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Research article

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

Chiung-Wen Hu, Jian-Lian Chen, Yu-Wen Hsu, Cheng-Chieh Yen, Mu-Rong Chao
Biochemical Journal Jan 01, 2015, 465 (1) 39-47; DOI: 10.1042/BJ20140844
Chiung-Wen Hu
Department of Public Health, Chung Shan Medical University, Taichung 402, TaiwanDepartment of Family and Community Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan
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Jian-Lian Chen
School of Pharmacy, China Medical University, Taichung 404, Taiwan
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Yu-Wen Hsu
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan
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Cheng-Chieh Yen
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan
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Mu-Rong Chao
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan
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  • For correspondence: chaomurong@gmail.commrchao@csmu.edu.tw
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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.

  • cadmium
  • Caenorhabditis elegans
  • decitabine
  • 5-hydroxymethyl-2′-deoxycytidine
  • 5-methyl-2′-deoxycytidine
  • liquid chromatography–tandem mass spectrometry

Abbreviations: AA, ammonium acetate; CV, coefficient of variation; DNMT, DNA methyltransferase; 5-hmdC, 5-hydroxymethyl-2′-deoxycytidine; 5-mdC, 5-methyl-2′-deoxycytidine; LOD, limit of detection; LOQ, limit of quantification; NGM, nematode growth medium; SPE, solid-phase extraction; TET, ten-to-eleven translocation; TIS, TurboIonSpray

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January 2015

Volume: 465 Issue: 1

Biochemical Journal: 465 (1)
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Trace analysis of methylated and hydroxymethylated cytosines in DNA by isotope-dilution LC–MS/MS: first evidence of DNA methylation in Caenorhabditis elegans
Chiung-Wen Hu, Jian-Lian Chen, Yu-Wen Hsu, Cheng-Chieh Yen, Mu-Rong Chao
Biochemical Journal Jan 2015, 465 (1) 39-47; DOI: 10.1042/BJ20140844
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Trace analysis of methylated and hydroxymethylated cytosines in DNA by isotope-dilution LC–MS/MS: first evidence of DNA methylation in Caenorhabditis elegans
Chiung-Wen Hu, Jian-Lian Chen, Yu-Wen Hsu, Cheng-Chieh Yen, Mu-Rong Chao
Biochemical Journal Jan 2015, 465 (1) 39-47; DOI: 10.1042/BJ20140844

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Keywords

cadmium
Caenorhabditis elegans
decitabine
5-hydroxymethyl-2′-deoxycytidine
5-methyl-2′-deoxycytidine
liquid chromatography–tandem mass spectrometry

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