DNA methylation is stable during replication and cell cycle arrest

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.

Figure 1. Sorting of fibroblasts based on Ki-67 expression and DNA content.

(A) Live cells were selected using forward scatter and side scatter. (B) Single cells were selected using pulse width. (C) Cells from actively proliferating culture were sorted into “G0”: 2N DNA and Ki67 negative, “G1”: 2N DNA and Ki67 positive, “G2/M”:4N DNA and Ki67 positive. (D) Cells from culture arrested by contact inhibition for one week were predominantly “G0”.

Figure 2. Global methylation is consistent after replication and cell cycle arrest.

(A) Mean methylation for each cell cycle phase from each donor profiled. (B) Distribution of high-frequency smoothed methylation values from CpGs with sufficient coverage from whole genome bisulfite sequencing (WGBS) for each donor profiled. (C) Distribution of high-frequency smoothed methylation values from all CpGs analyzed for each donor and cell cycle phase analyzed. Outliers have been removed for plotting. (D) Distribution of mean high-frequency smoothed methylation values across all donors from CpGs within and outside the regions previously identified as hypomethylated blocks in colon cancer for each phase profiled. Outliers have been removed for plotting.

Figure 3. Global methylation is highly correlated between cell cycle phases.

Shown is hexagonal binning of mean, high-frequency smoothed methylation values per CpG for all samples in G0 versus all samples in extended G0, all samples in G1 versus all samples in G0, all samples in G2 versus all samples in G1, all samples in extended G0 versus all samples in G2, for all samples from donor FB versus all samples from donor FA, for all samples from donor FC versus all samples from donor FB. The last two comparisons gives a measure of biological variability. For clarity, bins containing less than 20 CpGs are not shown.