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DNA Research Advance Access originally published online on January 11, 2006
DNA Research 2005 12(5):373-378; doi:10.1093/dnares/dsi013
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© The Author 2006. Kazusa DNA Research Institute
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XIST Repression in the Absence of DNMT1 and DNMT3B

Luciana R. Vasques1, Raquel Stabellini1, Fei Xue2, X. Cindy Tian2, Marina Soukoyan1 and Lygia V. Pereira1,*

1Depto Biologia and Centro de Estudos do Genoma Humano, Instituto de Biociências, Universidade de São Paulo São Paulo, SP 05508-900, Brazil
2Center for Regenerative Biology/Department of Animal Science, University of Connecticut Storrs, CT 06269, USA

Received 13 October 2004; revised 12 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
 References
 
X chromosome inactivation (XCI) in human and mice involves XIST/Xist gene expression from the inactive X (Xi) and repression from the active X (Xa). Repression of the XIST/Xist gene on the Xa has been associated with methylation of its 5' region. In mice, Dnmt1 has been shown to be involved in the methylation and transcriptional repression of Xist on Xa. We examined maintenance of XIST gene repression on Xa in HCT116 cell lines knockout for either DNMT1 or DNMT3B and for DNMT1 and DNMT3B simultaneously. Methylation of the XIST promoter and XIST transcriptional repression is sustained in DNMT1-, DNMT3B- and DNMT1/DNMT3B knockout cells. Despite global DNA demethylation, the double knockout cells present only partial demethylation of the XIST promoter, which is not sufficient for gene reactivation. In contrast, global DNA demethylation with 5-aza-2'-deoxycytidine leads to XIST expression. Therefore, in these human cells maintenance of XIST methylation is controlled differently than global genomic methylation and in the absence of both DNMT1 and DNMT3B.

Key words: X-chromosome inactivation; DNA-methyltransferase; XIST; epigenetic inheritance


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
 References
 
In mammals, dosage compensation of X-linked gene products between XY males and XX females is achieved by transcriptional inactivation of one X chromosome in females.1Go The Xist gene is expressed exclusively from the inactive X (Xi), and seems to trigger initiation of X chromosome inactivation (XCI) in cis.2Go It transcribes a 17.8 kb nuclear mRNA which is not translated.3Go In the early mouse embryo, low levels of Xist expression are detected from the single X and from both the Xs in the male and female embryo, respectively.4Go,5Go In female embryos, immediately prior to gastrulation, up-regulation of Xist RNA occurs on the one X chromosome to be inactivated.4Go

Repression of Xist/XIST on the active X (Xa) in males and females has been correlated with methylation of its 5' end.6Go,7Go This region is hypermethylated on the Xa, where Xist/XIST is repressed, and hypomethylated on the Xi, where Xist/XIST is expressed. These results suggest that methylation is involved in Xist/XIST gene silencing in humans7Go,8Go and mice.9Go,10Go Indeed, treatment of normal human fibroblasts and somatic cell hybrids containing one human Xa with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) leads to XIST demethylation and expression.11Go,12Go

To date, five different mammalian DNA (cytosine-5) methyltransferases (MTases) have been identified: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L.13Go–16Go Dnmt1 is constitutively expressed, has higher activity in hemi-methylated DNA and has been recognized as a major maintenance MTase (reviewed in Bestor17Go). Dnmt2 was isolated based on its homology to the pmt1p of fission yeast, and is expressed at low levels in all human and murine tissues.14Go Although neither de novo nor maintenance DNA MTase activity has been demonstrated for this protein in murine ES cells,18Go low in vitro activity of human DNMT2 was detected and it was specific for a loose DNA consensus sequence.19Go In contrast, Dnmt3a and Dnmt3b have been shown to be essential for de novo DNA MTase activity in murine ES cells and in early embryos, but not required for the maintenance of imprinted methylation patterns.20Go More recently, Dnmt3a was shown to be required for methylation of imprinted loci in germ cells.21Go In humans, mutations in DNMT3B cause ICF syndrome, characterized by the hypomethylation of pericentromeric repetitive DNA.22Go,23Go Finally, Dnmt3L was isolated based on its homology to Dnmt3a and Dnmt3b in the cysteine-rich region.16Go Like Dnmt3a, this protein is required for the establishment of genomic imprints during gametogenesis.24Go However, Dnmt3L lacks the catalytic domain common in the other MTases and it represses transcription by binding to the histone deacetylase HDAC1 protein rather than by methylating DNA.25Go,26Go

