Abstract

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.

Highlights

  • Let us know how access to this document benefits you Follow this and additional works at: https://jdc.jefferson.edu/dcbfp Part of the Medical Molecular Biology Commons

  • We characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines

  • 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

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Summary

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Let us know how access to this document benefits you Follow this and additional works at: https://jdc.jefferson.edu/dcbfp Part of the Medical Molecular Biology Commons. Recommended Citation Vető, Borbála; Szabó, Pál; Bacquet, Caroline; Apró, Anna; Hathy, Edit; Kiss, Judit; Réthelyi, János M.; Szeri, Flóra; Szüts, Dávid; and Arányi, Tamás, "Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells." (2018). 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. Abbreviations 5azadC, 5-aza-20-deoxycytidine; 5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; DNMT, DNA methyltransferase; IDH, isocitrate dehydrogenase; LC-MS/MS, liquid chromatography coupled with mass spectrometry; TET, Ten-eleven translocation. We have recently developed an alternative approach based on the liberation of nucleobases by formic acid to quantify genomic 5mC content Using this method, we have previously reported the highly dynamic nature of DNA methylation [22]. Cotreatment with decitabine and ascorbate allows a decitabine-dependent increase in 5hmC levels in HeLa cells

Cell culture
Drug treatments
Animals and animal studies
Primary mouse hepatocyte culture
MS measurement
Results and Discussion
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