title: The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts.
abstract: The methylation of cytosine at CpG dinucleotides (5 meC) 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 5 meC 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 5 meC 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 5 meC at 24-48 h after treatment. There was a marked trypsin-sensitive pool of 5 meC in these cells which was significantly increased after DNA damage. The increased levels of 5 meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-β staining. The relative amount of masked 5 meC 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 5 meC, with a significant fraction of 5 meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5 meC within the nuclei of fibroblasts in response to DNA damage.
authors.affiliation: Human Reproduction Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney 2065, Australia. Electronic address: selcencelik83@yahoo.com.
authors.forename: Selcen
authors.initials: S
authors.lastname: Çelik
authors.affiliation: Human Reproduction Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney 2065, Australia.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Human Reproduction Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney 2065, Australia.
authors.forename: Chris
authors.initials: C
authors.lastname: O'Neill
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D019175:DNA Methylation; D004317:Doxorubicin / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D006570:Heterochromatin / Q000235:genetics*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D014466:Ultraviolet Rays
chemical_list: D000903:Antibiotics, Antineoplastic; D006570:Heterochromatin; D004317:Doxorubicin; D003596:Cytosine
keywords: Antigen retrieval; Cytosine methylation; DNA damage; DNA repair; Epigenetics; Fibroblast; Genotoxic stress; Heterochromatin; Mouse; methyl-binding protein-1; trypsin; İmmune-staining