Figure 2
Dynamics of DNA methylation and its relationship with expression changes. (A) Heatmap showing expression levels on 0, 5, and 20 days for genes displaying significant methylation and expression changes (4,753 in total with FC ≥2 or FC ≤0.5; P ≤0.01 and FDR ≤0.05) (Additional file 7). (B) catterplots showing the relationship between the log2-transformed FC in expression and the log2-transformed FC in DNA methylation. Sixty-two percent of the hypomethylated genes are overexpressed (in blue); 55% of the hypermethylated genes are repressed (in red). (C) Correlation between methylation and expression data (slope from the linear regression between DNA methylation differences versus expression differences) for all differentially methylated genes organized by genomic location (first exon, TSS, 5′UTR, gene body, 3′UTR). (D) DNA methylation and expression dynamics of selected loci during monocyte-to-osteoclast differentiation. Methylation percentage determined by bisulfite pyrosequencing. Quantitative RT-PCR data relative to RPL38. DNA methylation and expression data are represented with a black line and a red line, respectively. (E) BrdU assay showing the percentage of replicating cells at different times. From days 1 to 4, only 9.46% of cells divide. (F) Effects of 5azadC treatment (50 nM, 500 nM) on osteoclastogenesis monitoring ACP5, CTSK, and CX3CR1 levels and TRAP staining over time. (G) Workflow for testing the presence of 5 hydroxymethylcytosine in hypomethylated genes. DNA was treated with a 5hmC-specific glucosyltransferase. Cytosines bearing a 5-hydroxymethyl are protected against MspI digestion, and the surrounding region can be amplified by qPCR. When no 5hmC is present, glucose is not transferred to C, DNA is cleaved at CCGG sites, and there is less qPCR amplification. Several controls are used to set the 0% and 100% content of 5hmC. (H) 5hmC content in several of the CpGs that are rapidly demethylated after RANKL and M-CSF stimulation of OC precursors.