Transient Down-regulation of DNMT1 Methyltransferase Leads to Activation and Stable Hypomethylation of MAGE-A1 in Melanoma Cells

MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression. MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression. Cytosines in CpG dinucleotides are often methylated in mammalian genomes. This epigenetic modification of DNA exerts a potent repressive effect on transcription by preventing the binding of transcription factors and by recruiting methyl-CpG-binding proteins, which in turn attract repressor complexes that modify chromatin structures by deacetylating or methylating specific residues on histones (1Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar). DNA methylation has an essential role in the inactivation of one of the two X chromosomes in female somatic cells (2Riggs A.D. Pfeifer G.P. Trends Genet. 1992; 8: 169-174Abstract Full Text PDF PubMed Scopus (282) Google Scholar), in the monoallelic silencing of parentally imprinted genes (3Ferguson-Smith A.C. Surani M.A. Science. 2001; 293: 1086-1089Crossref PubMed Scopus (355) Google Scholar), in the repression of genomic parasite sequences (4Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1532) Google Scholar), and in the regulation of tissue-specific gene expression (5Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (574) Google Scholar, 6Futscher B.W. Oshiro M.M. Wozniak R.J. Holtan N. Hanigan C.L. Duan H. Domann F.E. Nat. Genet. 2002; 31: 175-179Crossref PubMed Scopus (373) Google Scholar, 7De Smet C. Lurquin C. Lethé B. Martelange V. Boon T. Mol. Cell. Biol. 1999; 19: 7327-7335Crossref PubMed Scopus (511) Google Scholar). DNA methylation patterns are acquired during development by a process that involves a phase of extensive demethylation in the early embryo, followed by an active phase of de novo methylation (8Kafri T. Ariel M. Brandeis M. Shemer R. Urven L. McCarrey J. Cedar H. Razin A. Genes Dev. 1992; 6: 705-714Crossref PubMed Scopus (595) Google Scholar, 9Monk M. Boubelik M. Lehnert S. Development. 1987; 99: 371-382Crossref PubMed Google Scholar). Once established, methylation patterns are inherited through cell generations by a maintenance process that copies pre-existing methylation sites onto the newly synthesized strand (10Pfeifer G.P. Steigerwald S.D. Hansen R.S. Gartler S.M. Riggs A.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8252-8256Crossref PubMed Scopus (217) Google Scholar). In mammals, the transfer of a methyl group to cytosines is catalyzed by three DNA methyltransferases, namely DNMT1, 3The abbreviations used are: DNMT, DNA methyltransferase; MAGE-A1, melanoma antigen gene A1; RT-PCR; reverse transcription PCR; EGFP, enhanced green fluorescent protein; DNMT-AS, DNA methyltransferase antisense. DNMT3A, and DNMT3B (11Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar). The two latter enzymes have de novo methylation activity and are responsible for the establishment of DNA methylation patterns during embryonic development (12Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). Consistently, DNMT3A and -3B are highly expressed in the embryo, whereas they are expressed at low levels in adult somatic cells (13Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar). DNMT1, which has a preference for hemimethylated DNA, is the major maintenance methyltransferase (14Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar, 15Li E. Beard C. Jaenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1784) Google Scholar, 16Lei H. Oh S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar, 17Bestor T.H. EMBO J. 1992; 11: 2611-2617Crossref PubMed Scopus (392) Google Scholar, 18Pradhan S. Bacolla A. Wells R.D. Roberts R.J. J. Biol. Chem. 1999; 274: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). In embryonic cell lines, however, DNMT3A and DNMT3B appear to cooperate with DNMT1 to maintain methylation patterns (19Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (583) Google Scholar, 20Hattori N. Abe T. Suzuki M. Matsuyama T. Yoshida S. Li E. Shiota K. Genome Res. 2004; 14: 1733-1740Crossref PubMed Scopus (70) Google Scholar). Cancer cells show altered patterns of DNA methylation (21Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar). Normally unmethylated promoters often become densely methylated in tumor cells, and this results in silencing of critical genes such as tumor suppressor genes. In many tumors, including these same tumors, the global level of DNA methylation is usually decreased (22Feinberg A.P. Vogelstein B. Nature. 1983; 301: 89-92Crossref PubMed Scopus (1917) Google Scholar, 23Gama-Sosa M.A. Slagel V.A. Trewyn R. M. Res. 1983; 11: PubMed Scopus Google Scholar). a in which DNMT1 was by a J.G. A. L. Dausman J. H. Jaenisch R. Science. 2003; PubMed Scopus Google that hypomethylation tumor by genomic of the demethylation process in tumors is the activation of genes that are by DNA methylation A.P. B. Nat. 2004; PubMed Scopus Google Scholar). This is the for a group of germ line-specific genes that rely primarily on DNA methylation for silencing in normal somatic Smet C. Lurquin C. Lethé B. Martelange V. Boon T. Mol. Cell. Biol. 1999; 19: 7327-7335Crossref PubMed Scopus (511) Google Scholar, Smet C. Lurquin C. Boon T. