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Epithelial Cell Adhesion Molecule Regulation Is Associated with the Maintenance of the Undifferentiated Phenotype of Human Embryonic Stem Cells

2010; Elsevier BV; Volume: 285; Issue: 12 Linguagem: Inglês

10.1074/jbc.m109.077081

ISSN

1083-351X

Autores

Tung‐Ying Lu, Ruei‐Min Lu, Mei‐Ying Liao, John Yu, Chu-Hung Chung, Cheng-Fu Kao, Han‐Chung Wu,

Tópico(s)

Renal and related cancers

Resumo

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs. Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs. IntroductionHuman embryonic stem cells (hESCs) 4The abbreviations used are: hESChuman embryonic stem cellmAbmonoclonal antibodyMSPmethylation-specific PCRChIPchromatin immunoprecipitationH3K27lysine 27 of histone H3H3K27me3lysine 27 trimethylation of histone 3H3K4me3lysine 4 trimethylation of histone 3H3K9K14Aclysine 9/14 acetylation of histone 3H3K9me3lysine 9 trimethylation of histone 3H9-Diff.H9 cells differentiated at day 30PcGpolycomb groupESembryonic stemMEFmouse embryo fibroblastPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayRTreverse transcriptionQ-RTquantitative reverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseIPimmunoprecipitationshRNAshort hairpin RNA. are derived from the inner cell mass of blastocyst-stage embryos. They retain the unlimited proliferation and developmental pluripotency from their progenitors and are able to self-renew and give rise to differentiated progeny of all three germ layers (1.Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12103) Google Scholar). Therefore, hESCs have potential clinical applications and can be used to explore our knowledge of basic developmental biology. The identification of selectively expressed cell surface molecules is essential for the purification and characterization of pluripotent hESCs, playing an important role in helping us understand the mechanisms involved in stem cell differentiation and self-renewal.We recently established a very specific monoclonal antibody (mAb), OC98-1, against the cell surface protein EpCAM and found that EpCAM was highly expressed in undifferentiated hESCs. EpCAM (also known as 17-1A, GA733-2, KSA, ESA, and EGP-40) is a homophilic, calcium-independent cell adhesion molecule of 39–42 kDa (2.Litvinov S.V. Velders M.P. Bakker H.A. Fleuren G.J. Warnaar S.O. J. Cell Biol. 1994; 125: 437-446Crossref PubMed Scopus (461) Google Scholar, 3.Litvinov S.V. Balzar M. Winter M.J. Bakker H.A. Briaire-de Bruijn I.H. Prins F. Fleuren G.J. Warnaar S.O. J. Cell Biol. 1997; 139: 1337-1348Crossref PubMed Scopus (256) Google Scholar) expressed by most epithelial tissues and is abundantly and homogeneously expressed in human carcinomas. EpCAM is a type I transmembrane glycoprotein encoded by the TACSTD1 gene. The EpCAM protein consists of a total of 314 amino acids, containing an extracellular domain (EpEX) with a nidogen-like domain as well as thyroglobulin- and epidermal growth factor-like repeats (265 amino acids), a single transmembrane part, and a short intracellular domain (EpICD) of 26 amino acids. It is not structurally related to any of the major families of the adhesion molecules (cadherins, selectins, integrins, or cell adhesion molecules of the Ig superfamily) (4.Balzar M. Winter M.J. de Boer C.J. Litvinov S.V. J. Mol. Med. 1999; 77: 699-712Crossref PubMed Scopus (468) Google Scholar). The level of EpCAM expression has been correlated with dedifferentiation and malignant proliferation of epithelial cells (5.Litvinov S.V. van Driel W. van Rhijn C.M. Bakker H.A. van Krieken H. Fleuren G.J. Warnaar S.O. Am. J. Pathol. 1996; 148: 865-875PubMed Google Scholar, 6.Maetzel D. Denzel S. Mack B. Canis M. Went P. Benk M. Kieu C. Papior P. Baeuerle P.A. Munz M. Gires O. Nat. Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar). It is frequently detected in cancer-initiating cells (7.Al-Hajj M. Wicha M.S. Benito-Hernandez A. Morrison S.J. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3983-3988Crossref PubMed Scopus (8281) Google Scholar, 8.Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10158-10163Crossref PubMed Scopus (1747) Google Scholar) and tissue-specific normal stem or progenitor cells (9.Schmelzer E. Wauthier E. Reid L.M. Stem Cells. 2006; 24: 1852-1858Crossref PubMed Scopus (280) Google Scholar, 10.Dan Y.Y. Riehle K.J. Lazaro C. Teoh N. Haque J. Campbell J.S. Fausto N. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9912-9917Crossref PubMed Scopus (268) Google Scholar, 11.Schmelzer E. Zhang L. Bruce A. Wauthier E. Ludlow J. Yao H.L. Moss N. Melhem A. McClelland R. Turner W. Kulik M. Sherwood S. Tallheden T. Cheng N. Furth M.E. Reid L.M. J. Exp. Med. 2007; 204: 1973-1987Crossref PubMed Scopus (470) Google Scholar, 12.Anderson R. Schaible K. Heasman J. Wylie C. J. Reprod. Fertil. 1999; 116: 379-384Crossref PubMed Google Scholar, 13.Stingl J. Raouf A. Emerman J.T. Eaves C.J. J. Mammary Gland Biol. Neoplasia. 2005; 10: 49-59Crossref PubMed Scopus (125) Google Scholar). For example, EpCAM is expressed in the mammalian germ line (12.Anderson R. Schaible K. Heasman J. Wylie C. J. Reprod. Fertil. 1999; 116: 379-384Crossref PubMed Google Scholar) and is frequently present at the surface of human hepatic multipotent progenitors (9.Schmelzer E. Wauthier E. Reid L.M. Stem Cells. 2006; 24: 1852-1858Crossref PubMed Scopus (280) Google Scholar), hepatic stem cells (11.Schmelzer E. Zhang L. Bruce A. Wauthier E. Ludlow J. Yao H.L. Moss N. Melhem A. McClelland R. Turner W. Kulik M. Sherwood S. Tallheden T. Cheng N. Furth M.E. Reid L.M. J. Exp. Med. 2007; 204: 1973-1987Crossref PubMed Scopus (470) Google Scholar), and cancer stem cells (8.Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10158-10163Crossref PubMed Scopus (1747) Google Scholar). Very recently, EpCAM expression on ESCs has been reported by some studies (14.Sundberg M. Jansson L. Ketolainen J. Pihlajamäki H. Suuronen R. Skottman H. Inzunza J. Hovatta O. Narkilahti S. Stem Cell Res. 2009; 2: 113-124Crossref PubMed Scopus (79) Google Scholar, 15.Kolle G. Ho M. Zhou Q. Chy H.S. Krishnan K. Cloonan N. Bertoncello I. Laslett A.L. Grimmond S.M. Stem Cells. 2009; 27: 2446-2456Crossref PubMed Scopus (61) Google Scholar, 16.González B. Denzel S. Mack B. Conrad M. Gires O. Stem Cells. 2009; 27: 1782-1791Crossref PubMed Scopus (89) Google Scholar, 17.Ng V.Y. Ang S.N. Chan J.X. Choo A.B. Stem Cells. 2010; 28: 29-35Crossref PubMed Scopus (78) Google Scholar), suggesting that EpCAM might serve as a potential surface marker for these pluripotent cells.Little is known about molecular mechanisms underlying the regulation of EpCAM expression in hESC. For the past few years, more has been learned about the influence of DNA methylation and histone modifications on regulating gene expression and genome function. Several studies have discussed the DNA methylation status of EpCAM promoter in lung, colon, prostate, liver, bladder, ovary, and breast cancer cells and tissues (18.Tai K.Y. Shiah S.G. Shieh Y.S. Kao Y.R. Chi C.Y. Huang E. Lee H.S. Chang L.C. Yang P.C. Wu C.W. Oncogene. 2007; 26: 3989-3997Crossref PubMed Scopus (53) Google Scholar, 19.Spizzo G. Gastl G. Obrist P. Fong D. Haun M. Grünewald K. Parson W. Eichmann C. Millinger S. Fiegl H. Margreiter R. Amberger A. Cancer Lett. 2007; 246: 253-261Crossref PubMed Scopus (29) Google Scholar, 20.Yu G. Zhang X. Wang H. Rui D. Yin A. Qiu G. He Y. Oncol. Rep. 2008; 20: 1061-1067PubMed Google Scholar, 21.Shiah S.G. Chang L.C. Tai K.Y. Lee G.H. Wu C.W. Shieh Y.S. Oral Oncol. 2009; 45: e1-e8Crossref PubMed Scopus (22) Google Scholar). Post-translational modifications of histone tails, including phosphorylation, acetylation, ubiquitination, and methylation, have been validated as dynamic regulators of gene expression. In order to gain insight into the epigenetic transitions responsible for EpCAM expression in hESC, we studied the 5′-flanking region of EpCAM promoter by evaluating CpG status using methylation-specific PCR (MSP), bisulfite sequencing, and histone modification by chromatin immunoprecipitation (ChIP).The polycomb group (PcG) proteins are important chromatin modifiers that play a pivotal role in the epigenetic regulation of the development, differentiation, and maintenance of cell fates (22.Schuettengruber B. Chourrout D. Vervoort M. Leblanc B. Cavalli G. Cell. 2007; 128: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). Dynamic repression of developmental pathways by PcG may be required for maintaining ES cell pluripotency and plasticity during embryonic development (23.Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. Nature. 2006; 441: 349-353Crossref PubMed Scopus (2014) Google Scholar). The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalyzing the trimethylation of Lys27 on histone H3 (H3K27me3) (24.Schwartz Y.B. Pirrotta V. Nat. Rev. Genet. 2007; 8: 9-22Crossref PubMed Scopus (707) Google Scholar). Suppressor of Zeste 12 homolog (SUZ12), one of the PRC2 components, is essential for histone methyltransferase PRC2 activity on H3K27me3 methylation (25.Swigut T. Wysocka J. Cell. 2007; 131: 29-32Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 26.Cao R. Zhang Y. Mol. Cell. 2004; 15: 57-67Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 27.Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (656) Google Scholar). The recent identification of JmjC domain-containing histone lysine demethylase JMJD3 suggests that there may be positive and negative regulators simultaneously controlling chromatin structure dynamics through histone methylation mark alterations. JMJD3 specifically removes methyl marks of H3K27me3 in mammalian cells to antagonize PcG gene silencing and permit gene transcription. JMJD3 is highly expressed in ES cells and is responsible for the rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem cell differentiation (23.Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. Nature. 2006; 441: 349-353Crossref PubMed Scopus (2014) Google Scholar, 28.Agger K. Cloos P.A. Christensen J. Pasini D. Rose S. Rappsilber J. Issaeva I. Canaani E. Salcini A.E. Helin K. Nature. 2007; 449: 731-734Crossref PubMed Scopus (977) Google Scholar). These findings suggest that EpCAM may be regulated by both SUZ12 and JMJD3 during hESC differentiation.Understanding the downstream targets of EpCAM would help define the molecular function of this gene. However, such studies have been hindered by the obscure signaling mode of EpCAM until the very recent discovery of regulated intramembrane proteolysis and nuclear translocation of its intracellular domain EpICD. Released EpICD associates with FHL2, β-catenin, and Lef-1 and participates in gene regulation in the nucleus (6.Maetzel D. Denzel S. Mack B. Canis M. Went P. Benk M. Kieu C. Papior P. Baeuerle P.A. Munz M. Gires O. Nat. Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar). One of the EpCAM downstream targets, c-MYC, has been found to be regulated by EpCAM in both normal and cancer cells (29.Münz M. Kieu C. Mack B. Schmitt B. Zeidler R. Gires O. Oncogene. 2004; 23: 5748-5758Crossref PubMed Scopus (299) Google Scholar). c-MYC is a member of the four reprogramming factors involved in the induced pluripotent stem cell formation (30.Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2359) Google Scholar, 31.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14630) Google Scholar, 32.Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18453) Google Scholar, 33.Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. Nature. 2007; 448: 318-324Crossref PubMed Scopus (2202) Google Scholar, 34.Yu J. Hu K. Smuga-Otto K. Tian S. Stewart R. Slukvin II, Thomson J.A. Science. 2009; 324: 797-801Crossref PubMed Scopus (1767) Google Scholar). Understanding the regulation of EpCAM on c-MYC and even other reprogramming genes like OCT-4, NANOG, SOX2, and KLF4 in hESC may add to our understanding of how EpCAM contributes the long term maintenance of the ES cell phenotype.This study is a comprehensive analysis of EpCAM expression in undifferentiated hESCs using immunofluorescence microscopy, Western blotting, and flow cytometry. Loss of EpCAM expression in differentiated hESCs through epigenetic silencing is elucidated by ChIP. Because several reprogramming genes are under the regulation of EpCAM, we propose that EpCAM maintains hESC "stemness" through sustaining these reprogramming genes.DISCUSSIONThe recent successful use of somatic cells that can be directly reprogrammed into pluripotency and self-renewal stem cells (31.