Functional analyses of DMRs. (a) Analysis of histone modifications across DMRs using ChIP. Chromatin was prepared at the indicated time points and precipitated against monomethyl histone H3 lysine 4 (H3K4me1), dimethyl histone H3 lysine 4 (H3K4me2) and trimethyl histone H3 lysine 4 (H3K4me3) as well as against acetylated histones H3 and H4 (AcH3 and AcH4). The IgG background level is indicated by the violet line. DNA enrichment of the indicated time points is normalized to 5% input DNA and shown relative to monocyte (0 h) enrichment. Data represent mean values ± standard deviation of at least three independent ChIP experiments. (b) Selected regions were cloned upstream of a basic EF1 promoter into the CpG-free luciferase vector pCpGL. The indicated plasmids were in vitro SssI-methylated (mCpG) or unmethylated (CpG) and transiently transfected into THP-1 cells. Luciferase activity was normalized against the activity of a co-transfected Renilla construct and mean values ± standard deviation (n = 3) are shown relative to the unmethylated pCpGL-EF1 construct. Asterisks indicate significant differences between methylated and unmethylated plasmids (*P < 0.05 and **P < 0.01, paired Student's t-test). RLU, relative light units.