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, A. T. T. N. M. T. M. T. Cancer Sci. 2004; PubMed Scopus Google Scholar). these genes become in many types of tumors, they the expression of by P. Y. P. V. C. J. Boon T. 2002; PubMed Scopus Google Scholar). genes or gene specific expression in germ and cells have are or genes. The to DNA hypomethylation in tumors are In a we this by using the MAGE-A1 gene as a Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). We found that in tumor cells this gene, hypomethylated are not over the MAGE-A1 but are the of the In the MZ2-MEL melanoma cell for MAGE-A1 is hypomethylation the gene is to a region on the transcription This hypomethylation not appear to to a in vitro methylated MAGE-A1 sequences not demethylation into MZ2-MEL cells. in the same cells unmethylated MAGE-A1 transgenes were at sites in the of the This of de novo methylation appeared to depend on the level of transcriptional activity. on these we proposed that MAGE-A1 hypomethylation in tumors the of a past event of which would maintained by the presence of potent transcriptional Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). In the of the MAGE-A1 promoter region against de novo methylation was after of unmethylated In the we whether a previously methylated transgene would become permanently protected against induction of a transient demethylation this we an in vitro methylated MAGE-A1 into MZ2-MEL cells. We a transient demethylation phase by the cells for a of with antisense oligonucleotides to the DNA N. S. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. N. A. Nat. Genet. 2003; PubMed Scopus Google Scholar). By of and we found that depletion of DNMT1 resulted in transgene activation. oligonucleotides transgene in a of the cells. By using a transgene in which the of MAGE-A1 was by the to we were able to the cells in which transgene activation cells were for of demethylation and activation of the was by the Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google with and and by with the the with the same For of the the to to the was by G. Res. PubMed Scopus Google with and which a a or a the the MAGE-A1 and to the and a at was by using the and an antisense a with a The MAGE-A1 and were with and or with and and were to the in which of the had by and and contain a gene for of cells. For of and the of DNMT3A or DNMT3B was human MZ2-MEL cells using DNA The DNMT3A was into the the and The DNMT3B was into the the and were by In and of and were using the were methylated in vitro with the which cytosines in CpG as previously Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). methylation of the was by to with the The cell and of cell have previously Smet C. Lurquin C. Boon T. PubMed Scopus Google Scholar). The cells were in before transfection. were using the C. P. B. P. C. N. L. Boon T. 1992; PubMed Scopus Google with of DNA a in of human genomic of the in genomic DNA was previously to the of copies in MZ2-MEL cells Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). were in of for cell were by were in the or in the presence of during and expression of the transgene was tested by This to the of an in vitro methylated and an in vitro methylated in which the transgene was completely but was with were using the were on a and to a using the and transfer were in a and were into a with and and with an and and or with an were with or and using the for of DNMT3A and DNMT3B proteins, cells were with of or and were days after transfection. The level of DNMT3A and DNMT3B transcription in the cell population was by cells were in before transfection. were in of of and of antisense which were synthesized of cells were in were in this and after the The antisense sequences were with to N. S. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. N. A. Nat. Genet. 2003; PubMed Scopus Google Scholar). The of the is with the at For of cells in which DNMT1 depletion resulted in transgene cells were in of and were using transcription was on of using as previously P. Lethé B. M. Boon T. J. PubMed Scopus Google Scholar). of have Smet C. Lurquin C. Boon T. PubMed Scopus Google Scholar). For of and we used a but specific antisense or for have previously Smet C. Lurquin C. Boon T. PubMed Scopus Google Scholar). were as previously Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google that was and for are Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). and for are the same as used in for of Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). were and for DNMT1, and for DNMT3A, and for The sequences were for DNMT1, for DNMT3A, and for For specific were by known of in which the had were in or genomic was as previously Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). for the of of the MAGE-A1 promoter region were as Smet C. Lurquin C. Lethé B. Martelange V. Boon T. Mol. Cell. Biol. 1999; 19: 7327-7335Crossref PubMed Scopus (511) Google Scholar). For the we used the same with as antisense of an MZ2-MEL a MAGE-A1 an MZ2-MEL a methylated MAGE-A1 transgene, we MZ2-MEL cells with an in vitro methylated a of MAGE-A1 to to the transcription to the enhanced green fluorescent transcription were to to which was by the transgene and were we the transgene in these cells was completely but with the of the transgene was maintained in cells after not This that, although MZ2-MEL cells contain an unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 of DNMT1, DNMT3A, and DNMT3B by induce demethylation of the MAGE-A1 transgene in we to the maintenance DNA by the cells with specific antisense methylation maintenance has to DNMT1, that in tumor cells in this process H. T.H. Cancer Res. 2003; Google Scholar, H. A.P. C. S.B. Vogelstein B. Nature. 