On the evolutionary origin of eukaryotic DNA methyltransferases and Dnmt2

The Dnmt2 enzymes show strong amino acid sequence similarity with eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases. Yet, Dnmt2 enzymes from several species were shown to methylate tRNA-Asp and had been proposed that eukaryotic DNA methyltransferases evolved from a Dnmt2-like tRNA methyltransferase ancestor [Goll et al., 2006, Science, 311, 395-8]. It was the aim of this study to investigate if this hypothesis could be supported by evidence from sequence alignments. We present phylogenetic analyses based on sequence alignments of the methyltransferase catalytic domains of more than 2300 eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases and analyzed the distribution of DNA methyltransferases in eukaryotic species. The Dnmt2 homologues were reliably identified by an additional conserved CFT motif next to motif IX. All DNA methyltransferases and Dnmt2 enzymes were clearly separated from other RNA-(cytosine-C5)-methyltransferases. Our sequence alignments and phylogenetic analyses indicate that the last universal eukaryotic ancestor contained at least one member of the Dnmt1, Dnmt2 and Dnmt3 families of enzymes and additional RNA methyltransferases. The similarity of Dnmt2 enzymes with DNA methyltransferases and absence of similarity with RNA methyltransferases combined with their strong RNA methylation activity suggest that the ancestor of Dnmt2 was a DNA methyltransferase and an early Dnmt2 enzyme changed its substrate preference to tRNA. There is no phylogenetic evidence that Dnmt2 was the precursor of eukaryotic Dnmts. Most likely, the eukaryotic Dnmt1 and Dnmt3 families of DNA methyltransferases had an independent origin in the prokaryotic DNA methyltransferase sequence space.     

Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue

The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-l-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methylation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. ENZYMES: Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. STRUCTURED DIGITAL ABSTRACT: ? Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) ? Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction).     

Preferential methylation of unmethylated DNA by Mammalian de novo DNA methyltransferase Dnmt3a

DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-l-methionine.     

Dnmt2 is the most evolutionary conserved and enigmatic cytosine DNA methyltransferase in eukaryotes

Dnmt2 is the most strongly conserved cytosine DNA methyltransferase in eukaryotes. It has been found in all organisms possessing methyltransferases of the Dnmt1 and Dnmt3 families, whereas in many others Dnmt2 is the sole cytosine DNA methyltransferase. The Dnmt2 molecule contains all conserved motifs of cytosine DNA methyltransferases. It forms 3D complexes with DNA very similar to those of bacterial DNA methyltransferases and performs cytosine methylation by a catalytic mechanism common to all cytosine DNA methyltransferases. Catalytic activity of the purified Dnmt2 with DNA substrates is very low and could hardly be detected in direct biochemical assays. Dnmt2 is the sole cytosine DNA methyltransferase in Drosophila and other dipteran insects. Its overexpression as a transgene leads to DNA hypermethylation in all sequence contexts and to an extended life span. On the contrary, a null-mutation of the Dnmt2 gene leads to a diminished life span, though no evident anomalies in development are observed. Dnmt2 is also the sole cytosine DNA methyltransferase in several protists. Similar to Drosophila these protists have a very low level of DNA methylation. Some limited genome compartments, such as transposable sequences, are probably the methylation targets in these organisms. Dnmt2 does not participate in genome methylation in mammals, but seems to be an RNA methyltransferase modifying the 38th cytosine residue in anticodon loop of certain tRNAs. This modification enhances stability of tRNAs, especially in stressful conditions. Dnmt2 is the only enzyme known to perform RNA methylation by a catalytic mechanism characteristic of DNA methyltransferases. The Dnmt2 activity has been shown in mice to be necessary for paramutation establishment, though the precise mechanisms of its participation in this form of epigenetic heredity are unknown. It seems likely, that either of the two Dnmt2 activities could become a predominant one during the evolution of different species. The high level of the Dnmt2 evolutionary conservation proves its activity to have a significant adaptive value in natural environment.     

Developmentally regulated DNA methylation in Dictyostelium discoideum

Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.     

Target recognition,RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA^Asp(GUC) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA^Glu(CUC/UUC) and tRNA^Gly(GCC) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.     

5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.     

The Drosophila cytosine-5 methyltransferase Dnmt2 is associated with the nuclear matrix and can access DNA during mitosis

Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.     

The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity

The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases.     

The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2‐deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell‐autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA Asp^GTC, Gly^GCC, and Val^AAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2‐dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near‐cognate codons, thereby ensuring accurate polypeptide synthesis.     

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.     

A Dnmt2-like protein mediates DNA methylation in Drosophila

The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.     

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA^Asp methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA^Pro. Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.     

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.     

Plant DNA methyltransferases

DNA methylation is an important modification of DNA that plays a role in genome management and in regulating gene expression during development. Methylation is carried out by DNA methyltransferases which catalyse the transfer of a methyl group to bases within the DNA helix. Plants have at least three classes of cytosine methyltransferase which differ in protein structure and function. The METI family, homologues of the mouse Dnmt1 methyltransferase, most likely function as maintenance methyltransferases, but may also play a role in de novo methylation. The chromomethylases, which are unique to plants, may preferentially methylate DNA in heterochromatin; the remaining class, with similarity to Dnmt3 methyltransferases of mammals, are putative de novo methyltransferases. The various classes of methyltransferase may show differential activity on cytosines in different sequence contexts. Chromomethylases may preferentially methylate cytosines in CpNpG sequences while the Arabidopsis METI methyltransferase shows a preference for cytosines in CpG sequences. Additional proteins, for example DDM1, a member of the SNF2/SWI2 family of chromatin remodelling proteins, are also required for methylation of plant DNA.     

Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L

The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.