Frederick A. Dick

The retinoblastoma (RB) family of proteins are found in organisms as distantly related as humans, plants, and insects. These proteins play a key role in regulating advancement of the cell division cycle from the G1 to S-phases. This is achieved through negative regulation of two important positive regulators of cell cycle entry, E2F transcription factors and cyclin dependent kinases. In growth arrested cells transcriptional activity by E2Fs is repressed by RB proteins. Stimulation of cell cycle entry by growth factor signaling leads to activation of cyclin dependent kinases. They in turn phosphorylate and inactivate the RB family proteins, leading to E2F activation and additional cyclin dependent kinase activity. This propels the cell cycle irreversibly forward leading to DNA synthesis. This review will focus on the basic biochemistry and cell biology governing the regulation and activity of mammalian RB family proteins in cell cycle control.

DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G1 to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism. DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G1 to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism. Mammalian DNA (cytosine-5) methyltransferases Dnmt1, Dnmt3a, and Dnmt3b catalyze methylation of CpG dinucleotides in genomic DNA. Genetic studies of mice with mutations of these three Dnmt genes have shown that DNA methylation is essential for embryonic development, establishment and maintenance of allele-specific expression of imprinted genes, repression of inactivated X chromosome in female cells, and repression of endogenous viruses and transposable elements (1Chen T. Li E. Curr. Top. Dev. Biol. 2004; 60: 55-89Crossref PubMed Scopus (255) Google Scholar). Complete inactivation of Dnmt1 by gene targeting does not affect ES 1The abbreviations used are: ES, embryonic stem; MEF, mouse embryonic fibroblast; SA, senescence-associated; dpc, days post coitum. 1The abbreviations used are: ES, embryonic stem; MEF, mouse embryonic fibroblast; SA, senescence-associated; dpc, days post coitum. cell viability. However, Dnmt1-/- embryos die at ∼9.5 days post coitum (dpc), and mouse embryonic fibroblasts (MEFs) that lack Dnmt1 (generated by conditional deletion of Dnmt1) die after a few cell divisions. Inactivation of Dnmt1 in ES cells, embryos, and MEFs results in a genome-wide loss of DNA methylation (2Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3201) Google Scholar, 3Lei H. Oh S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development (Camb.). 1996; 122: 3195-3205Crossref PubMed Google Scholar, 4Jackson-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 (565) Google Scholar). Inactivation of Dnmt3a results in multiple organ defects and lethality of homozygous mice several weeks after birth, without significant changes in DNA methylation (5Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4449) Google Scholar). Disruption of Dnmt3b results in embryonic lethality at ∼13.5 dpc and hypomethylation of the centromeric minor satellite repeats. In addition, analysis of Dnmt3b-/- 9.5-dpc embryos demonstrates that Dnmt3b plays a major role in de novo methylation of the genome (5Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4449) Google Scholar). Similar to gene targeting of Dnmt1, inactivation of both Dnmt3a and Dnmt3b results in embryonic lethality at ∼9.5 dpc (5Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4449) Google Scholar). ES cells that lack both Dnmt3a and Dnmt3b progressively lose DNA methylation and after extended passage in culture lose almost all DNA methylation at all loci examined while expressing normal levels of Dnmt1 (6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar). Genetic studies of human DNA methyltransferases have been carried out in various cancer cell lines. It was shown that targeted disruption of DNMT1 or DNMT3b in the colon cancer cell line HCT116 had little effect on DNA methylation and cell growth, whereas inactivation of both DNMT1 and DNMT3b led to a genome-wide hypomethylation, expression of p16, and reduced cell growth (7Rhee I. Bachman K.E. Park B.H. Jair K.W. Yen R.W. Schuebel K.E. Cui H. Feinberg A.P. Lengauer C. Kinzler K.W. Baylin S.B. Vogelstein B. Nature. 