Evolution of DNMT2 in drosophilids: Evidence for positive and purifying selection and insights into new protein (pathways) interactions.

The DNA methyltransferase 2 (DNMT2) protein is the most conserved member of the DNA methyltransferase family. Nevertheless, its substrate specificity is still controversial and elusive. The genomic role and determinants of DNA methylation are poorly understood in invertebrates, and several mechanisms and associations are suggested. In Drosophila, the only known DNMT gene is Dnmt2. Here we present our findings from a wide search for Dnmt2 homologs in 68 species of Drosophilidae. We investigated its molecular evolution, and in our phylogenetic analyses the main clades of Drosophilidae species were recovered. We tested whether the Dnmt2 has evolved neutrally or under positive selection along the subgenera Drosophila and Sophophora and investigated positive selection in relation to several physicochemical properties. Despite of a major selective constraint on Dnmt2, we detected six sites under positive selection. Regarding the DNMT2 protein, 12 sites under positive-destabilizing selection were found, which suggests a selection that favors structural and functional shifts in the protein. The search for new potential protein partners with DNMT2 revealed 15 proteins with high evolutionary rate covariation (ERC), indicating a plurality of DNMT2 functions in different pathways. These events might represent signs of molecular adaptation, with molecular peculiarities arising from the diversity of evolutionary histories experienced by drosophilids.

Combining morphology and molecular data to improve Drosophila paulistorum (Diptera, Drosophilidae) taxonomic status.

The willistoni species subgroup has been the subject of several studies since the latter half of the past century and is considered a Neotropical model for evolutionary studies, given the many levels of reproductive isolation and different evolutionary stages occurring within them. Here we present for the first time a phylogenetic reconstruction combining morphological characters and molecular data obtained from 8 gene fragments (COI, COII, Cytb, Adh, Ddc, Hb, kl-3 and per). Some relationships were incongruent when comparing morphological and molecular data. Also, morphological data presented some unresolved polytomies, which could reflect the very recent divergence of the subgroup. The total evidence phylogenetic reconstruction presented well-supported relationships and summarized the results of all analyses. The diversification of the willistoni subgroup began about 7.3 Ma with the split of D. insularis while D.paulistorum complex has a much more recent diversification history, which began about 2.1 Ma and apparently has not completed the speciation process, since the average time to sister species separation is one million years, and some entities of the D. paulistorum complex diverge between 0.3 and 1 Ma. Based on the obtained data, we propose the categorization of the former "semispecies" of D. paulistorum as a subspecies and describe the subspecies D. paulistorum amazonian, D. paulistorum andeanbrazilian, D. paulistorum centroamerican, D. paulistorum interior, D. paulistorum orinocan and D. paulistorum transitional.

Evolution of DNMT2 in drosophilids: Evidence for positive and purifying selection and insights into new protein (pathways) interactions

Abstract The DNA methyltransferase 2 (DNMT2) protein is the most conserved member of the DNA methyltransferase family. Nevertheless, its substrate specificity is still controversial and elusive. The genomic role and determinants of DNA methylation are poorly understood in invertebrates, and several mechanisms and associations are suggested. In Drosophila, the only known DNMT gene is Dnmt2. Here we present our findings from a wide search for Dnmt2 homologs in 68 species of Drosophilidae. We investigated its molecular evolution, and in our phylogenetic analyses the main clades of Drosophilidae species were recovered. We tested whether the Dnmt2 has evolved neutrally or under positive selection along the subgenera Drosophila and Sophophora and investigated positive selection in relation to several physicochemical properties. Despite of a major selective constraint on Dnmt2, we detected six sites under positive selection. Regarding the DNMT2 protein, 12 sites under positive-destabilizing selection were found, which suggests a selection that favors structural and functional shifts in the protein. The search for new potential protein partners with DNMT2 revealed 15 proteins with high evolutionary rate covariation (ERC), indicating a plurality of DNMT2 functions in different pathways. These events might represent signs of molecular adaptation, with molecular peculiarities arising from the diversity of evolutionary histories experienced by drosophilids.

Linking epigenetic function to electrostatics: The DNMT2 structural model example.

The amino acid sequence of DNMT2 is very similar to the catalytic domains of bacterial and eukaryotic proteins. However, there is great variability in the region of recognition of the target sequence. While bacterial DNMT2 acts as a DNA methyltransferase, previous studies have indicated low DNA methylation activity in eukaryotic DNMT2, with preference by tRNA methylation. Drosophilids are known as DNMT2-only species and the DNA methylation phenomenon is a not elucidated case yet, as well as the ontogenetic and physiologic importance of DNMT2 for this species group. In addition, more recently study showed that methylation in the genome in Drosophila melanogaster is independent in relation to DNMT2. Despite these findings, Drosophilidae family has more than 4,200 species with great ecological diversity and historical evolution, thus we, therefore, aimed to examine the drosophilids DNMT2 in order to verify its conservation at the physicochemical and structural levels in a functional context. We examined the twenty-six DNMT2 models generated by molecular modelling and five crystallographic structures deposited in the Protein Data Bank (PDB) using different approaches. Our results showed that despite sequence and structural similarity between species close related, we found outstanding differences when they are analyzed in the context of surface distribution of electrostatic properties. The differences found in the electrostatic potentials may be linked with different affinities and processivity of DNMT2 for its different substrates (DNA, RNA or tRNA) and even for interactions with other proteins involved in the epigenetic mechanisms.