DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.
Epigenetic phenomena have been widely characterized in the genomes of vertebrates and DNA methylation is a key mechanism of epigenetic regulation. The DNA methylation systems of invertebrates and vertebrates show several notable differences. However, the evolutionary implications of those differences only recently began to be revealed. Our study investigated the recurrence of sex-specific methylation, as previously described for the species Drosophila willistoni, in other species of the Sophophora subgenus that present close evolutionary relationship. The MSRE and Southern blot techniques were used to analyze rDNA of some species of the willistoni, melanogaster, saltans and obscura groups of Drosophila and the results suggested that differential DNA methylation between sexes only occurs in Drosophila tropicalis and D. insularis, two sibling species of the willistoni subgroup. However, only using the MSRE technique we could detect sex-specific patterns of DNA methylation in all species of willistoni subgroup. These results indicate that DNA methylation may present important differences, even between closely related species, shedding new light on this Neotropical species complex.
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.
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