DNA methylation is an important epigenetic process. 5-methylcytosine (5mC) is one of most extensively studied DNA modifications due to its widespread distribution. 5-hydroxymethylcytosine (5hmC) is an oxidization product of 5mC mediated by ten eleven translocation (TET) family dioxygenases and an intermediate for DNA demethylation. As the “sixth” DNA base, 5hmC participates in many physiological activities including gene expression, somatic cell reprogramming, embryonic development and tumourigenesis . To date, quantification of epigenetic DNA modifications at the levels of subchromatin structures is limited. Matrix Attachment Regions (MARs) contain the DNA elements that specifically bind chromatin to the nuclear matrix in eukaryotes and involve in diseases. MARs are associated with boundaries of chromatin domains, and have the functions of insulating gene domains, facilitating replication, and enhancing transcription .<br/>Here, we exploited a high-salt extraction-based subchromatin fractionation approach for the isolation of MAR DNA and other fractions and further developed an approach of heavy stable isotope-diluted ultra-high-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 5hmdC in MARs decreased significantly in four tested cell lines compared to the contents in genomic DNA. This new method expands the application of LC-MS analysis in biological research, which may be helpful to better understanding of the functions of subchromatin structures and may provide potential disease biomarkers through subchromatin DNA modifications analysis.

Quantification of epigenetic DNA modifications in the subchromatin structure matrix attachment regions by stable isotope dilution UHPLC-MS/MS analysis

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ACS Fall 2022

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