Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation

<div><p>Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.</p></div

Global DNA methylation in genic and intergenic regions of irradiated versus non-irradiated fibroblast cultures.

<p>DNA methylation was assessed with Illumina 450K arrays in primary human fibroblasts at 1–24 h after irradiation with 2 Gy (upper panel) and at 6–72 h after 4 Gy (lower panel). The bars represent the average methylation of all analyzed CpGs that have been annotated to a particular category (promoter, 5' UTR, first exon, gene body, 3' UTR, intergenic). TSS200 is the region from transcription start site (TSS) to -200 bp, TSS1500 from -200 bp to -1,500 bp upstream of TSS. Data are presented as means over means.</p

Global DNA methylation and hydroxymethylation in irradiated versus non-irradiated fibroblast cultures.

<p>Global 5-mC and 5-hmC levels were measured by ELISA-based assays in primary human fibroblasts at 6 and 24 h after X-ray irradiation with 2 and 4 Gy, respectively. For each time point and dose, the number of analyzed cultures is given in parenthesis. Results are presented as mean (of different cultures) over means (triplicate measurements) ± standard error. Asterisk denotes a significant (p < 0.05) between-group difference.</p

DNA methylation of repetitive elements in irradiated versus non-irradiated fibroblast cultures.

<p>Global methylation of interspersed ALU and LINE-1 repeats, and α-satellite DNA was determined by bisulfite pyrosequencing in primary human fibroblasts at 6 and 24 h after irradiation with 2 and 4 Gray, respectively. For each time point and dose, the number of analyzed cultures is given in parenthesis. Results are presented as mean (of different cultures) over means (triplicate measurements) ± standard error. Asterisk denotes a significant (p < 0.05) between-group difference.</p

Box plots showing the distribution of repeat DNA methylation in single cells.

<p>DNA methylation of interspersed ALU and LINE-1 repeats, and α-satellite DNA was determined by bisulfite pyrosequencing in individual fibroblasts from two independent cultures at 24 h after irradiation with 2 Gy and 4 Gy, respectively. For each culture, time point, and dose, the number of analyzed cells is given in parenthesis. The median is represented by a horizontal line. The bottom of the box indicates the 25^th percentile, the top the 75^th percentile. Outliers are shown as circles and extreme outliers as stars.</p

Hypermethylation of the non-imprinted maternal <i>MEG3</i> and paternal <i>MEST</i> alleles is highly variable among normal individuals

<div><p>Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted <i>MEST</i> promoter and the paternally imprinted <i>MEG3</i> intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2–66%) of the paternal <i>MEST</i> and the maternal <i>MEG3</i> IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal <i>MEST</i> and paternal <i>MEG3</i> alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the <i>MEST</i> promoter and the <i>MEG3</i> IG DMR, and to a lesser extent, for the paternally imprinted secondary <i>MEG3</i> promoter and the maternally imprinted <i>PEG3</i> promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.</p></div

Mean FCB methylation and epimutation rates of maternal versus paternal alleles of the <i>MEG3</i> IG DMR, <i>MEG3</i> promoter, <i>MEST</i>, and <i>PEG3</i>.

<p>DBS was performed with the Illumina MiSeq. The non-imprinted allele of <i>MEG3</i> (both primary and secondary DMR) and <i>MEST</i> exhibited significantly higher epimutation rates than the imprinted allele.</p

Parental allele-specific methylation of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter.

<p>Mean methylation levels and standard deviations of the paternal vs. maternal alleles were determined by DBS with the Roche GSJunior in FCB, AB, and VAT. For both genes, the non-imprinted allele, which is expected to be completely unmethylated, showed an aberrantly high methylation in all analyzed tissues, whereas the imprinted allele showed the expected (90–100%) methylation.</p

Epimutation rates of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter in individual samples.

<p>The percentage of paternal (black dots) versus maternal alleles (gray dots) with >50% aberrantly (de)methylated CpGs was determined by DBS with the Roche GSJunior in individual FCB, AB, and VAT samples. With very few exceptions, the unmethylated alleles of the paternally imprinted <i>MEG3</i> and the maternally imprinted <i>MEST</i> genes displayed much higher epimutation rates than the methylated alleles.</p

Parental allele-specific epimutation rates of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter.

<p>The percentage of alleles with >50% aberrantly (de)methylated CpGs was demined by DBS with the Roche GSJunior in FCB, AB, and VAT samples. The unmethylated alleles of the paternally imprinted <i>MEG3</i> and the maternally imprinted <i>MEST</i> genes displayed significantly higher epimutation rates than the methylated alleles.</p