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14630) Google Scholar, 32.Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18453) Google Scholar) has opened a new means of investigating basic biology, developmental biology, and regenerative medicine. Ectopic transduction of terminally differentiated fibroblasts with the original four reprogramming transcription factors OCT-4, SOX2, KLF4, and c-MYC results in induced pluripotent stem cell formation morphologically and genetically (31.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14630) Google Scholar, 32.Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18453) Google Scholar). These four factors play a pivotal role in ES cell self-renewal and maintenance of pluripotency. However, until this study, it was unknown what was mutually regulating these four genes. This study found that EpICD could directly bind to the promoters of these four genes (Fig. 7) and sustain the expression of these genes in undifferentiated hESCs (Fig. 7). EpCAM expression in undifferentiated hESCs occurred through epigenetic regulation (FIGURE 5, FIGURE 6). Based on these findings, we propose that the maintenance of pluripotency by these four genes is controlled by the expression of EpCAM, which collaborates with these four factors to maintain self-renewal and pluripotency in ES cells.The expression of EpCAM has been used to recognize hepatic stem cells in fetal, postnatal, and adult humans (9.Schmelzer E. Wauthier E. Reid L.M. Stem Cells. 2006; 24: 1852-1858Crossref PubMed Scopus (280) Google Scholar, 10.Dan Y.Y. Riehle K.J. Lazaro C. Teoh N. Haque J. Campbell J.S. Fausto N. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9912-9917Crossref PubMed Scopus (268) Google Scholar, 11.Schmelzer E. Zhang L. Bruce A. Wauthier E. Ludlow J. Yao H.L. Moss N. Melhem A. McClelland R. Turner W. Kulik M. Sherwood S. Tallheden T. Cheng N. Furth M.E. Reid L.M. J. Exp. Med. 2007; 204: 1973-1987Crossref PubMed Scopus (470) Google Scholar) and is thought to be useful in the selection of cancer-initiating cells (7.Al-Hajj M. Wicha M.S. Benito-Hernandez A. Morrison S.J. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3983-3988Crossref PubMed Scopus (8281) Google Scholar, 8.Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10158-10163Crossref PubMed Scopus (1747) Google Scholar). Our study found EpCAM to be exclusively expressed in hESCs (FIGURE 1, FIGURE 2, FIGURE 3) and its expression to be correlated with the expression of OCT-4 (FIGURE 1, FIGURE 2), a known stem cell marker. This finding suggests that the cell surface expression property of EpCAM can be used to purify and enrich hESCs effectively, and it can be used as a surrogate for OCT-4 or potentially other hESC markers to simplify and improve the isolation and purity of hESCs. Our data can be compared with a recent publication showing EpCAM expression in undifferentiated hESCs (17.Ng V.Y. Ang S.N. Chan J.X. Choo A.B. Stem Cells. 2010; 28: 29-35Crossref PubMed Scopus (78) Google Scholar). Similar to that report, we show that EpCAM expression was restricted to undifferentiated hESCs, and its silencing was associated with cell differentiation. This property of EpCAM can make it useable as a surface marker for hESCs.Rigorous hESC isolation is a complicated process requiring various combinations of multiple cell surface markers. Nevertheless, many of the stem cell markers used nowadays cannot be used specifically on hESCs. For example, peanut agglutinin is only applicable as a cell surface marker in murine hematopoietic stem cells and human neural stem cells (56.Rietze R.L. Valcanis H. Brooker G.F. Thomas T. Voss A.K. Bartlett P.F. Nature. 2001; 412: 736-739Crossref PubMed Scopus (582) Google Scholar), and CD29 expression only can be used as a stem cell marker in murine skin or liver (10.Dan Y.Y. Riehle K.J. Lazaro C. Teoh N. Haque J. Campbell J.S. Fausto N. