Mammalian DNA methyltransferases show different subnuclear distributions

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.     

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.     

Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5′‐flanking region during mouse oogenesis and spermatogenesis

DNA methyltransferase1o (Dnmt1o), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the Dnmt1o 5′‐flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5′‐flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5′‐flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non‐CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non‐CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no‐CpG region in the 5′‐flanking region of Dnmt1o was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non‐CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non‐CpG sites in the Dnmt1o 5′‐upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage. Mol. Reprod. Dev. 80: 212–222, 2013. © 2013 Wiley Periodicals, Inc.     

A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA

Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.     

Cooperative activity of DNA methyltransferases for maintenance of symmetrical and non-symmetrical cytosine methylation in Arabidopsis thaliana

Maintenance of cytosine methylation in plants is controlled by three DNA methyltransferases. MET1 maintains CG methylation, and DRM1/2 and CMT3 act redundantly to enforce non-CG methylation. RPS, a repetitive hypermethylated DNA fragment from Petunia hybrida, attracts DNA methylation when transferred into Petunia or other species. In Arabidopsis thaliana, which does not contain any RPS homologues, RPS transgenes are efficiently methylated in all sequence contexts. To test which DNA methylation pathways regulate RPS methylation, we examined maintenance of RPS methylation in various mutant backgrounds. Surprisingly, CG methylation was lost in a drm1/2/cmt3 mutant, and non-CG methylation was almost completely eliminated in a met1 mutant. An unusual cooperative activity of all three DNA methyltransferases is therefore required for maintenance of both CG and non-CG methylation in RPS. Other unusual features of RPS methylation are the independence of its non-CG methylation from the RNA-directed DNA methylation (RdDM) pathway and the exceptional maintenance of methylation at a CC(m)TGG site in some epigenetic mutants. This is indicative of activity of a methylation system in plants that may have evolved from the DCM methylation system that controls CC(m)WGG methylation in bacteria. Our data suggest that strict separation of CG and non-CG methylation pathways does not apply to all target regions, and that caution is required in generalizing methylation data obtained for individual genomic regions.     

Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice

Genomic imprinting is regulated by differential methylation of the paternal and maternal genome. However, it remains unknown how parental imprinting is established during gametogenesis. In this study, we demonstrate that Dnmt3L, a protein sharing homology with DNA methyltransferases, Dnmt3a and Dnmt3b, but lacking enzymatic activity, is essential for the establishment of maternal methylation imprints and appropriate expression of maternally imprinted genes. We also show that Dnmt3L interacts with Dnmt3a and Dnmt3b and co-localizes with these enzymes in the nuclei of transfected cells, suggesting that Dnmt3L may regulate genomic imprinting via the Dnmt3 family enzymes. Consistent with this model, we show that [Dnmt3a(-/-), Dnmt3b(+/-)] mice also fail to establish maternal methylation imprints. In addition, both Dnmt3a and Dnmt3L are required for spermatogenesis. Together, our findings suggest that Dnmt3L may cooperate with Dnmt3 family methyltransferases to carry out de novo methylation of maternally imprinted genes in oocytes.     

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ~8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length.     

The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites

In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.     

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.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

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.     

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.     

CG dinucleotide periodicities recognized by the Dnmt3a–Dnmt3L complex are distinctive at retroelements and imprinted domains

The Dnmt3a and Dnmt3L genes are critical mediators of cytosine methylation during gametogenesis, with major actions noted at transposable elements and imprinted loci. The Dnmt3a–Dnmt3L complex was recently described to have preferential activity at CG dinucleotides located 8-10 bp apart. Because cytosine methylation is heterogeneously distributed in the genome, we tested whether this relative sequence preference explains the effects of mutation of the Dnmt3a and Dnmt3L genes using bioinformatic analysis. We found that the human and mouse genomes are significantly enriched in a CG dinucleotide periodicity of 2 bp, leading to an increased frequency of CGs spaced 8 bp apart that represent widespread targets for this protein complex. When we broke down the human and mouse genomes by annotation, we found that this significant 2-bp periodicity and increased 8-bp periodicity are maintained in Alu SINEs in both species. The 8-bp periodicity was mapped genome-wide, identifying enrichment at the promoters of both paternally and maternally methylated imprinted genes and at CG dinucleotide-enriched sequences. We conclude that CG dinucleotide periodicity helps to explain some but not all of the relative sequence specificity of mutations of Dnmt3a or Dnmt3L in the establishment of germline cytosine methylation patterns.