Figure 4

TET2 is required for active DNA demethylation in human monocytes. (A-C) mRNA (left panels) and protein expression (right panels) of TET2, MBD4, or TDG in monocytes left untreated or transfected with the corresponding TET2-, MBD4-, TDG-siRNA, or control siRNA after 27 h and 42 h of differentiation culture. qRT-PCR results were normalized to HPRT1 expression (n≥4, values are mean ± SD, * P <0.05 Student's T-test, paired, two-sided). Protein levels of TET2, MBD4, or TDG were analysed using western blotting (results are representative of n=3 independent experiments). (D) MassARRAY analysis of bisulfite-converted DNA at five 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 MassARRAY. Asterisks mark the CpGs that are shown in (E). Red arrows mark TET2-siRNA treated samples that show a specific decrease in demethylation. (DNase1L3 methylation did not change during the first 42 h, but spectra were of low quality and are not shown.) Methylation ratios of single CpG units for individual donors are also provided in Table S4 in Additional File 4. (E) Bar charts for MassARRAY results of the indicated CpG residues of actively demethylated (CCL13, USP20) or control loci (MMP7, HOXB1). Values are mean ± SD (n≥4; * P <0.05, *** P <0.001 Student's T-test, paired, two-sided).