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

Since 1986 till now, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to lost DNA methylation enzymes from its genomes. Here 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 (d3-5-mdC and d3-5-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 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether the DNA methylation/hydroxymethylation exists in C. elegans. Our work 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, while another epigenetic modification 5-hmdC is not detectable. Furthermore, we found that C. elegans DNA was hypomethylated or hypermethylated 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 active DNA methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. Our work highlights the importance that the evolutionary conservation of DNA methylation machinery in the nemat...

Source: BJ Gene - October 9, 2014 Category: Biochemistry Authors: C Hu, J Chen, Y Hsu, C Yen, M Chao Tags: BJ Biomolecules Source Type: research