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Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b 总被引:26,自引:4,他引:22
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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. 相似文献
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DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. 总被引:104,自引:0,他引:104
The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease. 相似文献
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Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells 总被引:16,自引:0,他引:16
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Jackson M Krassowska A Gilbert N Chevassut T Forrester L Ansell J Ramsahoye B 《Molecular and cellular biology》2004,24(20):8862-8871
It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1. 相似文献
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Lsh is involved in de novo methylation of DNA 总被引:5,自引:0,他引:5
Zhu H Geiman TM Xi S Jiang Q Schmidtmann A Chen T Li E Muegge K 《The EMBO journal》2006,25(2):335-345
Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh-/- embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA. 相似文献
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DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction 总被引:12,自引:0,他引:12
Suetake I Shinozaki F Miyagawa J Takeshima H Tajima S 《The Journal of biological chemistry》2004,279(26):27816-27823
In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity. 相似文献
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Arand J Spieler D Karius T Branco MR Meilinger D Meissner A Jenuwein T Xu G Leonhardt H Wolf V Walter J 《PLoS genetics》2012,8(6):e1002750
The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs. 相似文献
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In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation. 相似文献
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Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization 总被引:14,自引:0,他引:14
Dodge JE Okano M Dick F Tsujimoto N Chen T Wang S Ueda Y Dyson N Li E 《The Journal of biological chemistry》2005,280(18):17986-17991
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. 相似文献
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Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center 总被引:14,自引:0,他引:14
Xin Z Tachibana M Guggiari M Heard E Shinkai Y Wagstaff J 《The Journal of biological chemistry》2003,278(17):14996-15000
Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns. 相似文献
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Background
In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. 相似文献19.
Mouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are expected to be de novo-type DNA methyltransferases. In the present study, we found that exogenously expressed mouse Dnmt3a or Dnmt3b induced abnormal cell clusters at the gastrulation stage in Xenopus embryos. The abnormal cells were judged to be apoptotic from the positive staining with the TdT dUTP nucleotide end-labeling method and the rescue by hBcl-x(L), a Bcl-2 homologue. On the other hand, neither bacterial DNA (cytosine-5) methyltransferase nor Dnmt3b3, one of the three isoforms of Dnmt3b that has no DNA methylation activity, induced apoptosis. In addition, mutant Dnmt3a and the other two Dnmt3b isoforms, Dnmt3b1 and Dnmt3b2, which have no DNA methylation activity due to a change of the cysteine residue in the catalytic center to an alanine residue, retained the ability to induce apoptosis. This indicates that the apoptosis was not induced by DNA methylation activity. The domain of Dnmt3b1 (3b2) responsible for the apoptosis is the catalytic domain in the carboxyl-terminal half. 相似文献