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.     

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.     

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.     

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.     

The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue.

DNA methylation of cytosine residues is a widespread phenomenon and has been implicated in a number of biological processes in both prokaryotes and eukaryotes. This methylation occurs at the 5-position of cytosine and is catalyzed by a distinct family of conserved enzymes, the cytosine-5 methyltransferases (m5C-MTases). We have cloned a fission yeast gene pmt1+ (pombe methyltransferase) which encodes a protein that shares significant homology with both prokaryotic and eukaryotic m5C-MTases. All 10 conserved domains found in these enzymes are present in the pmt1 protein. This is the first m5C-MTase homologue cloned from a fungal species. Its presence is surprising, given the inability to detect DNA methylation in yeasts. Haploid cells lacking the pmt1+ gene are viable, indicating that pmt1+ is not an essential gene. Purified, bacterially produced pmt1 protein does not possess obvious methyltransferase activity in vitro. Thus the biological significance of the m5C-MTase homologue in fission yeast is currently unclear.     

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis

Background

DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known.

Results

We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants.

Conclusion

We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.     

Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.     

The cytosine N4-methyltransferase M.PvuII also modifies adenine residues

Methylation of DNA occurs at the C5 and N4 positions of cytosine and N6 of adenine. The chemistry of methylation is similar among methyltransferases specific for cytosine-N4 and adenine-N6. Moreover these enzymes have similar structures and active sites. Previously it has been demonstrated that the DNA-(adenine-N6)-methyltransferases M.EcoRV, M.EcoRI, E. coli dam and both domains of M.FokI also modify cytosine residues at the N4 position [Jeltsch et al., J. Biol. Chem. 274 (1999), 19538-19544]. Here we show that the cytosine-N4 methyltransferase M.PvuII, which modifies the second cytosine in CAGCTG sequences, also methylates adenine residues in CAGATG/CAGCTG substrates in which the target cytosine is replaced by adenine in one strand of the recognition sequence. Therefore, adenine-N6 and cytosine-N4 methyltransferases have overlapping target base specificities. These results demonstrate that the target base recognition by N-specific DNA methyltransferases is relaxed in many cases. Furthermore, it shows that the catalytic mechanisms of adenine-N6 and cytosine-N4 methyltransferases are very similar.     

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.     

Mechanism of CpG DNA Methyltransferases M.SssI and Dnmt3a Studied by DNA Containing 2-Aminopurine

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase–DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme–substrate complexes were observed.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

Expression of exogenous DNA methyltransferases: Application in molecular and cell biology

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.     

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.     

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.