Methylation is more variable in enhancer elements and DMSs are enriched in differentiation-induced neutrophil enhancers. (A) Box plot demonstrating the β-value variation of CpGs investigated with the 450K methylation array. All sites present on the 450K platform (filtered for detection P < .01) were divided in groups based on location in CpG island, shore, shelf, or open sea. Each group was further divided based on location in enhancer regions (white) or outside enhancer regions (gray). The log₁₀ of the standard deviations of probe β-values for all replicates were plotted. P values (Student t test) for comparison between enhancer and not enhancer for each group are indicated between boxes. E, enhancer; noE, not enhancer; SD, standard deviation. (B-D) Bar graphs displaying the relative distribution of 450K methylation array probes/DMSs in enhancers identified by bidirectional transcription in CAGE analysis. Overlapping 450K array probes, ie, percentages of 450K probes located in enhancers, are shown in dark gray (total 477 112 probes) and percentages of DMSs located in enhancers are shown in light gray (total 10 156 probes). Absolute numbers are given at the top of each bar. P values were calculated by Fisher’s exact test. (B) Overlap in all enhancers identified as being active (expressed) in neutrophils. (C) Comparing enhancer data for CD34⁺ cells vs neutrophils. The graph displays the relative number of overlapping probes in enhancers specifically expressed/up-regulated in (left) CD34⁺ cells or in enhancers specifically expressed/up-regulated in neutrophils. (D) Same as in C, but for the CD133⁺ vs neutrophils comparison. (E) Expression (ie, bidirectional transcription) changes of enhancers with decreased methylation in neutrophils compared with progenitor cells. A total of 294 CAGE-derived enhancers active in CD133⁺ cells, CD34⁺ cells or neutrophils overlapped with DMSs showing decreases in β-value during differentiation. The expression of these enhancers was measured by CAGE. Box plot displays log₂ fold change between neutrophils and (left) CD133⁺ cells or (right) neutrophils and CD34⁺ cells. P values were 1.16 × 10⁻³⁵ and 4.24 × 10⁻⁴¹, respectively (1-sided Wilcoxon signed-rank test). (F) Expression changes of genes associated with differentially methylated enhancers. A total of 105 of the enhancers in E were associated with genes. Box plot shows expression (log₂ fold change) of genes associated with enhancers with DMSs that decrease during differentiation in PMN cells compared with CMP, GMP, and PMC cells. P values were 1.031 × 10⁻¹⁰, 1.694 × 10⁻¹⁰, and 2.17 × 10⁻⁹ using the 1-sided Wilcoxon signed-rank test based on the average of replicates. Expression was measured by microarray analysis of 5 biological replicate samples. Genes were required to have 3 of 5 replicates with a detection P < .01 in ≥1 cell type.