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9912-9917Crossref PubMed Scopus (268) Google Scholar, 57.Shackleton M. Vaillant F. Simpson K.J. Stingl J. Smyth G.K. Asselin-Labat M.L. Wu L. Lindeman G.J. Visvader J.E. Nature. 2006; 439: 84-88Crossref PubMed Scopus (1580) Google Scholar). CD133 (prominin-1) has recently emerged as a major somatic stem cell or progenitor marker (58.Huttner H.B. Janich P. Köhrmann M. Jászai J. Siebzehnrubl F. Blümcke I. Suttorp M. Gahr M. Kuhnt D. Nimsky C. Krex D. Schackert G. Löwenbrück K. Reichmann H. Jüttler E. Hacke W. Schellinger P.D. Schwab S. Wilsch-Bräuninger M. Marzesco A.M. Corbeil D. Stem Cells. 2008; 26: 698-705Crossref PubMed Scopus (81) Google Scholar). Thus, there is a need to expand the current repertoire of hESC markers to enable hESC studies. Recognition by hESC-selective cell surface molecules is required in order to specifically isolate pure hESCs. Particularly, the combination of viable markers has made it possible to separate multiple hESC populations at each distinct differentiation stage and yielded important insights into stem cell differentiation. Because EpCAM is not expressed by differentiated cells, this molecule is probably exerted at the stem or multipotent progenitor cell stage.The results of studies over the past few years have suggested that epigenetic mechanisms play a key role in these fundamental processes of self-renewal, maintenance of pluripotency, and lineage specification (23.Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. 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Non-transcribed genes in ESCs are repressed by the PcG in mice and humans with the promoters of these genes enriched with repressive histone H3K27 trimethylation. Our investigation on the epigenetic regulations of EpCAM expression during differentiation indicated that the expression of EpCAM was not regulated by DNA methylation (Fig. 4), which would lead to permanent silencing of the gene. Instead, we found that during differentiation, there is a drastic reduction in histone active markers, such as H3K4 trimethylation and H3K9 acetylation, and clear enhancement of repressive markers H3K9 and H3K27 trimethylation at the promoter of EpCAM (Fig. 5). In addition, EpCAM was not controlled by bivalent chromatin modifications (61.Azuara V. Perry P. Sauer S. Spivakov M. J⊘rgensen H.F. John R.M. Gouti M. Casanova M. Warnes G. Merkenschlager M. Fisher A.G. Nat. Cell Biol. 2006; 8: 532-538Crossref PubMed Scopus (1048) Google Scholar, 62.Bernstein B.E. Mikkelsen T.S. Xie X. Kamal M. Huebert D.J. Cuff J. Fry B. Meissner A. Wernig M. Plath K. Jaenisch R. Wagschal A. Feil R. Schreiber S.L. Lander E.S. Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3990) Google Scholar) because H3K4 and H3K27 trimethylation did not coexist at its promoter before or after the differentiation (Fig. 5), indicating that EpCAM does not belong to the category of lineage commitment or cell fate determination genes (62.Bernstein B.E. Mikkelsen T.S. Xie X. Kamal M. Huebert D.J. Cuff J. Fry B. Meissner A. Wernig M. Plath K. Jaenisch R. Wagschal A. Feil R. Schreiber S.L. Lander E.S. Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3990) Google Scholar, 63.Mikkelsen T.S. Ku M. Jaffe D.B. Issac B. Lieberman E. Giannoukos G. Alvarez P. Brockman W. Kim T.K. Koche R.P. Lee W. Mendenhall E. O'Donovan A. Presser A. Russ C. Xie X. Meissner A. Wernig M. Jaenisch R. Nusbaum C. Lander E.S. Bernstein B.E. Nature. 2007; 448: 553-560Crossref PubMed Scopus (3201) Google Scholar). Our findings reflect the fact that EpCAM is virtually reintroduced in mature tissues (64.Trzpis M. McLaughlin P.M. de Leij L.M. Harmsen M.C. Am. J. Pathol. 