Epigenetic Control of Mouse Oct-4 Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells

Abstract

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 and the chromatin structure of Oct-4 enhancer/promoter region in both ES and TS cells. and were from The ES cell from mice E. H. N. J. Dev. Biol. 1994; Google Scholar), was by H. and in a W. A Scholar). The cells were by the of S. D. N. Nature. PubMed Scopus Google TS cells were from mice according to the previously (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). the of these TS cells, were on cells in the of and (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). TS cells from mice were in the by in embryonic with and on culture cells were in with fetal to with 5-aza-2′-deoxycytidine methylation TS cells were for and for days in or with or trichostatin A inhibitor of cells were in with or for days with TSA, cells were for and to or with or for mice were from The Jackson were on a and to and of the on which a was found was as days Dnmt1n/n embryos and were from mice and at dpc, in and at of the and CpG the and CpG of the genomic DNA with a by the of DNA from cells and tissues of mice and Dnmt1n/n was as previously (6Ohgane J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). was with and in with was by and for at the and were to of and and the was at for in The DNA was the DNA and the was with at for The was by to a of The DNA was in and by as were with or to the methylation status of Oct-4 upstream only to by the from genomic DNA to or and from methylated DNA by the The of were by The were and by the of The were cloned into and were for Analysis of Oct-4 Expression by was with of first was with to genomic The of was into first with and for was with and reverse to Oct-4 whereas of was with and reverse In the of Oct-4, were under the of were under the of region of the Oct-4 gene including the proximal enhancer/promoter to was by from the genomic DNA of and the was cloned into of the reporter was the which found in methylated the was with P. D. A. S. A. Nucleic Acids Res. 1990; PubMed Scopus Google in the of at for of the methylation was by to cells at in a were for and were with reporter the activity, of a having a was into the cells. In a a of of the reporter and of expression was into cells. The of both were a according to the were in Chromatin chromatin immunoprecipitation assay was and Cell. 1998; Full Text Full Text PDF PubMed Scopus Google In cells were with and with for at to form was on and with the at was with of the DNA from the for The of was with The were used to the Oct-4 proximal promoter and mouse A and and and at of and of Oct-4 by with Oct-4 gene is expressed only in restricted of cells. The expression of Oct-4 was not in TS cells or in cells, by analyses the Oct-4 gene was in somatic tissues and cells not Oct-4 in TS cells with 5-aza-dC, inhibitor of DNA in a In the Oct-4 expression was also in TS cells with inhibitor of that Oct-4 gene activity is in the suppressed in TS cells by DNA methylation and chromatin The expression of Oct-4 in cells, was not by with or the with and the Oct-4 gene in cells DNA or was not to Oct-4 transcription in cells. the that the mechanism of the Oct-4 gene in Oct-4 non-expressing cells DNA this was the the region of the Oct-4 gene is to hypermethylated in cells or tissues in which the Oct-4 gene is DNA of the of the Oct-4 Oct-4 gene has a distal and proximal which the cell type-specific expression of the Oct-4 gene G. A. K. M. K. Schöler H.R. PubMed Google Scholar, T. N. M. K. K. M. K. Schöler H.R. Y. Dev. 1999; PubMed Google Scholar). The is in Oct-4 expression in preimplantation embryos, germ cells, ES and embryonic germ cells G. A. K. 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Dev. 1999; PubMed Google Scholar). is no CpG at the of the Oct-4 the promoter region is in CpG is one and and in the DE, whereas has a for and at and In the promoter is one at We the DNA methylation status of the Oct-4 regulatory region by on these in ES cells, TS cells, and In ES cells, for were not with indicating that the genomic DNA of ES cells was at including and after in ES cells. it is that the Oct-4 locus was hypomethylated a of regulatory regions in ES cells In in TS cells all were to The of CpG methylation was that the Oct-4 upstream region of TS cells was methylated that of ES cells in the in which the Oct-4 gene is also not all of were indicating that the Oct-4 is methylated the regulatory regions in this tissue and is a the and distal regions of the Oct-4 gene the was hypomethylated in ES cells that express the Oct-4 whereas TS cells and the which do not express Oct-4, have methylated DNA of the Oct-4 investigated the methylation status of CpG upstream of the Oct-4 gene the promoter region to from the as by genomic CpGs in this and the CpG is in regions upstream of the Oct-4 gene in In ES cells, the of CpG methylation was only and the region is In the region was methylated in TS cells, the methylation of we that the promoter region of Oct-4 is hypomethylated in ES cells but hypermethylated in TS cells and these with of the it is that Oct-4 gene expression is DNA gene of Oct-4 by DNA promoter activity of Oct-4 the region was G. A. K. M. K. Schöler H.R. PubMed Google Scholar). We the reporter with the region of Oct-4 the and promoter regions to The Oct-4 reporter activity to that of the The activity of Oct-4 was by in vitro DNA DNA methylation at the Oct-4 regulatory region in Oct-4 of the reporter with expression also caused These that the transcription of the Oct-4 gene was CpG Expression of Oct-4 in has been in Dnmt1n/n mice, which activity compared with type E. R. Cell. Full Text PDF PubMed Scopus Google Scholar). We that the Oct-4 gene in mice the Oct-4 promoter region is also in these We the methylation status of the Oct-4 promoter region and the expression of Oct-4 in the placenta of Dnmt1n/n mice at E. R. Cell. Full Text PDF PubMed Scopus Google Scholar). methylated CpGs in the Oct-4 promoter region only of the CpGs in the placenta of Dnmt1n/n mice, whereas methylated CpGs in the placenta of type the expression of Oct-4 was in the Dnmt1n/n placenta but not in type placenta Expression of the Oct-4 gene was in type embryos to the of germ cells that express the Oct-4 gene. expression of Oct-4 in the mutant embryo to in the DNA in the expression of Oct-4 gene. Chromatin of the Oct-4 in ES and TS of CpG with the of transcription A. Cell. Full Text PDF PubMed Scopus Google and of and the transcription A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). DNA methylation chromatin by with and A. Nature. 1998; PubMed Scopus Google Scholar). we the status in ES cells and TS cells at the and promoter region of the Oct-4 gene by chromatin immunoprecipitation assay that and We of from the Oct-4 and promoter regions assay of in ES cells was that of TS cells and that the status of in the Oct-4 region was in ES cells in TS cells. Furthermore, the status of in ES cells was revealed to that of TS cells, indicating that the Oct-4 promoter region was in ES cells compared with TS cells and the of at both the Oct-4 and promoter was in ES cells in TS cells. DNA methylation status is linked to the chromatin structure of the Oct-4 upstream We and on the (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, 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. 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Full Text PDF PubMed Scopus Google Scholar). a Oct-4 expression and epigenetic by DNA methylation and chromatin was in the mammalian all cells differentiate changes in DNA the differentiation of a cell type is with the activation of a of genes and the of the Oct-4 gene is a for regulation DNA methylation early In the the that the Oct-4 gene has T-DMRs and that DNA methylation is critical for the regulation of Oct-4 gene expression. and DNA methylation pattern is specific as reported previously T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar). differentiation with the and methylation of the genomic DNA to form the tissue type-specific methylation pattern T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar, J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. 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