The role of Dnmt1 in the process of DNA methylation and XCI has been extensively studied in mice. Murine ES cells deficient for Dnmt1 show high levels of global DNA demethylation, which in turn leads to biallelic expression of imprinted genes.27Go,28Go In addition, upon differentiation, these cells fail to repress Xist expression, a phenomenon correlated to lack of proper methylation of the 5' region of the Xist gene.6Go These results demonstrate that Dnmt1 activity is causally involved in global DNA methylation and in transcriptional repression of imprinted genes and of the Xist gene.5Go,6Go

The role of the human homologue DNMT1 in controlling gene expression was investigated in the human carcinoma cell line HCT116 knockout for the DNMT1 gene by homologous recombination.29Go Surprisingly, the authors showed that despite a greatly decreased DNMT1 activity the cells presented only a 20% reduction in overall DNA methylation, restricted to specific regions of the genome. Using the same approach, Rhee et al.30Go generated HCT116 cell lines deficient for DNMT3B and for both DNMT1 and DNMT3B. While DNMT3B knockout cells retained >97% of genomic 5-methylcytosine (m5C), the double knockout (DKO) cells presented ~95% reduction in the m5C content. This in turn leads to transcriptional activation of TIMP-3, the imprinted IGF2 allele and the wild-type p16INK4a allele. The authors thus concluded that DNMT1 and DNMT3B cooperate to maintain global DNA methylation and gene silencing in those human cancer cells.

These data indicate that human MTases may be involved in DNA methylation differently than their murine counterparts. That prompted us to investigate the methylation status of XIST in the absence of DNMT1 and DNMT3B activity in the HCT116 knockout cells.


   2. Materials and Methods
Top
 Abstract
 1. Introduction
 2. Materials and Methods
3. Results and Discussion
 Acknowledgements
 References
 
2.1. Cell culture
Parental HCT116 cell line, two independent DNMT1 knockout clones (1C1 and 9A), one DNMT3B knockout clone (3bKO) and one DNMT1/DNMT3B DKO clone were kindly provided by Drs B. Vogelstein and K. Schuebel.29Go,30Go These cells contain one normal X and one normal Y chromosomes. Cells were cultured without selection in McCoy media supplemented with 10% fetal calf serum and penicilin/streptomycin (Invitrogen) at 37°C/5% CO2. Treatment with 5-aza-dC (Sigma) was as follows: cells at the mid-log phase in 100 mm culture dishes were supplemented with fresh media containing 10 µM of 5-aza-dC. Fresh media with 5-aza-dC was added every 24 h for 48 h. Cells were allowed to recover from treatment for 48 h in media without the drug before harvesting. Treatment was carried out in duplicate plates. DKO cells were treated for up to 72 h with 30 µM 5-aza-dC.

2.2. Analysis of XIST gene expression
RNA was isolated from treated and untreated pooled cells with the Trizol reagent according to manufacturer's instructions (Invitrogen). Analysis of XIST gene expression was performed by northern blotting with 20 µg of total RNA as described previously31Go using a probe from the most 5' XIST cDNA clone Hbc1a.32Go Normal female fibroblasts were used as a positive control for XIST expression and G3PDH cDNA probe as an internal control. DNMT1 and DNMT3B gene activity were evaluated by hybridization of northern blots with radiolabeled DNMT1 (data not shown) and DNMT3B cDNAs, respectively. Lack of DNMT1 expression was confirmed by RT–PCR.

2.3. Analysis of XIST 5' end methylation
DNA was isolated from cells as described previously.31Go Analysis of methylation of the 5' end of the XIST gene was performed by Southern blotting with the most 5' XIST cDNA clone Hbc1a as the probe, as described previously.7Go,12Go An aliquot of 5–10 µg of genomic DNA was digested with 100 U each of EcoRV and one of the methylation-sensitive restriction enzymes HhaI and AvaI, and with 100 U each of PstI and the methylation-sensitive restriction enzyme SacII (Amersham-Pharmacia).


