DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

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.     

Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b

The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3' long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.     

Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain

DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N‐terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi‐methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N‐terminal regulatory domain. By deletion analysis and co‐immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N‐terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex has a bipartite interaction interface and is formed in a head‐to‐head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi‐methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co‐ordinated. J. Cell. Biochem. 106: 521–528, 2009. © 2009 Wiley‐Liss, Inc.     

Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.     

Lsh is involved in de novo methylation of DNA

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.     

Chromatin targeting of de novo DNA methyltransferases by the PWWP domain

DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both enzymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible for the catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWP-mediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation in satellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.     

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b

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.     

Interactions within the mammalian DNA methyltransferase family

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.     

Genomic imprinting and epigenetic reprogramming: unearthing the garden of forking paths

Genomic imprinting, an epigenetic form of gene regulation, determines the parent-dependent gene expression of marked or imprinted genes during gametogenesis and embryonic development. Imprinting involves differential allele DNA methylation in one sex cell lineage but not in the other. Egg and sperm each contributes the same DNA sequences to the zygote but epigenetic imprinting of a subset of genes determines that only one of the parent alleles are expressed relative to the parental origin. Primordial germ cells inherit biallelically imprinted genes from maternal and paternal origin and erase their imprints to start de novo monoallelic imprinting during gametogenesis. Epigenetic paternalization is an ongoing process in the mitotically-dividing spermatogonial stem cell and derived meiotically-dividing spermatocyte progeny to endow sperm with imprinted alleles. Epigenetic maternalization is restricted to the oocyte growth phase of folliculogenesis and is unrelated to DNA replication since it takes place while the oocyte remains in the diplotene stage of meiotic prophase I. Sperm and oocyte genomic methylation patterns depend on the activity of DNA methyltransferases (Dnmt). A variant of Dnmt1, designated Dnmt1o, accumulates in oocyte nuclei during the follicular growth phase. Dnmt3L, an isoform of Dnmt3a and Dnmt3b, but lacking enzymatic activity, interacts with Dnmt2a and Dnmt3b and is required for spermatogenesis. In the mouse early zygote, the male pronucleus is demethylated within 4 h of fertilization. Global demethylation takes place gradually up to the morula stage. In the blastocyst, de novo methylation is reestablished in the inner cell mass but not in the trophectoderm. Both the significance of genomic imprinting and the severe developmental defects caused by disrupted Dnmt activity, point to a need for a better understanding of the causes of low cloning efficiency by somatic nuclear transfer to enucleated ovulated oocyte.     

Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.     

Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts.

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI-38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI-38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI-38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI-38 cells. These studies indicate that reduced genome-wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene-localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase.