Open AccessInhibition of DNA methyltransferase leads to increased genomic 5‐hydroxymethylcytosine levels in hematopoietic cells
Author(s) -
Vető Borbála,
Szabó Pál,
Bacquet Caroline,
Apró Anna,
Hathy Edit,
Kiss Judit,
Réthelyi János M.,
Szeri Flóra,
Szüts Dávid,
Arányi Tamás
Publication year - 2018
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.12392
Subject(s) - 5 methylcytosine , 5 hydroxymethylcytosine , dna methylation , epigenetics , dna methyltransferase , methyltransferase , dna , microbiology and biotechnology , cell culture , haematopoiesis , gene silencing , chemistry , genomic dna , biology , methylation , biochemistry , gene expression , gene , genetics , stem cell
5‐Hydroxymethylcytosine (5hmC) is produced from 5‐methylcytosine (5mC) by Ten‐eleven translocation ( TET ) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC ‐ MS / MS ‐based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5‐aza‐2′‐deoxycytidine without significantly influencing 5mC levels.