   3. Results and Discussion
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
Acknowledgements
 References
 
3.1. Maintenance of XIST repression in the absence of DNMT1
XIST gene activity was assayed by northern blot analysis of total RNA from the parental HCT116 cell line and from the DNMT1 knockout clone 9A (Fig. 1). Although XIST RNA was detected from control female cells, no XIST expression was detected from either the parental cell line or the DNMT1 knockout cells (Fig. 1A, lanes 1, 2 and 9). Lack of DNMT1 expression in the DNMT1 knockout cells was confirmed by RT–PCR (Fig. 1B).


Figure 1
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  		Analysis of XIST expression in different MTase knockout HCT116 cells. (A) Northern blot analysis of HCT116 clones. cDNA probes are indicated on the left of the corresponding panels. (B) RT–PCR analysis of DNMT1 expression. Plus signs, RT added; minus signs, RT omitted; DKO, DNMT1/DNMT3B double knockout cells; F, human female fibroblast cell line. DNMT1 and DNMT3B genotypes and 5-aza-dC treatment are indicated above the lanes.

 
Lack of XIST expression in the DNMT1 knockout cells could be due to the maintenance of methylation of XIST 5' region, or alternatively due to the absence of other factors required for XIST induction after demethylation. Therefore, methylation status of the methylation-sensitive restriction enzyme HhaI, AvaI and SacII sites at XIST 5' end was analyzed by Southern blotting (Figs 2 and 3). Unlike PCR-based assays, this approach allows detection of partial DNA demethylation patterns. Our results show that Xa-specific methylation of the 5' end of XIST is retained in the DNMT1 knockout cells (Figs 2B and 3B). These data demonstrate maintenance of XIST methylation and transcriptional repression in the absence of DNMT1 activity.


Figure 2
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  		Methylation status of the XIST 5' end. (A) Scheme of restriction patterns of the 5'end of XIST in the Xa and Xi. Single and double asterisks indicate methylated HhaI and AvaI sites, respectively. (B) Southern blotting analysis: genomic DNA from HCT116 clones and fibroblasts from a normal female (F) was digested with EcoRV and AvaI or with EcoRV and HhaI as indicated. DNMT1 and DNMT3B genotypes and 5-aza-dC treatment are indicated above the lanes; sizes in kb, 0.6 kb bands correspond to unmethylated XIST alleles. The HhaI site remains methylated in the DKO cells and 5-aza-dC treatment leads to demethylation of both HhaI and AvaI sites on HCT116 cells (see text).

 

Figure 3
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  		Methylation status of SacII sites at XIST 5' end. (A) Scheme of restriction patterns of the 5' end of XIST on the Xa, Xi and on the partially methylated X chromosome of DKO cells (XDKO). Asterisks indicate methylated SacII sites; plus sign indicates the partially methylated SacI(1) site at the XDKO. (B) Southern blotting: genomic DNA from HCT116 clones and from fibroblasts from a normal female (F) was digested with PstI and SacII. DNMT1 and DNMT3B genotypes are indicated above the lanes; sizes in bp. Absence of the 240/170 bp bands indicates that the SacII(2) site remains methylated in the DKO cells (see text).

 
3.2. Maintenance of XIST repression in the absence of both DNMT1 and DNMT3B
XIST expression was analyzed in the DNMT3B knockout and also in the DNMT1/DNMT3B DKO cells. Northern blot analysis revealed lack of XIST expression in DNMT3B knockout cells and, surprisingly, also in the DKO cells, which are known to present ~95% reduction in the m5C content30Go (Fig. 1). Lack of DNMT3B and of both DNMT3B and DNMT1 expression was confirmed in the DNMT3B knockout and DKO cells, respectively (Fig. 1).

Methylation analysis of the XIST gene in different MTase knockout HCT116 cells was performed (Figs 2 and 3). DNMT3B knockout cells maintained the four methylated sites present in the parental HCT116 (Figs 2B and 3B). In the DKO cells, maintenance of methylation of the HhaI and the downstream SacII [SacII(2)] sites was observed (Figs 2B and 3B). However, the AvaI site was completely unmethylated, as indicated by the presence of the 600 bp band and absence of the 3.2 kb band (Fig. 2B), and the upstream SacII [SacII(1)] site was only partially unmethylated, as indicated by the presence of the 800 bp band and absence of the 240 and 170 bp bands (Fig. 3B). Our results show that, while causing global DNA demethylation, lack of both DNMT1 and DNMT3B activity was not sufficient to disrupt maintenance of methylation specifically on the HhaI and SacII(2) sites at the 5'end of XIST.

