The Exit of Mouse Embryonic Fibroblasts from the Cell-Cycle Changes the Nature of Solvent Exposure of the 5′-Methylcytosine Epitope within Chromatin

Abstract

The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.

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

“…Careful design and validation of immunodetection of DNA methylation is required to optimizeoptimise epitope retrieval in each system under analysis. Conditions for testing in each system include technical conditions, such as the antibody incubation time and temperature required to achieve equilibrium binding conditions [37], the form and extent of antigen retrieval procedures [14][15][16]38,39], and the nature of the blocking protocol used to minimize non-specific binding and maximize, the form and extent of antigen retrieval procedures , and the nature of the two proteins that regulate methylation activity, UHRF1 and GADD45A, are also recruited to sites of repair and can catalyse selective methylation of the promoter-distal segment of the damaged DNA [34]. DNMT1 accumulation occurs in UV-induced lesions [35] and was detected in micronuclei in doxorubicin-treated cells [36].…”

“…Conventionally, the detection of 5meC has relied upon denaturation of chromatin by brief acid treatment to cause solvent exposure of 5meC. We have recently shown, however, that this form of epitope retrieval leaves a large amount5meC masked from immunodetection [14][15][16]. This was most evident in the newly fertilized mouse embryo where the use of conventional immuno-staining methods showed a progressive loss of 5meC staining resulting in an appearance of almost complete demethylation by the time of the first cell division [17][18][19].…”

“…This T methodologyrevealed that there were only relatively minor changes in the levels of 5meC in the early embryo, and did not support claims of active global demethylation in the zygote. Analysis of mouse embryonic fibroblasts (MEFs) showed that these cells also had a degree oftrypsin-sensitive masking of 5meC [14]. While the near complete masking of 5meC seen in the zygote was not evidentevident in MEFs, there was still a large trypsinsensitive pool of 5meC.…”

“…This pool was predominantly associated with the heterochromatic fraction of the genome. Most notably, it was found that 5meC associated with heterochromatin varied with the growth disposition of cells [14]. For instance, proliferative cells displayed a relatively greater accumulation of 5meC within the trypsin-sensitive pool than quiescent cells, and this was in large part associated with changes in the heterochromatin fraction between these two growth states (14).…”

Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge

“…5A). The use of acid and trypsin enzyme resulted in increased antigenicity of both 5meC (Li and O'Neill, 2012;Li and O'Neill, 2013;Çelik et al, 2014) and MBD1 (Çelik et al, 2014) in embryos and embryonic fibroblasts of mouse but not of 5hmC (Li and O'Neill, 2013) in embryos (Fig. 5A).…”