amplified by PCR, and the fragments were cloned into pGL3-Basic vector (Promega). Primers were designed as follows: –358 Luc forward, 5'-GGGGTACCCCACTTGACCTGAAACTT-3'; –358 Luc reverse, 5'-GAAGATCTTCCCACAGAAAGAGCAAGA-3'; –983 Luc forward, 5'-GGGGTACCCCACTCACTTATCTGTGAGCAC-3'; –983 Luc reverse, 5'-GAAGATCTTCCCACAGAAAGAGCAAGA-3'.
Amplification of the reporter constructs was performed using dam–, dcm– bacterial strain, SCS110 (Stratagene, La Jolla, CA), which lacks two methylases found in E. coli. A methylated reporter was obtained by incubating the construct with 3 U/µg of SssI methylase (New England BioLabs) in the presence of 160 µM of S-adenosylmethionine at 37 °C for 3 h. The construct was resistant to Hha I digestion, thereby confirming completion of the methylation reaction.
Embryonic stem (ES) cells plated at 1.4 x 105 cells per well in a 24-well dish were transiently transfected with 0.25 µg of the luciferase reporter constructs with Effectene Transfection Reagent (Qiagen). Trophoblast stem (TS) cells were replaced on 24-well plates at 5 x 104 cells per well 1 day prior to transfection, and were cultured without feeder cells in the absence of FGF4 and heparin to induce differentiation. All constructs (0.8 µg) were transfected into the cells by using Lipofectamine 2000 (Invitrogen). To normalize firefly luciferase activity of the reporter constructs, 1/10 (mol ratio) of internal control plasmid expressing Renilla luciferase (pRL-TK vector, Promega) was co-transfected into the cells. Promoter activity was measured at 48 h or 72 h after the transfection, in ES or TS cells, respectively. The activities of both luciferases were determined by means of a Dual-Luciferase Reporter System (Promega) according to the manufacturer's instructions. Assays were performed 3 times each in triplicate, and all results are shown as means ± S.E.
Chromatin immunoprecipitation (ChIP) assays
The ChIP assays were performed using anti-acetylated histone H3 and H4 antibodies (Catalog no. 06-599 and no. 06-598, respectively; Upstate Biotechnology, Lake Placid, NY) and anti-trimethylated H3-K4 (Catalog no. ab8580; Abcam), dimethylated H3-K4, H3-K9 and H3-K27 antibodies (Catalog no. 07-030, no. 07-452 and no. 07-212, respectively; Upstate Biotechnology) according to the manufacturer's instructions. Normal rabbit IgG (Catalog no. 12-370; Upstate Biotechnology) was used as a negative control to verify immunoprecipitation specificity. In brief, cells were rinsed twice with PBS and treated with 1% formaldehyde for 20 min at room temperature to form DNA-protein cross-links. Samples were sonicated on ice until chromatin fragments became 200–1000 bp in size and incubated with antibodies at 4 °C overnight. The PCR amplification was performed under the following conditions: 95 °C for 10 min, 31 or 33 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, final extension 72 °C for 10 min. Primers were designed as follows: distal T-DMR forward, 5'-GGTTAGAGTGCTTTCACTCAC-3'; distal T-DMR reverse, 5'-GCTGGCTTCAGACTTACTGC-3'; proximal T-DMR forward, 5'-GGAAGTGTCTTTAGATCAGAGG-3'; proximal T-DMR reverse, 5'-CCAAATCAGCCTATCTGAAGG-3', Oct-4 forward, 5'-GTGAGGTGTCCGGTGACCCAAGGCAG-3'; Oct-4 reverse, 5'-CGGCTCACCTAGGGACGGTTTCACC-3'. The amount of each product was evaluated with an ethidium bromide stained gel-image using NIH IMAGE 1.61 software.
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Acknowledgements |
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This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Grant-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology of Japan 15080202 to K.S. and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (N.H.).
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Footnotes |
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aThese authors have contributed equally to this work.
