EVI1 binding sites are overrepresented in the hypermethylated promoter regions of EVI1 AMLs and EVI1 binds these hypermethylated promoters in vivo. (A) Motif analysis of the in EVI1 AML differentially methylated genes showed a significant overrepresentation (yellow in the heatmap color key) of three 7-bp motifs. Per bar, each 7-bp optimized motif is shown. The underlined sequences overlap with the first or second EVI1-binding domain. The number of genes that harbored the representative motifs in their promoter sequences is depicted per bar. (B) Further analysis of the hypermethylated and hypomethylated genes reveals 2 overrepresented motifs in the promoter regions of the hypermethylated genes and 1 overrepresented in hypomethylated genes. (C) Schematic representation of the EVI1 nuclear zinc-finger protein, with the binding sequence of the first and second EVI1 zinc finger domains. The overlapping motifs overrepresented in hypermethylated genes are underlined in, respectively, blue and green. (D) Quantitative PCR of ChIP in the EVI1-positive (EVI1+) SB1960CB cell line and the EVI1-negative (EVI1-) MOLM13 cell line using EVI1 and IgG antibody. Percentage of amount of input material is shown. The mean and SD of 3 independent experiments is shown.