Abstract
DNA methylation is an epigenetic modification with important functions in development. Large-scale loss of DNA methylation is a hallmark of cancer. Recent work has identified large genomic blocks of hypomethylation associated with cancer, EBV transformation and replicative senescence, all of which change the proportion of actively proliferating cells within the population measured. We asked if replication or cell-cycle arrest affects the global levels of methylation or leads to hypomethylated blocks as observed in other settings. We used fluorescence activated cell sorting to isolate primary dermal fibroblasts in G0, G1 and G2 based on DNA content and Ki67 staining. We additionally examined G0 cells arrested by contact inhibition for one week to determine the effects of extended arrest. We analyzed genome wide DNA methylation from sorted cells using whole genome bisulfite sequencing. This analysis demonstrated no global changes or large-scale hypomethylated blocks in any of the examined cell cycle phases, indicating that global levels of methylation are stable with replication and arrest.
Highlights
DNA methylation is an epigenetic modification with important functions in development
DNA methylation is an epigenetic modification that has important functions in mammalian development. It consists of addition of a 5’ methyl group to the cytosine base. This modification is most frequently found in the context of CpG dinucleotides, where it can be placed by three methyltransferase enzymes: DNMT1, the maintenance methyltransferase that re-establishes the methylation pattern following DNA replication, and DNMT3a and DNMT3b, which function in de novo methylation[1]
We sought to identify potential genome scale changes in DNA methylation associated with cell proliferation
Summary
DNA methylation is an epigenetic modification with important functions in development. We analyzed genome wide DNA methylation from sorted cells using whole genome bisulfite sequencing This analysis demonstrated no global changes or large-scale hypomethylated blocks in any of the examined cell cycle phases, indicating that global levels of methylation are stable with replication and arrest. A more recent study using flow cytometry to quantify 5-methylcytosine (5 mC) in two cancer cell lines found no differences in the ratio of 5 mC staining to DNA content between G1 and S phase, but noted a lag in early G2/M before the maximum levels of 5 mC were observed[16] While these studies provide valuable insights into the methylation of newly synthesized DNA, both studies focus on changes during DNA synthesis, and neither provides region specific data. We have previously reported the presence of large hypomethylated domains, encompassing up to two thirds of the genome, termed “hypomethylated blocks”, in colon cancer samples and associated www.nature.com/scientificreports/
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