First evidence of methylation in the genome of Drosophila willistoni

Abstract

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.

Evolution of Gene Sequence in Response to Chromosomal Location

“…Chemical analysis then revealed the existence of a small amount of cytosine methylation for a short period during early development (Gowher et al 2000;Lyko et al 2000). The finding of methylated DNA and genes coding for putative DNA methyltransferases and methyl-DNA-binding proteins in other invertebrate species, including within the same Drosophila genus, supports the possibility of the existence of a methylated component of the genome of D. melanogaster (Garcia et al 2007;Schaefer and Lyko 2007). Methylated cytosine residues are known to be especially prone to mutation, undergoing spontaneous deamination to T (Shen et al 1994).…”

Sex-specific methylation in Drosophila: an investigation of the Sophophora subgenus

“…Contrasting with vertebrate cells, which steadily maintain their methylation patterns throughout development, DNA methylation in D. melanogaster seems to be a transient epigenetic signal observed in the early development stages (Hung et al 1999;Tweedie et al 1999;Lyko et al 2000;Lyko 2001;Kunert et al 2003). A different picture was revealed by Garcia et al (2007) in Drosophila willistoni. When the DNA of adult males and females of this species was cleaved with enzymes sensitive to methylation by MSRE, sex-specific patterns of fragments were observed.…”

“…Recent findings from Clark et al (2007), Schaeffer et al (2008), Bhutkar et al (2008) and Vicario et al (2007) point to the singularity of Drosophila willistoni, among the other 11 species that have their genomes completely sequenced, with respect to several characteristics. The methylation pattern found by Garcia et al (2007) in D. willistoni seems to provide yet another piece of evidence of this uniqueness.…”

“…When the DNA of adult males and females of this species was cleaved with enzymes sensitive to methylation by MSRE, sex-specific patterns of fragments were observed. The analysis of the sequences forming these bands revealed the presence of ribosomal genes (Garcia et al 2007). …”

“…This idea may be corroborated by comparing the dDnmt2 amino acid sequences of D. willistoni and of D. melanogaster, in which the sequence conservation common to these both species only occurs in the domain responsible for DNA methylation. The domain responsible for the recognition of the target sequence to be methylated is highly variable, indicating that substantial variations may occur even between species of the same genus (Garcia et al 2007). …”