On the evolutionary origin of eukaryotic DNA methyltransferases and Dnmt2

Abstract

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.

Figure 1. Multiple sequence alignment of the conserved amino acid sequence motifs from representative sequences of prokaryotic and eukaryotic DNA- and RNA-(cytosine-C5)-methyltransferases.

The motif numbers are indicated on top of the sequence alignment. Note that the CFT motif is present only in Dnmt2.

Figure 2. Consensus phylogenetic tree of the eukaryotic DNA-(cytosine C5)-MTases and Dnmt2 proteins constructed from 100 generated bootstrap trees.

The bootstrap values of the branch points are indicated. Branch points with less than 30% incidence among the generated trees were collapsed representing that the phylogeny at this point cannot be reliably inferred.

Figure 3. Consensus phylogenetic tree of prokaryotic and eukaryotic DNA-(cytosine C5)-MTases and Dnmt2 proteins constructed from 100 generated bootstrap trees.

The bootstrap values of the branch points are indicated. Branch points with less than 30% incidence among the generated trees were collapsed representing that the phylogeny at this point cannot be reliably inferred.

Figure 4. Compilation of different topologies 100 alternative the phylogenetic trees of prokaryotic and eukaryotic DNA-(cytosine C5)-MTases and Dnmt2 proteins generated during the bootstrapping analysis of the tree shown in Fig. 3.

Figure 5. 3D clustering of methyltransferase sequences based on pairwise BLAST similarity scores prepared using CLANS .

A) Clustering of 2935 sequences of prokaryotic and eukaryotic DNA-(cytosine C5)-MTases and Dnmt2 proteins. B) Clustering of the sequences of prokaryotic and eukaryotic DNA-(cytosine C5)-MTases and Dnmt2 proteins including sequences of RNA-(cytosine C5)-MTases.

Figure 6. Distribution of the methyltransferases in different eukaryotic species.

The tree was prepared using NCBI taxonomy and Interactive Tree Of Life. Dnmts were categorized into the Dnmt1 (1, colored red), Dnmt3 (3, colored blue), Chromomethylase (C, colored orange) and Dim2 families (D, colored green). The Dnmt1 enzymes were subdivided into animal, plant and fungi subgroups, Dnmt3 enzymes into the canonical Dnmt3 enzymes and the plant DRM enzymes. Lineages that underwent loss of Dnmt2 are indicated by green crosses. Dnmt1 orthologues are found in all the branches of eukaryotes. Chromomethylases are related to Dnmt1 enzyme appearing in the plant lineage only (indicated by the orange arrow). Dnmt3 enzymes apparently have been lost in the fungal lineage (indicated by a blue cross).

Figure 7. Consensus model of the phylogeny of DNA methyltransferases and Dnmt2 indicating that Dnmt1 and Dnmt2/3 enzymes have an independent origin in the prokaryotic DNA methyltransferases.