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 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

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

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

Additional file 4: Table S3. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Multivariate analyses (adjusting for maternal BMI, gestational age, and fetal sex): CpG methylation of candidate genes in GDM versus control FCB samples. (DOC 68 kb

Additional file 2: Figure S1. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Estimation of blood cell composition based on 450K methylation array profiles. Blue box plots show the distribution of cell types in GDM cord blood and red box plots in control samples. The median is represented by a horizontal line. The bottom of the box indicates the 25th percentile and the top the 75th percentile. Outliers are shown as circles. (DOC 146 kb

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

Additional file 1: Table S1. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Primers for pyrosequencing. (DOC 52 kb

Epigenetic dysregulation in the developing Down syndrome cortex

<p>Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3–11 times more hyper- than hypo-methylated sites. Reduced <i>NRSF/REST</i> expression due to upregulation of <i>DYRK1A</i> (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of <i>DNMT3L</i> (on chromosome 21q22.4) could lead to <i>de novo</i> methylation in neuroprogenitors, which then persists in the fetal DS brain where <i>DNMT3A</i> and <i>DNMT3B</i> become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (<i>PCDHG</i>) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of <i>PCDHG</i> subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased <i>PCDHG</i> expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of <i>PCDHG</i> and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions.</p