2007; 171: 386-395Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), especially in epithelia cells. Therefore, our results suggest that, to facilitate the dynamic expression pattern of EpCAM during development, the chromatin state of its promoter is elaborately modulated by histone-modifying enzymes, such as SUZ12 and JMJD3, but not DNA methylation (Fig. 6) in response to hESC differentiation, which sustains the plasticity of EpCAM expression. SUZ12 has been found to be a key component of polycomb complex, PRC2 (26.Cao R. Zhang Y. Mol. Cell. 2004; 15: 57-67Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 27.Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (656) Google Scholar), and essential for the activity of H3K27 methyltransferase. Conversely, JMJD3, a newly identified histone demethylase for H3K27 methylation, is able to remove the methylation mark from H3 lysine 27 and control cell differentiation (65.De Santa F. Totaro M.G. Prosperini E. Notarbartolo S. Testa G. Natoli G. Cell. 2007; 130: 1083-1094Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). We found SUZ12 and JMJD3 to be dynamically associated with EpCAM promoter (Fig. 6). The methylation patterns of H3 Lys27 also changed accordingly (Fig. 5). ChIP combination resulted from the enzyme bindings and the changes of the corresponding histone mark, suggesting that EpCAM is part of a polycomb-mediated differentiation program in human ES cells.Our study found that EpCAM played an intricate role in the regulation of the ES cell state. This may be achieved by modulating the expression of the EpCAM downstream target gene c-MYC, which, as other studies have reported, is involved in governing cell proliferation and dedifferentiation (29.Münz M. Kieu C. Mack B. Schmitt B. Zeidler R. Gires O. Oncogene. 2004; 23: 5748-5758Crossref PubMed Scopus (299) Google Scholar, 50.Cartwright P. McLean C. Sheppard A. Rivett D. Jones K. Dalton S. Development. 2005; 132: 885-896Crossref PubMed Scopus (583) Google Scholar). Both c-MYC and EpCAM are controlled by the Wnt signaling cascade (51.He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4047) Google Scholar, 66.Yamashita T. Budhu A. Forgues M. Wang X.W. Cancer Res. 2007; 67: 10831-10839Crossref PubMed Scopus (351) Google Scholar, 67.van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. Tjon-Pon-Fong M. Moerer P. van den Born M. Soete G. Pals S. Eilers M. Medema R. Clevers H. Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1717) Google Scholar, 68.Muncan V. Sansom O.J. Tertoolen L. Phesse T.J. Begthel H. Sancho E. Cole A.M. Gregorieff A. de Alboran I.M. Clevers H. Clarke A.R. Mol. Cell. Biol. 2006; 26: 8418-8426Crossref PubMed Scopus (202) Google Scholar), in which signal transduction by EpICD interacts with β-catenin/Lef-1 in cohort to regulate c-MYC expression (6.Maetzel D. Denzel S. Mack B. Canis M. Went P. Benk M. Kieu C. Papior P. Baeuerle P.A. Munz M. Gires O. Nat. Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar). The canonical Wnt signaling pathway has emerged as a critical regulator of ES and hematopoietic lineage stem cells in other studies (69.Reya T. Duncan A.W. Ailles L. Domen J. Scherer D.C. Willert K. Hintz L. Nusse R. Weissman I.L. Nature. 2003; 423: 409-414Crossref PubMed Scopus (1778) Google Scholar, 70.Sato N. 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Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar) up-regulates c-MYC, OCT-4, NANOG, SOX2, and KLF4 (FIGURE 7, FIGURE 8), indicating that EpCAM targeting of these ES cell fate genes may not occur exclusively through Wnt signaling, suggesting that there may be several pathways orchestrating the maintenance of the physical state of ES cells. Further investigations are required to clarify the relationship between Wnt signaling and OCT-4, NANOG, SOX2, and KLF4, because the results of such studies may further our understanding the nature of pluripotency and of self-renewal signals in hESCs. IntroductionHuman embryonic stem cells (hESCs) 4The abbreviations used are: hESChuman embryonic stem cellmAbmonoclonal antibodyMSPmethylation-specific PCRChIPchromatin immunoprecipitationH3K27lysine 27 of histone H3H3K27me3lysine 27 trimethylation of histone 3H3K4me3lysine 4 trimethylation of histone 3H3K9K14Aclysine 9/14 acetylation of histone 3H3K9me3lysine 9 trimethylation of histone 3H9-Diff.H9 cells differentiated at day 30PcGpolycomb groupESembryonic stemMEFmouse embryo fibroblastPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayRTreverse transcriptionQ-RTquantitative reverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseIPimmunoprecipitationshRNAshort hairpin RNA. are derived from the inner cell mass of blastocyst-stage embryos. They retain the unlimited proliferation and developmental pluripotency from their progenitors and are able to self-renew and give rise to differentiated progeny of all three germ layers (1.Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12103) Google Scholar). Therefore, hESCs have potential clinical applications and can be used to explore our knowledge of basic developmental biology. The identification of selectively expressed cell surface molecules is essential for the purification and characterization of pluripotent hESCs, playing an important role in helping us understand the mechanisms involved in stem cell differentiation and self-renewal.We recently established a very specific monoclonal antibody (mAb), OC98-1, against the cell surface protein EpCAM and found that EpCAM was highly expressed in undifferentiated hESCs. EpCAM (also known as 17-1A, GA733-2, KSA, ESA, and EGP-40) is a homophilic, calcium-independent cell adhesion molecule of 39–42 kDa (2.Litvinov S.V. Velders M.P. Bakker H.A. Fleuren G.J. Warnaar S.O. J. 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Med. 1999; 77: 699-712Crossref PubMed Scopus (468) Google Scholar). The level of EpCAM expression has been correlated with dedifferentiation and malignant proliferation of epithelial cells (5.Litvinov S.V. van Driel W. van Rhijn C.M. Bakker H.A. van Krieken H. Fleuren G.J. Warnaar S.O. Am. J. Pathol. 1996; 148: 865-875PubMed Google Scholar, 6.Maetzel D. Denzel S. Mack B. Canis M. Went P. Benk M. Kieu C. Papior P. Baeuerle P.A. Munz M. Gires O. Nat. Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar). It is frequently detected in cancer-initiating cells (7.Al-Hajj M. Wicha M.S. Benito-Hernandez A. Morrison S.J. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3983-3988Crossref PubMed Scopus (8281) Google Scholar, 8.Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Proc. Natl. Acad. Sci. 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Schaible K. Heasman J. Wylie C. J. Reprod. Fertil. 1999; 116: 379-384Crossref PubMed Google Scholar) and is frequently present at the surface of human hepatic multipotent progenitors (9.Schmelzer E. Wauthier E. Reid L.M. Stem Cells. 2006; 24: 1852-1858Crossref PubMed Scopus (280) Google Scholar), hepatic stem cells (11.Schmelzer E. Zhang L. Bruce A. Wauthier E. Ludlow J. Yao H.L. Moss N. Melhem A. McClelland R. Turner W. Kulik M. Sherwood S. Tallheden T. Cheng N. Furth M.E. Reid L.M. J. Exp. Med. 2007; 204: 1973-1987Crossref PubMed Scopus (470) Google Scholar), and cancer stem cells (8.Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10158-10163Crossref PubMed Scopus (1747) Google Scholar). Very recently, EpCAM expression on ESCs has been reported by some studies (14.Sundberg M. Jansson L. Ketolainen J. Pihlajamäki H. Suuronen R. Skottman H. 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Several studies have discussed the DNA methylation status of EpCAM promoter in lung, colon, prostate, liver, bladder, ovary, and breast cancer cells and tissues (18.Tai K.Y. Shiah S.G. Shieh Y.S. Kao Y.R. Chi C.Y. Huang E. Lee H.S. Chang L.C. Yang P.C. Wu C.W. Oncogene. 2007; 26: 3989-3997Crossref PubMed Scopus (53) Google Scholar, 19.Spizzo G. Gastl G. Obrist P. Fong D. Haun M. Grünewald K. Parson W. Eichmann C. Millinger S. Fiegl H. Margreiter R. Amberger A. Cancer Lett. 2007; 246: 253-261Crossref PubMed Scopus (29) Google Scholar, 20.Yu G. Zhang X. Wang H. Rui D. Yin A. Qiu G. He Y. Oncol. Rep. 2008; 20: 1061-1067PubMed Google Scholar, 21.Shiah S.G. Chang L.C. Tai K.Y. Lee G.H. Wu C.W. Shieh Y.S. Oral Oncol. 