The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts

• Immune-detectable 5meC within two pools retrieved after acid and trypsin after acid.
• The size of the trypsin-sensitive pool varied in response to DNA damage.
• This was associated with changes in the level of HP1-β within heterochromatic foci.
• Epitope retrieval method detected remarkable dynamism in 5meC׳s level and location.

The methylation of cytosine at CpG dinucleotides (5meC) is an important epigenetic mechanism that governs genome stability and gene expression. Important ontological and pathological transitions are associated with marked global changes in detectable levels of methylation. We have previously found two pools of immune-detectable 5meC exist within cells, a pool that can be detected after acid treatment of fixed cells to denature chromatin and another large but variable pool that requires a further tryptic digestion step for complete epitope retrieval. The trypsin-sensitive pool has been shown to be largely associated with the heterochromatic fraction (by a heterochromatin marker, HP1-β) of the genome, and the size of this pool varies with the growth disposition of cells. Since DNA damage imposes large changes on chromatin structure the present study analyzed how such changes influences the faithful immunological detection of 5meC within mouse embryonic fibroblasts. DNA damage was induced by either UV-irradiation or doxorubicin treatment, each of which resulted in increased levels of immune-detectable 5meC at 24–48 h after treatment. There was a marked trypsin-sensitive pool of 5meC in these cells which was significantly increased after DNA damage. The increased levels of 5meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-β staining. The relative amount of masked 5meC after DNA damage was positively associated with increased levels of HP1-β. The methyl binding protein, MBD1, was a less reliable measure of changes in 5meC, with a significant fraction of 5meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5meC within the nuclei of fibroblasts in response to DNA damage.