* Correspondence: E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Bird, A. & Wolffe, A. (1999) Methylation-induced repression—belts, braces, and chromatin. Cell 99, 451–454.[CrossRef][Medline]
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. & Smith, A. (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655.[CrossRef][Medline]
Cheng, J.C., Yoo, C.B., Weisenberger, D.J., Chuang, J., Wozniak, C., Liang, G., Marquez, V.E., Greer, S., Orntoft, T.F., Thykjaer, T. & Jones, P.A. (2004) Preferential response of cancer cells to zebularine. Cancer Cell 6, 151–158.[CrossRef][Medline]
Cho, J., Kimyra, H., Mimami, T., Ohgane, J., Hattori, N., Tanaka, S. & Shiota, K. (2001) DNA methylation regulates placental lactogen I gene expression. Endocrinology 142, 3389–3396.
Eberharter, A. & Becker, P.B. (2002) Histone acetylation: a switch between repressive and permissive chromatin. Second in press series on chromatin dynamics. EMBO Rep. 3, 224–229.[CrossRef][Medline]
Ezeh, U.I., Turek, P.J., Reijo, R.A. & Clark, A.T. (2005) Human embryonic stem cell genes Oct4, Nanog, Stellar, and Gdf3 are expressed in both seminoma and breast carcinoma. Cancer 104, 2255–2265.[CrossRef][Medline]
Fazzari, M.J. & Greally, J.M. (2004) Epigenomics: beyond CpG islands. Nat. Rev. Genet. 5, 446–455.[CrossRef][Medline]
Feldman, N., Gerson, A., Fang, J., Li, E., Zhang, Y., Shinkai, Y., Cedar, H. & Bergman, Y. (2006) G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat. Cell Biol. 8, 188–194.[CrossRef][Medline]
Gu, P., LeMenuet, D., Chung, A.C., Mancini, M., Wheeler, D.A. & Cooney, A.J. (2005) Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation. Mol. Cell. Biol. 251, 8507–8519.
Hattori, N., Nishino, K., Ko, Y.G., Ohgane, J., Tanaka, S. & Shiota, K. (2004) Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J. Biol. Chem. 279, 17063–17069.
Hayashi, K., Yoshida, K. & Matsui, Y. (2005) A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature 438, 374–378.[CrossRef][Medline]
Ikegami, K., Iwatani, M., Suzuki, M., Tachibana, M., Shinkai, Y., Tanaka, S., Greally, J.M., Yagi, S., Hattori, N. & Shiota, K. (2007) Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embyonic stem cells. Genes Cells 12, 1–11.
Imamura, T., Ohgane, J., Ito, S., Ogawa, T., Hattori, N., Tanaka, S. & Shiota, K. (2001) CpG island of rat sphingosine kinase-1 gene: tissue-dependent DNA methylation status and multiple alternative first exons. Genomics 76, 117–125.[CrossRef][Medline]
Jenuwein, T. & Allis, C.D. (2001) Translating the histone code. Science 293, 1074–1080.
Kawase, E., Suemori, H., Takahashi, N., Okazaki, K., Hashimoto, K. & Nakatsuji, N. (1994) Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int. J. Dev. Biol. 38, 385–390.[Medline]
Kim, M.S., Blake, M., Baek, J.H., Kohlhagen, G., Pommier, Y. & Carrier, F. (2003) Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res. 63, 7291–7300.
Ko, Y.G., Nishino, K., Hattori, N., Arai, Y., Tanaka, S. & Shiota, K. (2005) Stage-by-stage change in DNA methylation status of Dnmt1 locus during mouse early development. J. Biol. Chem. 280, 9627–9634.
Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H., Nakatsuji, N. & Tada, T. (2005) Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25, 2475–2485.
Lachner, M. & Jenuwein, T. (2002) The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 14, 286–298.[CrossRef][Medline]
Li, E. (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662–673.[CrossRef][Medline]
Lin, T., Chao, C., Saito, S., Mazur, S.J., Murphy, M.E., Appella, E. & Xu, Y. (2005) p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat. Cell Biol. 7, 165–171.[CrossRef][Medline]
Loh, Y.H., Wu, Q., Chew, J.L., et al. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–440.[CrossRef][Medline]
Martin, G.R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultures in medium conditions by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7636.