2002; 416: 552-556Crossref PubMed Scopus (1033) Google Scholar, 8Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (370) Google Scholar). However, more recent studies showed that inactivation of DNMT1 alone by antisense RNA or RNA interference methods in HCT116 cells and other human cancer cells resulted in loss of DNA methylation and inhibition of cell growth (9Robert M.F. Morin S. Beaulieu N. Gauthier F. Chute I.C. Barsalou A. MacLeod A.R. Nat. Genet. 2003; 33: 61-65Crossref PubMed Scopus (518) Google Scholar). Additional studies are necessary to resolve these discrepancies. Studies of tumor cell lines and primary tumor tissues provide compelling evidence that aberrant change in DNA methylation patterns is a hallmark of cancer. Hypermethylation of CpG-containing gene promoters often targets putative tumor suppressor genes such as p16, RASSF1A, BRCA1, and pRb and is associated with their silencing. Hypomethylation of bulk genomic DNA such as pericentromeric repeats is also frequently observed in tumor cells (10Jones P.A. Baylin S.B. Nat. Rev. Genet. 2002; 3: 415-428Crossref PubMed Google Scholar, 11Baylin S.B. Bestor T.H. Cancer Cell. 2002; 1: 299-305Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). However, the mechanism by which hypomethylation contributes to tumorigenesis remains largely unknown. In this study, we use MEF cells derived from Dnmt3a or Dnmt3b knock-out mice to study how DNA hypomethylation might affect cell growth and chromosome stability. We show that inactivation of Dnmt3b by either constitutive or conditional gene targeting in MEF cells results in global DNA hypomethylation, premature senescence or spontaneous immortalization, and chromosomal instability. In contrast, disruption of Dnmt3a appears to have no effect on these processes in MEF cells. MEF Cell Culture—MEFs were isolated from 12.5- or 13.5-dpc embryos of 129SvJae × C57BL/6 hybrid background and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Each MEF line was expanded to three 10-cm dishes (except those Dnmt3b-/- cultures that grew slowly and were harvested from the initial 10-cm dish) and then frozen in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 10% Me2SO and labeled as passage 1 (P1). Conditional inactivation of Dnmt3a or Dnmt3b was carried out in vitro by multiple infections of primary MEFs derived from Dnmt3a-/2loxP or Dnmt3b-/2loxP embryos with high-titer Cre adenovirus. Cre-mediated recombination was confirmed by Southern blot and Western blot analyses. Transfection of Dnmt3a or Dnmt3b expression vectors in MEF cells was carried out using Lipofectamine (Invitrogen) and selected with blasticidin as described previously (6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar). DNA Methylation Analysis—Genomic DNA was digested with the methylation-sensitive enzyme HpaII and analyzed by Southern hybridization using probes for minor satellite repeats and the endogenous C-type retrovirus. Bisulfite sequencing analysis of the p21 proximal promoter was done as described previously (12Dodge J.E. List A.F. Futscher B.W. Int. J. Cancer. 1998; 78: 561-567Crossref PubMed Scopus (65) Google Scholar), using the following oligonucleotides as nested PCR primers: F1 (5′-GATGTATGTGGTTTTGTTGGTG-3′), F2 (5′-GAGAATAGTTTAGGTGTGGGGG-3′), R1 (5′-CACCCACTAAACTCAACICATTAC-3′), and R2 (5′-CIAAAAAAAACTATTATTCCCTACCAC-3′) (I = inosine). Spontaneous Immortalization and SA-β-Galactosidase Staining Assays—The growth of MEFs was assayed following a standard 3T9 protocol. Briefly, 900,000 cells were plated in a 6-cm dish and grown in a 37 °C incubator with 5% CO2 for 3 days, and the procedure was repeated until wild-type MEFs started to show immortalized growth. All counts were done in duplicate, and the population doublings, which were calculated using the log formula (13Blasko M.A. Lee H.W. Hande M.P. Samper E. Lansdorp P.M. DePinho R.A. Greider C.W. Cell. 1997; 91: 25-34Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar), were plotted on the y axis versus days of culture on the x axis. SA-β-galactosidase staining was performed as described