We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or "maintenance," of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre-and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset ؊/؊ blastocysts showed defective growth of the inner cell mass and, in contrast to the ϳ65% recovery of wild-type and Eset ؉/؊ ES cells, no Eset ؊/؊ ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset ؊/؊ blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

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 G 1 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 (1).Complete inactivation of Dnmt1 by gene targeting does not affect ES 1 cell viability. However, Dnmt1Ϫ/Ϫ embryos die at ϳ9.5 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 (2-4). Inactivation of Dnmt3a results in multiple organ defects and lethality of homozygous mice several weeks after birth, without significant changes in DNA methylation (5). Disruption of Dnmt3b results in embryonic lethality at ϳ13.5 dpc and hypomethylation of the centromeric minor satellite repeats. In addition, analysis of Dnmt3bϪ/Ϫ 9.5-dpc embryos demonstrates that Dnmt3b plays a major role in de novo methylation of the genome (5). Similar to gene targeting of Dnmt1, inactivation of both Dnmt3a and Dnmt3b results in embryonic lethality at ϳ9.5 dpc (5). 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 (6).Genetic studies of human DNA methyltransferases have...