A Dnmt2-like protein mediates DNA methylation inDrosophila

Abstract

The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation,we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.

Genome methylation in D. melanogaster is found at specific short motifs and is independent of DNMT2 activity

“…Consistent with previous reports of cytosine methylation in Drosophila (Lyko et al 2000a;Kunert et al 2003), we find that methylation occurs in CA-and CT-rich contexts. However our results show that, in the relatively highly methylated regions we have identified, methylation is found at specific 4-to 5-base sequence motifs that lack guanine (Fig.…”

“…The relative rarity of methylation in Drosophila, even at stage 5 where it has been shown to be most abundant (Lyko et al 2000a), has made it a subject of controversy. Some have recently suggested that it may be absent, or its presence is spurious and a reflection of nonspecific activity by MT2 (Marhold et al 2004;Schaefer and Lyko 2010a;Raddatz et al 2013); the near-normal phenotype of flies lacking functional MT2 has supported this point of view (Kunert et al 2003;Phalke et al 2009;Schaefer and Lyko 2010b). Although we find that methylation can be present on a substantial proportion of alleles within methylated regions, methylation is virtually never present on more than half of the alleles.…”

“…Drosophila was long thought to lack cytosine methylation, but identification of a Drosophila homolog of DNA methyltransferase 2 (MT2, also known as DNMT2) (Hung et al 1999;Lyko et al 2000b) prompted studies that established the presence of small amounts of genomic cytosine methylation in early embryogenesis (Gowher et al 2000;Lyko et al 2000a;Kunert et al 2003). Despite this, the pattern and function of cytosine methylation in Drosophila has never been clear, and recently considerable doubt has been expressed as to the presence of any significant quantity of genomic methylation (Goll et al 2006;Schaefer and Lyko 2010a;Zemach et al 2010;Raddatz et al 2013).…”

“…Despite this, the pattern and function of cytosine methylation in Drosophila has never been clear, and recently considerable doubt has been expressed as to the presence of any significant quantity of genomic methylation (Goll et al 2006;Schaefer and Lyko 2010a;Zemach et al 2010;Raddatz et al 2013). Inactivation of Mt2 does not visibly alter embryonic development (Kunert et al 2003), although it can suppress position-effect variegation (Phalke et al 2009). No clear pattern of genomic cytosine methylation has been established by any study, and genomewide bisulfite sequencing studies of wild type Drosophila embryos at an average depth of 5.6-fold (Zemach et al 2010) and 32-fold (Raddatz et al 2013) did not report methylated cytosines.…”

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae

“…Recent data indicate that histone H3 methylation may be a sign for Dnmts (Jackson et al, 2002;Kunert et al, 2003;Tamaru and Selker, 2001).…”