2009; 45: e1-e8Crossref PubMed Scopus (22) Google Scholar). Post-translational modifications of histone tails, including phosphorylation, acetylation, ubiquitination, and methylation, have been validated as dynamic regulators of gene expression. In order to gain insight into the epigenetic transitions responsible for EpCAM expression in hESC, we studied the 5′-flanking region of EpCAM promoter by evaluating CpG status using methylation-specific PCR (MSP), bisulfite sequencing, and histone modification by chromatin immunoprecipitation (ChIP).The polycomb group (PcG) proteins are important chromatin modifiers that play a pivotal role in the epigenetic regulation of the development, differentiation, and maintenance of cell fates (22.Schuettengruber B. Chourrout D. Vervoort M. Leblanc B. Cavalli G. Cell. 2007; 128: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). Dynamic repression of developmental pathways by PcG may be required for maintaining ES cell pluripotency and plasticity during embryonic development (23.Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. 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The recent identification of JmjC domain-containing histone lysine demethylase JMJD3 suggests that there may be positive and negative regulators simultaneously controlling chromatin structure dynamics through histone methylation mark alterations. JMJD3 specifically removes methyl marks of H3K27me3 in mammalian cells to antagonize PcG gene silencing and permit gene transcription. JMJD3 is highly expressed in ES cells and is responsible for the rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem cell differentiation (23.Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. Nature. 2006; 441: 349-353Crossref PubMed Scopus (2014) Google Scholar, 28.Agger K. Cloos P.A. Christensen J. Pasini D. Rose S. Rappsilber J. Issaeva I. Canaani E. Salcini A.E. Helin K. Nature. 2007; 449: 731-734Crossref PubMed Scopus (977) Google Scholar). These findings suggest that EpCAM may be regulated by both SUZ12 and JMJD3 during hESC differentiation.Understanding the downstream targets of EpCAM would help define the molecular function of this gene. However, such studies have been hindered by the obscure signaling mode of EpCAM until the very recent discovery of regulated intramembrane proteolysis and nuclear translocation of its intracellular domain EpICD. Released EpICD associates with FHL2, β-catenin, and Lef-1 and participates in gene regulation in the nucleus (6.Maetzel D. Denzel S. Mack B. Canis M. Went P. Benk M. Kieu C. Papior P. Baeuerle P.A. Munz M. Gires O. Nat. Cell Biol. 2009; 11: 162-171Crossref PubMed Scopus (535) Google Scholar). One of the EpCAM downstream targets, c-MYC, has been found to be regulated by EpCAM in both normal and cancer cells (29.Münz M. Kieu C. Mack B. Schmitt B. Zeidler R. Gires O. Oncogene. 2004; 23: 5748-5758Crossref PubMed Scopus (299) Google Scholar). c-MYC is a member of the four reprogramming factors involved in the induced pluripotent stem cell formation (30.Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2359) Google Scholar, 31.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14630) Google Scholar, 32.Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18453) Google Scholar, 33.Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. Nature. 2007; 448: 318-324Crossref PubMed Scopus (2202) Google Scholar, 34.Yu J. Hu K. Smuga-Otto K. Tian S. Stewart R. Slukvin II, Thomson J.A. Science. 2009; 324: 797-801Crossref PubMed Scopus (1767) Google Scholar). Understanding the regulation of EpCAM on c-MYC and even other reprogramming genes like OCT-4, NANOG, SOX2, and KLF4 in hESC may add to our understanding of how EpCAM contributes the long term maintenance of the ES cell phenotype.This study is a comprehensive analysis of EpCAM expression in undifferentiated hESCs using immunofluorescence microscopy, Western blotting, and flow cytometry. Loss of EpCAM expression in differentiated hESCs through epigenetic silencing is elucidated by ChIP. Because several reprogramming genes are under the regulation of EpCAM, we propose that EpCAM maintains hESC "stemness" through sustaining these reprogramming genes.

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