Figure 1.

High-throughput methylation comparison between monocytes (MOs) and derived osteoclasts (OCs). (A) Heatmap including the data for three paired samples of MOs (MO D1, D2, D3) and their derived OCs (OC D1, D2, D3) harvested on day 21. The heatmap includes all CpG-containing probes displaying significant methylation changes (8,028 in total with FC ≥2 or FC ≤0.5; P ≤0.01 and FDR ≤0.05) (Additional file 2). Scale shown at the bottom, whereby beta values (that is, the ratio of the methylated probe intensity to the overall intensity, where overall intensity is the sum of methylated and unmethylated probe intensities) ranging from 0 (unmethylated, blue) to 1 (completely methylated, red). (B) Scatterplot showing the mean methylation profile of three matching MO/OC pairs. Genes with significant differences (FC >2, FDR <0.05) in averaged results from the three pairs of samples are highlighted in red (hypermethylated) or blue (hypomethylated). (C) Distribution of differentially methylated CpGs among genomic regions (promoter, gene bodies, 3′UTR, and intergenic) in different subsets of CpGs (hypomethylated, hypermethylated). (D) Gene ontology enrichment analysis of hypomethylated and hypermethylated CpGs showing the most important categories. (E) Technical validation of the array data by bisulfite pyrosequencing of modified DNA. BS pyrosequencing of three representative hypomethylated genes (ACP5, CTSK, and TM7SF4) and one hypermethylated gene (CX3CR1) from the array data are shown. A representation showing the excellent correlation between array data (beta values) and pyrosequencing data (% methylation) including the data for the four genes (right panel). (F) Cluster analysis of contiguous differentially methylated regions (<500 bp). Two examples of regions with more than nine consecutive CpGs differentially methylated are shown. (G) Analysis of methylation levels in repetitive elements (Sat2, D4Z4, NBL2) and ribosomal RNA genes (18S and 28S regions) as obtained from bisulfite sequencing analysis.