3.3. 5-aza-dC-mediated DNA demethylation induces XIST expression in HCT116 parental and knockout cells
In order to investigate the effect of global demethylation on the maintenance of XIST transcriptional repression in HCT116 cells, these cells were treated with 5-aza-dC. Northern blot analysis revealed partial reactivation of the XIST gene in treated parental and all knockout HCT116 cells (Fig. 1). 5-aza-dC treatment of DKO cells was less effective even at higher drug concentrations and longer treatment time (data not shown), probably due to the slower growth rate of these cells, as previously reported by Rhee et al.30Go Our results show that XIST expression from the Xa is induced by global DNA demethylation in HCT116 parental and knockout cells, as reported in human fibroblasts and in somatic cell hybrids containing the human Xa.11Go,12Go It is interesting to note that 5-aza-dC treatment also leads to higher expression of DNMT3B in HCT116 parental and DNMT1 knockout cells (Fig. 1A).

The methylation status of the 5' region of the XIST gene in the 5-aza-dC treated HCT116 cells was analyzed (Figs 2 and 3). Partial demethylation of the AvaI, HhaI and the two SacII sites was observed in the treated cells expressing XIST (Figs 2B and 3B). Therefore, our data show that DNA methylation is involved in the control of XIST expression from the Xa in HCT116 cells, as already shown for a normal human cell line.12Go Moreover, these results show that the HhaI and SacII(2) methylation sites are essential for XIST transcriptional control.

In mice, Dnmt1 is required for the maintenance of global DNA methylation,27Go,28Go and for the establishment of Xist methylation and maintenance of Xist repression on the Xa.6Go Therefore, Dnmt1 has been recognized as the major mammalian MTase involved in both imprinting and XCI. Rhee et al.29Go have challenged this idea by generating DNMT1 knockout HCT116 cells that retained most of the overall genomic methylation. Subsequently, Rhee et al.30Go showed that maintenance of global DNA methylation in HCT116 cells was lost only in the DNMT1/DNMT3B DKO cells, suggesting a cooperation between these two MTases. More recently, Ting et al.33Go demonstrated that inhibition of DNMT1 expression by small interfering RNA (siRNA) in HCT116 cells did not affect global DNA methylation, corroborating the results of Rhee et al.29Go In addition, using the same siRNA approach in an epithelial ovarian cancer cell line, Leu et al.34Go observed that although DNMT1 has an important role in maintaining DNA methylation, deficiency of both DNMT1 and DNMT3B leads to a 2-fold increase in global DNA demethylation than DNMT1 deficiency alone. These data suggest that if indeed DNMT1 is a major maintenance MTase in humans, there must be other compensatory pathways for lack of DNMT1 expression in those human cancer cells.

Nevertheless, our results in the DNMT1/DNMT3B DKO cells demonstrate that, despite global DNA demethylation, XIST gene methylation and repression in the Xa can be maintained in the absence of both DNMT1 and DNMT3B activity. DKO cells present ~5% of the normal levels of m5C,30Go which we show are partially targeted to the XIST gene, specifically to the HhaI and SacII(2) sites, which appear to be sufficient for XIST repression.

Recently, lower expression of XIST in recurrent versus primary ovarian tumors has been reported, suggesting that directly or indirectly this gene may be important for the control of cell growth.35Go Thus, one cannot exclude that during the derivation of the DNMT1/DNMT3B DKO cells there could have been a selective advantage of those that maintained XIST methylation and transcriptional repression. Characterization of loss of hypermethylated CpG islands in the DKO cells leads to the identification of silenced tumor suppressor genes.36Go Conversely, the identification of the genomic regions that maintain DNA methylation in the DNMT1/DNMT3B DKO cells may point to genes whose transcriptional repression is essential for cell growth and/or viability.

In addition, since the 5-aza-dC experiments showed that XIST repression is dependent on DNA methylation in the HCT116 cells, our data indicate that MTases other than DNMT1 and DNMT3B must be involved in the process of XIST methylation in these cells. Currently, DNMT2 and DNMT3A are the only other known candidate MTases for this process. However, as in Arabidopsis, where at least 10 different MTase genes have been identified (reviewed in Martienssen and Colot37Go), other unidentified DNA MTases may exist in mammals.

