Fig. 2

Analysis of the differently methylated CpGs. A Volcano plot representing the differentially methylated CpGs (M-values) associated with the differentiation of MSCs to osteoblasts. Significant (adjusted p ≤ 0.05) changes in CpG methylation (red). B Heatmap and clustering of the methylation values (M-values) of 2 462 CpGs differently methylated for MSCs differentiated to osteoblast. yellow: hypomethylated; red: hypermethylated. C Distribution of CpGs according to their location in relation to CpG islands. The number of CpGs were normalised according to the total CpGs in the array and the total amount of significant CpGs in each group (hypo- and hyper-methylated). Green line represents the percentage of hypo-methylated CpGs. Red line represents the percentage of hyper-methylated CpGs. Boxes identify a significant relationship between methylation and location. Broken grey line represents the expected distribution of the CpGs. ** p < 0.01, **** p < 0.0001. D Distribution of the differentially methylated CpGs according to the different chromatin states. The bars represent the percentage of significantly hypo- (green) and hyper- (red) differentially methylated CpGs normalised according to the number of CpGs present in each state (on the array). E Analysis of the methylation profile of all the CpGs studied in the array that overlap different chromatin states. Violin plots representing the 15 different chromatin states with methylation level represented from 0 to 1. For the statistical analysis a cut off of 0.1% in the difference of methylation was applied. The insulator state (p = 2.78E−2) and the heterochromatin state (p = 6.33E−37) were more methylated in OB than in MSC. The TXF elongation (p = 9.82E−30), TXF transition (p = 1.31E−09), and weak enhancer (p = 2.45E−06) states were less methylated in OB than in MSC. *** p < 0.001