Fig. 3

DNA hydroxymethylation changes in MSCs of two different human age groups. a Box plots showing the Δß levels of both hyperhydroxymethylated (upper) and hypohydroxymethylated (lower) CpG sites for the young and older groups sampled. b Unsupervised hierarchical clustering and heatmap showing the Δß values for the 785 hyper5hmC and 846 hypo5hmC from all the samples. In order to cluster both sites and samples the Manhattan distance metric and Ward clustering method were used. Whenever possible, samples are arranged by age. A blue to yellow scale is used to represent the Δß values, from 0 (blue) to a maximum value of 0.43 (yellow). In order to enhance the visual representation, negative Δß values were set to 0 prior to clustering. c Two dimensional density plot showing the relationship between Δß values of youngest (two years old; 2-yo) and oldest (89 years old; 89-yo) MSCs. d Examples of two of the aging-specific d5hmCs, which were further validated by pyrosequencing in eleven samples. For each of the genes of interest, a box plot of the percentage of 5-hydroxymethylation of the CpG site of interest obtained for each sample is shown. The gene on the left (VWA2) shows an age-dependent hypohydroxymethylation tendency, while the gene on the right (KCNQ1) shows hyperhydroxymethylation with respect to age. Negative values were adjusted to 0. e Venn diagram showing the overlap of the number of differentially hyper- and hypohydroxymethylated CpG sites and differentially hyper- and hypo5methylated CpG sites