previously (14Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelly C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5655) Google Scholar). Analysis of Metaphase and Anaphase Chromosomes—Metaphase chromosome spreads were prepared following standard procedures. Briefly, after treatment with Colcemid for ∼3 h, cells were harvested, treated twice with 75 mm KCl for 15 min each time, and fixed in methanol:glacial acetic acid (3:1) at room temperature for 1 h. The cell suspension was then dropped onto a microscope slide, and chromosomes were visualized with a light microscope. For anaphase bridge analysis, asynchronous cells grown on chambered slide glass were fixed, stained for mitotic spindle with anti-γ-tubulin antibody (T-6557 monoclonal antibody clone GTU-88; Sigma) followed by Alexa 488-conjugated goat anti-mouse secondary antibody (Molecular Probe), and counterstained for DNA with 4′,6′-diamidino-2-phenylindole. Anaphase cells were visualized by fluorescence microscopy. Cell Cycle, Cell Proliferation, and DNA Damage Checkpoint Assays— For cell cycle and cell proliferation analysis, cells were stained with propidium iodide and anti-bromodeoxyuridine antibodies and analyzed by flow cytometry on a Becton Dickinson FACScan. To induce DNA damage, the cells were dosed with 10 grays of ionizing radiation from a cesium source. For Western blot analysis, proteins were isolated 2 h after irradiation for p53 or 18 h after irradiation for p21. Antibodies against p21 (F-5) and p53 (Pab240) were obtained from Santa Cruz Biotechnology and Chemicon International, Inc., respectively. Dnmt3b Is Required for the Maintenance of DNA Methylation in MEF Cells—We have previously shown that Dnmt3a and Dnmt3b are required for de novo methylation of the mouse genome during early embryogenesis and for both de novo methylation and stable maintenance of DNA methylation patterns in ES cells (5Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4449) Google Scholar, 6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar). To examine whether Dnmt3a and Dnmt3b are required for maintaining DNA methylation in somatic cells after the hypermethylated state of the genome is established, we obtained MEF cells for conditional inactivation of these genes. Primary MEF cell cultures were established from embryos that carry one null allele and one conditional allele of Dnmt3a or Dnmt3b. Conditional inactivation of Dnmt3a or Dnmt3b was achieved by infecting the corresponding MEFs with Cre adenovirus. The conditional alleles, which contain two loxP sites flanking the exons that encode the catalytic regions of Dnmt3a or Dnmt3b, had equivalent functions to their wild-type alleles, whereas Cre-mediated deletion of catalytic motifs produced functionally null alleles (Fig. 1). The Dnmt3a conditional allele has been described previously (15Kaneda M. Okano M. Hata K. Sado T. Tsujimoto N. Li E. Sasaki H. Nature. 2004; 429: 900-903Crossref PubMed Scopus (1016) Google Scholar) (Fig. 1A). The Dnmt3b conditional allele with two loxP sites flanking the exons encoding the PC and ENV catalytic motifs is shown in Fig. 1B. The conditional alleles (2P), the Cre-mediated deletion alleles (1P), and the null alleles (-) could be distinguished by Southern blot analysis (Fig. 1, C and D). Both Dnmt3a and Dnmt3b proteins were expressed in wild-type primary MEF cells. Constitutive or conditional inactivation of Dnmt3a or Dnmt3b abolished the corresponding protein product (Fig. 1, E and F). To determine global DNA methylation levels, genomic DNA was digested with the restriction enzyme MspI, which DNA of whether it is or or with which DNA. The digested DNA was analyzed by followed by staining (Fig. and Southern blot hybridization with the endogenous C-type DNA (Fig. The of endogenous throughout the mouse genome were in both wild-type ES and MEF cells (Fig. and All Dnmt3a or immortalized primary and immortalized MEF cells not show changes in DNA methylation of C-type DNA (Fig. as with wild-type MEF cells, with the for ES cells and 9.5-dpc embryos (5Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4449) Google Scholar). In contrast, primary Dnmt3b-/- MEFs showed a of