Trace analysis of methylated and hydroxymethylated cytosines in DNA by isotope-dilution LC–MS/MS: first evidence of DNA methylation in Caenorhabditis elegans

Abstract

From 1986 to the present, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to have lost DNA methylation enzymes from its genomes. In the present study, we report the development of a sensitive and selective assay based on LC–MS/MS to simultaneously measure 5-methyl-2′-deoxycytidine (5-mdC) and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC) in DNA hydrolysates. With the use of isotope internal standards ([²H₃]5-mdC and [²H₃]5-hmdC) and online solid-phase extraction, the detection limits of 5-mdC and 5-hmdC were estimated to be 0.01 and 0.02 pg respectively, which correspond to a 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether DNA methylation/hydroxymethylation exists in C. elegans. The present study for the first time demonstrates that 5-mdC is present in C. elegans genomic DNA (0.0019–0.0033% of cytosine methylated) using LC–MS/MS, whereas another epigenetic modification, 5-hmdC, is not detectable. Furthermore, we found that C. elegans DNA was hypo- or hyper-methylated in a dose-dependent manner by the DNA methyltransferase (DNMT)-inhibiting drug decitabine (5-aza-2′-deoxycytidine) or cadmium respectively. Our data support the possible existence of an active DNA-methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. The present study highlights the importance of re-evaluating the evolutionary conservation of DNA-methylation machinery in nematodes which were traditionally considered to lack functional DNA methylation.