Matise, M.P., Auerbach, W. & Joyner, A.L. (2000) Production of targeted embryonic stem cell clones. In: Gene Targeting: a Practical Approach (ed. A.L. Joyner), pp. 102–132. New York: Oxford University Press Inc.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M. & Yamanaka, S. (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642.[CrossRef][Medline]
Monk, M. & Holding, C. (2001) Human embryonic genes re-expressed in cancer cells. Oncogene 20, 8085–8091.[CrossRef][Medline]
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J.C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 9424–9428.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H. & Smith, A. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391.[CrossRef][Medline]
Nishino, K., Hattori, N., Tanaka, S. & Shiota, K. (2004) DNA methylation-mediated control of Sry gene expression in mouse gonadal development. J. Biol. Chem. 279, 22306–22313.
Niwa, H., Miyazaki, J. & Smith, A.G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376.[CrossRef][Medline]
O’Neill, L.P., VerMilyea, M.D. & Turner, B.M. (2006) Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat. Genet. 38, 835–841.[CrossRef][Medline]
Ohgane, J., Aikawa, J., Ogura, A., Hattori, N., Ogawa, T. & Shiota, K. (1998) Analysis of CpG island of trophoblast giant cells by restriction landmark genomic scanning. Dev. Genet. 22, 132–140.[CrossRef][Medline]
Pereira, L., Yi, F. & Merrill, B.J. (2006) Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell. Biol. 26, 7479–7491.
Robertson, K.D. (2005) DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610.[CrossRef][Medline]
Rodda, D.J., Chew, J.L., Lim, L.H., Loh, Y.H., Wang, B., Ng, H.H. & Robson, P. (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol. Chem. 280, 24731–24737.
Selker, E.U., Freitag, M., Kothe, G.O., Margolin, B.S., Rountree, M.R., Allis, C.D. & Tamaru, H. (2002) Induction and maintenance of nonsymmetrical DNA methylation in Neurospora. Proc. Natl. Acad. Sci. USA 99 (Suppl. 4), 16485–16490.
Shiota, K., Kogo, Y., Ohgane, J., Imamura, T., Urano, A., Nishino, K., Tanaka, S. & Hattori, N. (2002) Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells 7, 961–969.[Abstract]
Suzuki, A., Raya, A., Kawakami, Y., Morita, M., Matsui, T., Nakashima, K., Gage, F.H., Rodriguez-Esteban, C. & Izpisua Belmonte, J.C. (2006) Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc. Natl. Acad. Sci. USA 103, 10294–10299.
Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H. & Shinkai, Y. (2002) G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779–1791.
Tai, M.H., Chang, C.C., Kiupel, M., Webster, J.D., Olson, L.K. & Trosko, J.E. (2005) Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 26, 495–502.
Tamaru, H., Zhang, X., McMillen, D., Singh, P.B., Nakayama, J., Grewal, S.I., Allis, C.D., Cheng, X. & Selker, E.U. (2003) Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat. Genet. 34, 75–79.[CrossRef][Medline]
Tanaka, S., Kunath, T., Hadjantonakis, A.-K., Nagy, A. & Rossant, J. (1998) Promotion of trophoblast stem cell proliferation by FGF-4. Science 282, 2072–2075.
Tomikawa, J., Fukatsu, K., Tanaka, S. & Shiota, K. (2006) DNA methylation-dependent epigenetic regulation of dimethylarginine dimethylaminohydrolase 2 gene in trophoblast cell lineage. J. Biol. Chem. 281, 12163–12169.
Tsuji-Takayama, K., Inoue, T., Ijiri, Y., Otani, T., Motoda, R., Nakamura, S. & Orita, K. (2004) Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells. Biochem. Biophys. Res. Commun. 323, 86–90.[CrossRef][Medline]
Ushijima, T. (2005) Detection and interpretation of altered methylation patterns in cancer cells. Nat. Rev. Cancer 5, 223–231.[CrossRef][Medline]
Williams, 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. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687.[CrossRef][Medline]
Received: 1 September 2006
Accepted: 13 December 2006
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