In conclusion, the observed discrepancy between maintenance of methylation of global DNA and of the XIST gene in the DKO HCT116 cells is unexpected since in mice these processes are mediated by the same enzyme, namely Dnmt1.27Go,28Go In the human DKO cells, although global DNA methylation is disrupted, XIST methylation at the HhaI and SacII(2) sites is maintained. Therefore, our findings suggest that in humans different epigenetic mechanisms may control global and XIST gene expression and that XIST methylation can be mediated by other unknown factors. Additional experiments in normal human somatic cells using alternative gene inactivation approaches, such as siRNA, are required to confirm our observations and identify these factors.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
References
 
The authors thank Bert Vogelstein and Kornel E. Schuebel for the different MTase deficient cell lines and for the scientific support, Angela V. Morgante for helpful comments and discussion and Lígia Vieira for technical assistance with cells. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).


   Footnotes
 
*To whom correspondence should be addressed. Tel. +55-11-3091-7476, Fax. +55-11-3091-7553, E-mail: LPEREIRA{at}USP.BR

Communicated by Mitsuo Oshimura


   References
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
 References
 

  1. Lyon, M. F. 1961, Gene action in the X chromosome of the mouse (Mus musculus L.), Nature, 190, 372–373.[CrossRef][Medline]
  2. Heard, E., Clerc, P., Avner, P. 1997, X-chromosome inactivation on mammals, Annu. Rev. Genet., 31, 571–610.[CrossRef][ISI][Medline]
  3. Hong, Y. K., Ontiveros, S. D., Chen, C., Strauss, W. M. 1999, A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation, Proc. Natl Acad. Sci. USA, 96, 6829–6834.[Abstract/Free Full Text]
  4. Kay, G. F., Penny, G. D., Patel, D., Ashworth, A., Brockdorff, N., Rastan, S. 1993, Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation, Cell, 72, 171–182.[CrossRef][ISI][Medline]
  5. Panning, B. and Jaenisch, R. 1996, DNA hypomethylation can activate Xist expression and silence X-linked genes, Genes Dev., 10, 1991–2002.[Abstract/Free Full Text]
  6. Beard, C., Li, E., Jaenisch, R. 1995, Loss of methylation activates Xist in somatic but not in embryonic cells, Genes Dev., 9, 2325–2334.[Abstract/Free Full Text]
  7. Hendrich, B. D., Brown, C. J., Willard, H. F. 1993, Evolutionary conservation of possible functional domains of the human and murine XIST genes, Hum. Mol. Genet., 2, 663–672.[Abstract/Free Full Text]
  8. Lafreniere, R. G. and Willard, H. F. 1993, Pulsed-field map of Xq13 in the region of the human X inactivation center, Genomics, 17, 502–506.[CrossRef][ISI][Medline]
  9. Heard, E., Simmler, M. C., Larin, Z., et al. 1993, Physical mapping and YAC contig analysis of the region surrounding Xist on the mouse X chromosome, Genomics, 15, 559–569.[CrossRef][ISI][Medline]
  10. Norris, D. P., Patel, D., Kay, G. F., et al. 1994, Evidence that random and imprinted Xist expression is controlled by preemptive methylation, Cell, 77, 41–51.[CrossRef][ISI][Medline]
  11. Tinker, A. V. and Brown, C. J. 1998, Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation, Nucleic Acids Res., 26, 2935–2940.[Abstract/Free Full Text]
  12. Hansen, R. S., Canfield, T. K., Stanek, A. M., Keitges, E. A., Gartler, M. 1998, Reactivation of XIST in normal fibroblasts and a somatic cell hybrid: abnormal localization of XIST RNA in hybrid cells, Proc. Natl Acad. Sci. USA, 95, 5133–5138.[Abstract/Free Full Text]
  13. Bestor, T., Laudano, A., Mattaliano, R., Ingram, V. 1988, Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells: the carboxy-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases, J. Mol. Biol., 203, 971–983.[CrossRef][ISI][Medline]
  14. Yoder, J. A. and Bestor, T. H. 1998, A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast, Hum. Mol. Genet., 7, 279–284.[Abstract/Free Full Text]