2002; PubMed Scopus Google Scholar). We therefore tested the effect of of the three in this we used the antisense oligonucleotides and previously by N. S. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. N. A. Nat. Genet. 2003; PubMed Scopus Google Scholar). We the level of expression of the three in by The results showed that the of DNMT1 in these cells is and that of DNMT3A and DNMT3B, We on a a at a to the DNMT1 and not of a of the the of of DNMT3A and DNMT3B is to the low level of transcription of the genes in The in that were to levels of DNMT3A and or DNMT3B and of of and expression these that DNMT1 is the major DNA methyltransferase in the of the has previously in cell N. S. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. N. A. Nat. Genet. 2003; PubMed Scopus Google Scholar), we the ability of these antisense oligonucleotides to expression of gene in cells. this the cells were with of the three or with an and the level of expression of the three was after transfection. in the gene with a in the expression In the had effect on expression We by that the in DNMT1 by the was with depletion of the of the in that the expression of the gene in we tested ability to induce of the methylated In the of activation of was depletion of DNMT1 or DNMT3A and The two latter enzymes were they are known to (12Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). DNA demethylation of methylation maintenance by a We therefore maintained depletion of the for days by the cells with the antisense cells that had in this during and days were and by to the level of expression of the expression was in cells as with cells of DNMT3A and -3B or cells This at after days of DNMT1 the level of expression of the transgene in was that in MZ2-MEL cells that were with the unmethylated This that with resulted in activation of the transgene in a of the cells or of the transgene in cells. the results that DNMT1 is in repression of the whether DNMT1 exerts the same on an gene, we the effect of DNMT1 and depletion on the activation of gene which is in MZ2-MEL cells. the transgene, in cells of the DNMT1 whether activation of the transgene depletion of DNMT1 with demethylation of the promoter this DNA was cells that were or for days with or We the genomic to the methylation of CpG sites and to the transcription this region was previously to critical for transcription of the MAGE-A1 gene Smet C. Lurquin C. Lethé B. Martelange V. Boon T. Mol. Cell. Biol. 1999; 19: 7327-7335Crossref PubMed Scopus (511) Google Scholar). MAGE-A1 sequences the transgene as they the MAGE-A1 by the presence of a in the of the transgene was methylated in cells level of methylation was in cells of DNMT3A and which was with the lack of transgene activation in this group of cells the transgene showed demethylation in cells. in these cells sequences of the unmethylated these that DNMT1 is in methylation maintenance the transgene, they that to the not to an demethylation of the transgene in cells. by of DNMT1 and DNMT3A the that the low level of transgene demethylation and activation that we DNMT1 depletion was to the presence of DNMT3A and two enzymes de novo methylation activity to the CpG sites that methylation by lack of the maintenance methyltransferase that depletion of DNMT1 and DNMT3B in tumor cell results in gene demethylation and as with depletion of DNMT1 H. T.H. Cancer Res. 2003; Google Scholar, H. A.P. C. S.B. Vogelstein B. Nature. 2002; PubMed Scopus Google Scholar). cells that had to the for days were into and during days with and and or a of three The effect of these on of the transgene was by the level of expression. in of DNMT3A DNMT3B in cells of DNMT1 not the level of activation of the We results for the gene is therefore that the low level of demethylation by is to depletion of DNMT1 in cells by DNMT3A and cells in which DNMT1 is as was previously that of DNMT1 induces cell and (5Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (574) Google Scholar, S. A. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. Res. 2003; 31: PubMed Scopus Google Scholar). of in the by DNMT1 to isolate a of cells in which with resulted in demethylation and activation of the a the green fluorescent we to cells in which the transgene was by of fluorescent cells. We were however, to cells the not We to and a MAGE-A1 the This construct, was methylated in vitro and into MZ2-MEL cells. the we a cell as a transgene that with cells were to during days This resulted in in DNMT1 and in but activation of the that this activation was with demethylation of the transgene as by of methylation in of sequences days of and cells were into cells after days of cells that had with the showed of a population in that the of cells of the cells. The cell population showed a level of expression as with the cells Consistently, showed that resulted in the of cells an unmethylated transgene, as sequences the cell population and unmethylated The expression of DNMT1 in the population to a level with that before with these that DNMT1 depletion resulted in transgene activation in a of the cells that through the by the of of of a cell population in which the transgene has down-regulation of DNMT1 to whether hypomethylation and activation of the transgene would maintained in the of further of this were the cell were in to of cells, and was after days of that expression was in at these days further maintenance of the active three