Figure 2.

5hmC deposition precedes active DNA demethylation in human monocytes. (A) Positions of regions (purple) measured by MALDI-TOF analysis of bisulfite converted DNA (MassARRAY, in B) and of primers (red) used for hMeDIP qPCR (in C) are shown relative to positions of CpG dinucleotides (green) and neighboring genes (blue). Tracks were generated using the UCSC Genome Browser. (B) MassARRAY analysis of bisulfite-converted DNA at four loci that show active DNA demethylation during monocyte to DC differentiation, as well as for two control regions (values are mean of n≥4). Data are presented as heatmaps. The methylation content (including both 5mC and 5hmC) is indicated by coloring (yellow: no methylation, dark blue: 100% methylation) with each box representing a single CpG dinucleotide and each row representing the succession of CpGs measured. Grey boxes indicate CpGs that were not detected by MALDI-TOF MS. Red asterisks mark the CpGs that are shown in (C). Methylation ratios of single CpG units for individual donors are also provided in Table S2 in Additional File 2. (C) Dynamics of DNA methylation (5mC + 5hmC) and hydroxymethylation (5hmC) during monocytic differentiation. DNA methylation levels of single CpGs as measured by MassARRAY (open squares) are compared with 5hmC enrichment (measured by hMeDIP, red squares) at the same loci shown in (B) (n≥4, values are mean + or - SD). Exact genomic positions of analyzed CpG residues are given in Table S3 in Additional File 3.