Epigenetic regulation of CXXC5. (A) Schematic figure of methylation analysis by pyrosequencing within the CXXC5 gene and median methylation values (with range) for AML samples (n = 46) and healthy controls (normal BM CD34⁺ cells, n = 6). CDS, coding region; CpG, CpG island; E1-3, 3 CXXC5 exons. Four different regions in the CXXC5 promoter (#1-3) and intron 1 (#4) were analyzed: (#1) −1343 to −1213 (2 CpGs); (#2) −914 to −741 (3 CpGs); (#3) −749 to −593 (5 CpGs); (#4) +1955 to +2179 (4 CpGs). The previous study of Treppendahl et al¹⁷  investigated a site further upstream in the CXXC5 promoter (−20 440 to −20 330, personal communication Marianne Treppendahl, Department of Hematology, Rigshospitalet Copenhagen, Denmark). Methylation of region #2 was significantly higher in AML than in healthy controls (P = .04); methylation of region #3 was significantly lower (P = .001). No difference between AML and controls was observed in the other 2 regions. P values were calculated by the Mann-Whitney U test. (B) Upregulation of CXXC5 after demethylation of region #2. U937 and NB4 cells were treated with 5-Aza-2′-deoxycytidine (DAC) for 96 hours and CXXC5 expression was determined by RT-qPCR. Values are normalized to dimethyl sulfoxide control. Mean values of 2 independent experiments with standard deviations are shown (*P ≤ .05 by the Mann-Whitney U test). Demethylation of region #2 was confirmed by pyrosequencing and mean methylation percentages of 2 independent experiments are shown.