These data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, our data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity.

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.

Previous studies have shown that the Dnmt3b gene encodes multiple variants via alternative splicing. However, only one form of Dnmt3a has been identified to date. We report here the discovery of a small form of Dnmt3a, denoted Dnmt3a2, from both human and mouse. The transcript encoding Dnmt3a2 is initiated from a downstream intronic promoter. As a result, the Dnmt3a2 protein lacks the N-terminal 223 (human) or 219 (mouse) amino acid residues of the full-length Dnmt3a. Recombinant Dnmt3a2 protein displayed similar cytosine methyltransferase activity as Dnmt3a in vitro. However, Dnmt3a and Dnmt3a2 exhibited strikingly different subcellular localization patterns. Unlike Dnmt3a, which was concentrated on heterochromatin, Dnmt3a2 displayed a localization pattern suggestive of euchromatin association. Dnmt3a2 is the predominant form in embryonic stem cells and embryonal carcinoma cells and can also be detected from testis, ovary, thymus, and spleen, whereas Dnmt3a is expressed at low levels ubiquitously. Comparison of human embryonal carcinoma cell lines with breast/ovarian cancer cell lines indicates that DNMT3A2 expression correlates with high de novo methylation activity. These findings suggest that Dnmt3a and Dnmt3a2 may have distinct DNA targets and different functions in development.

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.

It is generally accepted that the diagnosis of hepatitis B virus (HBV) infection is based on the detection of serum hepatitis B surface antigen (HBsAg), and the disappearance of this antigen indicates the clearance of HBV. However, recent studies have shown that HBV DNA was frequently detected in patients with chronic liver disease who were negative for HBsAg, but positive for antibodies to core antigens (antiHBc). 1-4 More recently, occult HBV infection has been reported in patients with chronic hepatitis C liver disease. 5 The possibility of sustained HBV infection in anti-HBc-positive individuals is also supported by previous studies showing that traces of HBV are often detectable in the blood for many years after clinical recovery from acute hepatitis despite the presence of serum antibodies against HBV and HBV-specific cytotoxic T lymphocytes. 6,7 Moreover, we recently showed the transmission of HBV from anti-HBc-positive healthy donors to almost all recipients via liver grafts in living related liver transplantation (LRLT). 8 Several investigators have also described HBV transmission from anti-HBc-positive donors via liver transplants. [9][10][11][12] We have also shown that more than 10% of the general population of Japan are estimated to be positive for anti-HBc, which implies a high risk of viral transmission to recipients via liver transplants. 13 Furthermore, it is possible that viral reactivation could occur in latent HBV carriers without HBsAg under certain immunosuppressive conditions. 14 However, little is known about the molecular mechanism of viral persistence in such cases.Here we investigated the molecular forms of latently infected HBV in the liver of healthy individuals who lacked any of the serological markers for viral antigen, circulating viral genomes in their sera, and ongoing liver dysfunction. Generation of covalently closed circular (ccc)DNA and pregenomic RNA in the host hepatocytes indicates the presence of ongoing viral replication, 15,16 and recently several new polymerase chain reaction (PCR) approaches have been developed to discriminate cccDNA from relaxed circular (rc)DNA molecules in viral particles. [17][18][19][20][21] In this study, we applied the following methods to investigate the molecular forms of HBV genomes in the liver tissue of healthy individuals: a highly selective PCR assay using specific primers for cccDNA forms; treatment of DNA samples with mung bean nuclease before amplification with primer sets across the nick region; size fractionation of DNA samples before detection of HBV DNA; and reverse-transcription polymerase chain reaction (RT-PCR) followed by Southern blotting assay for detection of HBV RNA. To characterize the latently infected HBV, we also analyzed the nucleic acid changes in the core (C) and pre-C region of the infected HBV genomes in those cases that lacked evidence of hepatic inflammation.Our findings showed that the replicative forms of HBV, including cccDNA and intermediate RNA, were present in the

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...

p73 has been identi®ed as a protein which shares signi®cant homology with the tumor suppressor p53. We found two new types of splicing variant mRNAs for p73 expressed in MCF-7 cells which we named p73g and e. Sequence analysis revealed that these mRNAs encode variant p73 proteins bearing distinct carboxy-terminal structures, which are also di erent from the previously reported variants p73a and b. The mRNAs encoding p73g and e as well as a and b were con®rmed to be expressed in normal human tissues in varied patterns. All of these splicing variants activated promoter with the p53-binding consensus sequence, but to di erent degrees. Furthermore, suppressive e ects of p73a, g and e, but not b, on endogenous p53 activity were observed when transiently expressed in HepG2 and MCF-7 cells. These results suggested that the carboxy-terminal regions of p73 which were altered by alternative splicing a ect these transactivation abilities and modulate the functions of p73 molecules.

ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome is a rare autosomal recessive disease caused by mutations in the DNA methyltransferase gene DNMT3B. To investigate the function of Dnmt3b in mouse development and to create animal models for ICF syndrome, we have generated three mutant alleles of Dnmt3b in mice: one carrying a deletion of the catalytic domain (null allele) and two carrying ICF-like missense mutations in the catalytic domain. The Dnmt3b null allele results in embryonic lethality from E14.5 to E16.5 with multiple tissue defects, including liver hypotrophy, ventricular septal defect and haemorrhage. By contrast, mice homozygous for the ICF mutations develop to term and some survive to adulthood. These mice show phenotypes that are reminiscent of ICF patients, including hypomethylation of repetitive sequences, low body weight, distinct cranial facial anomalies and T cell death by apoptosis. These results indicate that Dnmt3b plays an essential role at different stages of mouse development, and that ICF missense mutations cause partial loss of function. These mutant mice will be useful for further elucidation of the pathogenic and molecular mechanisms underlying ICF syndrome.