methylation of C-type DNA (Fig. Dnmt3b-/- MEFs also showed the of as primary Dnmt3b-/- MEFs (Fig. 15 and whereas MEFs showed normal levels of DNA methylation in C-type DNA (Fig. We also examined the effect of inactivation of both Dnmt3a and Dnmt3b on DNA methylation in MEF cells. MEFs showed normal levels of DNA methylation (Fig. inactivation of the Dnmt3b allele resulted in hypomethylation to that observed in MEFs (Fig. MEFs = 2) that were in culture for levels of DNA methylation to those in cells (Fig. with and in to the loss of DNA methylation in ES cells (Fig. (6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar). a MEFs with Cre and for the of wild-type levels of DNA methylation (Fig. These results suggest that Dnmt3b is required for the maintenance of global DNA in MEF cells, whereas Dnmt3a is of Dnmt3b, but Dnmt3a, MEF analysis of wild-type MEF lines showed that after ∼3 weeks in senescence had as by both a in cell proliferation and (Fig. and growth was observed for and MEF cells = not In contrast, of 10 Dnmt3b-/- MEF cultures the wild-type of proliferation in early followed by a in these MEF lines showed either = 3) or spontaneous immortalization = a MEF lines = 2) senescence and MEF lines = 2) showed almost growth to that of MEF cells, that Dnmt3b is not required for the proliferation of cells (Fig. of Dnmt3b in Dnmt3b-/- MEFs that loss of Dnmt3b resulted in in proliferation in we whether of Dnmt3b Dnmt3b-deficient MEFs affect the proliferation of these cells. We Dnmt3b or (6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar) immortalized MEF line and to stable expressing were obtained and expression of the wild-type Dnmt3b resulted in growth of Dnmt3b or in the stable was by analysis (Fig. of the Dnmt3b the centromeric minor satellite in expressing Dnmt3b, but not in those expressing (Fig. 3T9 growth showed that reduced proliferation with the of Dnmt3b (Fig. SA-β-galactosidase was in of cells that expressed Dnmt3b, but not in cells that expressed (Fig. In addition, cells were observed in but not in (Fig. with that of Dnmt3a had no effect on of Dnmt3a MEFs not growth not of Dnmt3b, but Dnmt3a, Is with performed analysis of primary and MEFs were previously described to be and as a We that Dnmt3b-/- MEFs more cells wild-type or MEFs (Fig. not with this analysis that Dnmt3b-/- MEFs grown in asynchronous cultures had of cells wild-type MEFs not Dnmt3b-/- MEFs also showed increased of wild-type MEFs of Dnmt3b-/- MEFs chromosomes (Fig. we one MEF line that and and this with the of anaphase bridge in of the (Fig. and was also observed in one of the two MEF lines In contrast, of the Dnmt3a MEFs examined chromosomal (Fig. Interestingly, anaphase bridge was in the that had Dnmt3b but it was in the (Fig. Inactivation of Dnmt3b in of p21 determine the by which Dnmt3b may we examined the of the G1 to S-phase checkpoint that is largely by the p53 MEF cells were treated with or without 10 grays of and h was by analysis was 1 h to Similar to wild-type both primary and immortalized Dnmt3b-/- MEFs showed reduced proliferation in to DNA by whereas as a to after DNA (Fig. analysis that immortalized Dnmt3b-/- and MEF lines had increased levels of p53 protein 2 h after DNA (Fig. These results that the G1 to S-phase checkpoint is intact in Dnmt3b-deficient MEF cells. We then examined the protein levels of a major of G1 in Dnmt3b-deficient MEF cells 18 h after all three spontaneously immortalized Dnmt3b-/- MEF lines showed increased levels of p21 in to DNA damage, cells, had levels of p21 protein (Fig. their lack of p53 (Fig. levels of p21 protein were observed in one Dnmt3b-/- line one and one line (Fig. that of gene promoters is associated with we performed genomic sequencing of the p21 proximal promoter in and Similar to the of that have examined the methylation of the p21 proximal we not DNA methylation in the in of these not that of p21 promoter CpG is not the mechanism of the p21 observed in Dnmt3b-deficient MEFs S. