Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species

Abstract

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC-MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast.

Figure 1

(A) Quantification methods involving hydrolysis of nucleic acids (method A) are unable to distinguish whether modified cytosine is of RNA or DNA origin. Structural representation of RNA-derived 5-methyl-cytidine and DNA-derived 5-methyl-deoxycytidine. (B) Quantification of deoxycytidine and 5me-deoxycytidine specifically derived from DNA. Top: Chromatograms corresponding to deoxycytidine, as measured by LC-SRM in DNA samples obtained from A. thaliana, M. musculus, E. coli (K12 derivates DH5alpha and the methyltransferase mutant GM2929), D. melanogaster (w118), as well as two yeast species (S. cerevisiae BY4741 and S. pombe 972h-). The chromatographic peak represents deoxycytidine. Bottom: The chromatographic peak represents 5me-deoxycytidine; the illustrated chromatogram is normalized to the corresponding deoxycytidine signal as shown in the top panel. Inset bottom panel: (left) calibration curve for 5me-deoxycytidine resulting in a linear correlation spanning over 3 orders of magnitude; limit of detection is estimated at 250 attomol; (right) external calibration (increasing amount of standard in solvent) and standard addition (increasing amount of standard spiked into 100 ng DNA digest prepared from S. cerevisiae BY4741). The calibration curve for 5me-deoxycytidine at the lower concentration limit excludes significant matrix effects on quantification and LOD. (C) Methylation levels in genomic DNA purified from six different species. The number of 5me-deoxycytidine is expressed as percentage of unmodified deoxycytidine for each species (n = 3 from biological triplicates, error bars = ± SD); n.d. = not detected.