were during in in after this of three maintained expression of the Consistently, on two of these that hypomethylation of the transgene promoter was completely maintained over these results that hypomethylation and activation of the MAGE-A1 promoter acquired a past event of human tumors show hypomethylation of but the to this epigenetic this we used gene MAGE-A1 as a the promoter region of this gene is a of DNA demethylation in cells. This is the in melanoma cell MAGE-A1 is hypomethylated and We showed in that in vitro methylated MAGE-A1 transgenes not become demethylated after into MZ2-MEL cells, that these cells have not acquired a activity to the gene Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). these not the that MZ2-MEL cells a demethylation process of to transgene demethylation in a therefore of the cells. The that this is not the we not cell activation of the methylated transgene the by the presence of in the We therefore that MAGE-A1 hypomethylation and activation in MZ2-MEL results a past event of is to that MAGE-A1 activation was in a of P. C. P. Lurquin C. E. B. A. Boon T. Science. PubMed Scopus Google Scholar), that the demethylation process during tumor in and is not a of in vitro of a activity to a of tumor cells, as by the that hypomethylated tumor cell usually not show further demethylation of during Science. 1983; PubMed Scopus Google Scholar, E. Mol. Cell. Biol. PubMed Scopus Google Scholar, D. M. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). tumor cell often show but de novo methylation activity J. A. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). Consistently, that the level of expression of the DNA in tumor cells is or that in normal cells Lee Cancer Res. 2001; Google Scholar, S. R. Cancer Res. 2003; 9: Google Scholar). The of an process of demethylation in the activation of MAGE-A1 that hypomethylation is maintained the promoter region of the gene after the of the transient demethylation In of that MZ2-MEL cells contain transcription factors of de novo methylation the promoter region of MAGE-A1 transgenes that were unmethylated before transfection. The of the transgene were methylation after transfection. This that MZ2-MEL cells de novo methylating activity Smet C. A. Boon T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). We have that, induction of a transient demethylation phase, previously methylated MAGE-A1 transgenes become permanently protected against results that in MZ2-MEL cells a transient phase of DNA demethylation to convert a methylated and MAGE-A1 gene into a permanently hypomethylated and active one. on we the of activation of MAGE-A1 in the the cells would a transient phase of epigenetic to demethylation of the MAGE-A1 would able to to the unmethylated promoter In cells factors that a level of transcriptional activation to the region against remethylation, the promoter of MAGE-A1 would maintained at the de novo methylation activity. This hypomethylation in tumors is maintained methylation and hypomethylated and sequences The the of the in the methylation maintenance of gene The of DNMT1 and DNMT3B to methylation maintenance in tumor cells has a that of DNMT1 in and cell to induce and global DNA demethylation S. N. A. Nat. Genet. 2003; PubMed Scopus Google Scholar, M. P. N. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In that in tumor cells DNA in the methylation maintenance In cells for of the DNMT1 gene resulted in DNA whereas of DNMT1 and DNMT3B resulted in of methylation H. A.P. C. S.B. Vogelstein B. Nature. 2002; PubMed Scopus Google Scholar, C. Herman J.G. Vogelstein B. S.B. Nature. 2000; PubMed Scopus (373) Google Scholar, Suzuki H. S.B. Nat. Genet. 2004; PubMed Scopus Google Scholar). In an cell H. T.H. Cancer Res. 2003; Google showed that demethylation of tumor suppressor genes was DNMT1 and DNMT3B were We have that in MZ2-MEL cells depletion of DNMT1 to induce demethylation and activation of MAGE-A1 transgenes and of the gene is that DNMT1 depletion to induce of the tumor suppressor gene Vertino P.M. Cancer Res. 2000; Google Scholar), which we found to and in MZ2-MEL cells not DNMT1 therefore to have a predominant role for methylation maintenance in MZ2-MEL cells. these that the of the DNA to methylation maintenance one tumor cell to This depend on the of the as by the low level DNMT3A and DNMT3B expression in MZ2-MEL cells. transient depletion of DNMT1, of cells, which contain a selectable transgene, in factors to this low level of of cells with an unmethylated and active that, the of DNMT1 that we after antisense the was in cells to transgene This is by the results that after days of with of the transgene promoter sequences had unmethylated is that cells in which DNMT1 was and therefore the transgene was were during has that of DNMT1 induces cell and (5Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (574) Google Scholar, S. A. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. Res. 2003; 31: PubMed Scopus Google Scholar). The cell which a methylated transgene to to a to cells in which demethylation and activation of the transgene This cell used to the of factors to DNA methylation maintenance in MZ2-MEL cells. factors methyl-CpG-binding and enzymes such as and methyltransferases, which appear to with DNA to the maintenance of D. Science. 2003; 301: PubMed Scopus Google Scholar). the factors in methylation maintenance to the responsible for the DNA demethylation process in We S. for and for We for