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Maintenance of a hypermethylated state of the genome is essential for and studies have that Dnmt1 is the major maintenance we have shown that Dnmt3a and Dnmt3b also a role in maintaining DNA methylation patterns in ES cells (6Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (572) Google Scholar). In this study, we show that conditional inactivation of Dnmt3b results in of DNA methylation in MEF cells, that the of DNA methyltransferases is required for maintaining normal DNA methylation levels in somatic cells as In to the largely of Dnmt3a and Dnmt3b in ES cells, Dnmt3a does not to for the loss of Dnmt3b in that both Dnmt3a and Dnmt3b are expressed in it is that associated with genomic loci to Dnmt3a but does not affect their to Dnmt3b. However, we the that Dnmt3a is in the maintenance of DNA methylation in other cell types or during of is that MEF cells, ES cells, not show loss of methylation in suggesting that in the absence of Dnmt3a and Dnmt3b, Dnmt1 is of maintaining a of DNA methylation in MEFs in ES cells. these results suggest that the of the Dnmt1 and of DNA methyltransferases to the maintenance of global methylation in a cell and Inactivation of Dnmt1 has been shown to global and in MEF cells 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 (565) Google Scholar). In this study, we that Dnmt3b-deficient MEF cells global and but show wild-type MEF cells, which senescence after ∼3 weeks in the of Dnmt3b-/- MEF lines of senescence and spontaneously immortalized because to during the of Dnmt3b-/- MEF lines of show proliferation and We analyzed of the p53 that are to cell We showed that the p53 protein could be by DNA in primary as as immortalized Dnmt3b-deficient Interestingly, the expression of the kinase inhibitor p21 was in a p53-independent It remains to be whether of p21 is a for the premature senescence observed in the of Dnmt3b-deficient MEF lines. levels of p21 are of G1 in Dnmt3b-deficient cells, suggesting that the may be Additional studies are necessary to determine the by which Dnmt3b cell It is Dnmt3b-deficient cells spontaneous immortalization, whereas premature The background of the MEF lines does not to for the because all the MEF cells were isolated from embryos of 129SvJae × C57BL/6 hybrid and and female cells showed no in is that genomic in the Dnmt3b-deficient MEF in studies provide a for disruption of which a of the of has been shown to global hypomethylation and proliferation of MEF cells K. T. T. K. Dev. 2001; PubMed Scopus Google Scholar, T. J. S. E. D. K. Cancer 2003; Google Scholar). Dnmt3b, has been in maintaining E. S. K. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar, K.E. Baylin S.B. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). the that and as a of global hypomethylation, might be the of premature senescence observed in Dnmt3b-deficient MEF cells. of proliferation was for to Dnmt3b-deficient patterns of growth, on of which contain two of the genome, are to spontaneous immortalization and whereas which contain two of the genome, premature senescence and These have been to loss of L. S. G. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). a role for Dnmt3b in the maintenance of genomic has to be it is that expression of imprinted genes, to DNA might determine whether Dnmt3b-deficient MEF cells spontaneous immortalization or premature studies have established a in DNA methylation and cancer. of promoter CpG of tumor suppressor genes a that inactivation of tumor suppressor genes how DNA hypomethylation contributes to tumorigenesis remains largely (10Jones P.A. Baylin S.B. Nat. Rev. Genet. 2002; 3: 415-428Crossref PubMed Google Scholar, U. Beard C. L. Jaenisch R. Nature. 1998; PubMed Scopus Google Scholar, A. F. A. Jaenisch R. 2003; PubMed Scopus Google Scholar, F. J.G. A. L. Dausman J. H. Jaenisch R. 2003; PubMed Scopus Google Scholar). Our results show that inactivation of Dnmt3b in MEFs results in hypomethylation and chromosomal instability, which in turn may to spontaneous It remains to be whether immortalized Dnmt3b-/- MEFs are to DNMT3b is expressed at levels in human somatic it is that of DNMT3b a in genome-wide hypomethylation and chromosomal instability. It also be to examine whether of normal cells to of DNMT3b expression and DNA Additional studies are necessary to the role of DNMT3b in and cancer