Quantification of Epigenetic DNA Modifications in the Subchromatin Structure Matrix Attachment Regions by Stable Isotope Dilution UHPLC-MS/MS Analysis.

To date, subchromatin structure-based quantification of epigenetic DNA modifications is limited. Matrix attachment regions (MARs), an important subchromatin structure, contain DNA elements that specifically bind chromatin to the nuclear matrix in eukaryotes and are involved in a number of diseases. Here, we exploited a high-salt extraction-based subchromatin fractionation approach for the isolation of MAR DNA and other fractions and further developed heavy stable isotope-diluted ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the specific quantification of epigenetic DNA modifications in the subchromatin structures. By this approach, we showed for the first time that the content of a DNA demethylation intermediate, 5-hydroxymethylcytosine (5hmdC), in MARs decreased significantly in four tested cell lines compared to the contents in genomic DNA. In particular, the content of DNA 5hmdC in the MARs of 293T cell lines decreased the most at approximately 41.09%. Together, our findings implicate that MAR DNA is less sensitive than genomic DNA to DNA demethylation.