Resumen:
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-20-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-20-deoxycytidine without significantly
influencing 5mC levels.