  15. Okano, M., Xie, S., Li, E. 1998, Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases, Nat. Genet., 19, 219–220.[CrossRef][ISI][Medline]
  16. Aapola, U., Kawasaki, K., Scott, H. S., et al. 2000, Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family, Genomics, 65, 293–298.[CrossRef][ISI][Medline]
  17. Bestor, T. H. 2000, The DNA methyltransferases of mammals, Hum. Mol. Genet., 9, 2395–2402.[Abstract/Free Full Text]
  18. Okano, M., Xie, S., Li, E. 1998, Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells, Nucleic Acids Res., 26, 2536–2540.[Abstract/Free Full Text]
  19. Hermann, A., Schmitt, S., Jeltsch, A. 2003, The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity, J Biol. Chem., 34, 31717–31721.
  20. Okano, M., Bell, D. W., Haber, D. A., Li, E. 1999, DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development, Cell, 99, 247–257.[CrossRef][ISI][Medline]
  21. Kaneda, M., Okano, M., Hata, K., et al. 2004, Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting, Nature, 429, 900–903.[CrossRef][Medline]
  22. Jeanpierre, M., Turfeau, C., Aurias, A., et al. 1993, An embryonic-like methylation pattern of classical satellite DNA is observed in ICF syndrome, Hum. Mol. Genet., 2, 731–735.[Abstract/Free Full Text]
  23. Xu, G.-L., Bestor, T. H., Bourc'his, D., et al. 1999, Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene, Nature, 402, 187–191.[CrossRef][Medline]
  24. Bourc'his, D., Xu, G. L., Lin, C. S., Bollman, B., Bestor, T. H. 2001, Dnmt3L and the establishment of maternal genomic imprints, Science, 294, 2536–2539.[Abstract/Free Full Text]
  25. Aapola, U., Liiv, I., Peterson, P. 2002, Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity, Nucleic Acids Res., 30, 3602–3608.[Abstract/Free Full Text]
  26. Deplus, R., Brenner, C., Burgers, W. A., et al. 2002, Dnmt3L is a transcriptional repressor that recruits histone deacetylase, Nucleic Acids Res., 30, 3831–3838.[Abstract/Free Full Text]
  27. Li, E., Bestor, T. H., Jaenish, R. 1992, Targeted mutation of the DNA methyltransferase gene results in embryonic lethality, Cell, 69, 915–926.[CrossRef][ISI][Medline]
  28. Li, E., Beard, C., Jaenisch, R. 1993, Role for DNA methylation in genomic imprinting, Nature, 190, 362–365.
  29. Rhee, I., Jair, K., Yen, R. C., et al. 2000, CpG methylation is maintained in human cancer cells lacking DNMT1, Nature, 404, 1003–1007.[CrossRef][Medline]
  30. Rhee, I., Bachman, K. E., Park, B. H., et al. 2002, DNMT1 and DNMT3B cooperate to silence genes in human cancer cells, Nature, 416, 552–556.[CrossRef][Medline]
  31. Sambrook, E., Fritsch, E. F., Maniatis, T. 1989, Molecular Cloning: A Laboratory Manual, New York Cold Spring Harbor Laboratory, Cold Spring Harbor.
  32. Brown, C. J., Ballabio, A., Rupert, J. L., et al. 1991, A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome, Nature, 349, 38–44.[CrossRef][Medline]
  33. Ting, A. H., Jair, K. W., Suzuki, H., Yen, R. W., Baylin, S. B., Schuebel, K. E. 2004, CpG island hypermethylation is maintained in human colorectal cancer cells after RNAi-mediated depletion of DNMT1, Nat. Genet., 36, 582–584.[CrossRef][ISI][Medline]
  34. Leu, Y. W., Rahmatpanah, F., Shi, H., et al. 2003, Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation, Cancer Res., 63, 6110–6115.[Abstract/Free Full Text]
  35. Huang, K. C., Rao, P. H., Lau, C. C., et al. 2002, Relationship of XIST expression and responses of ovarian cancer to chemotherapy, Mol. Cancer Ther., 1, 769–776.[Abstract/Free Full Text]
  36. Paz, M. F., Wei, S., Cigudosa, J. C., et al. 2003, Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases, Hum. Mol. Genet., 12, 2209–2219.[Abstract/Free Full Text]
  37. Martienssen, R. A. and Colot, V. 2001, DNA methylation and epigenetic inheritance in plants and filamentous fungi, Science, 293, 1070–1074.[Abstract/Free Full Text]

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