(A) Genome-wide changes in DNA methylation during HSC commitment. Red dots represent loci with significantly lower methylation at the developmentally later stage, i.e., loci demethylated during the respective transition (P < 0.05, t test). (B) Significant changes in DNA cytosine methylation at the transition from LTHSC to STHSC (outer circle), STHSC to CMP (middle circle), and CMP to MEP (inner circle) are plotted in relation to the genomic position. Chromosomes are plotted along the ideogram. Red bars denote significantly demethylated loci; green bars denote significant increase in methylation at the respective loci. (C) SAM was used to define an epigenetic signature based on loci that undergo the most significant methylation changes during HSPC differentiation. The epigenetic signature (561 loci) distinguishes HSPC subsets in hierarchical clustering analysis. Log₂-transformed HpaII/MspI ratios (color code next to the heat map) of all 561 loci are shown for the 4 analyzed differentiation stages (indicated above the heat map) from 3 healthy human individuals. Trees result from Euclidean clustering of this signature. Associated genes are listed in Supplemental Table 4. (D) Ingenuity pathway analysis highlights functional implications of gene enrichment analysis of the epigenetic stem cell commitment-associated signature. 62 genes significantly associated (P < 0.05 after Benjamini-Hochberg correction) with Ingenuity “Top bio functions” were entered into pathway generation. Top 5 “Canonical pathways” (“AML signaling,” “Molecular mechanisms of cancer,” “Glioblastoma multiforme signaling,” “Pancreatic adenocarcinoma signaling,” “Glucocorticoid receptor signaling”) and the top 3 characteristics of “Function and disease” (“Differentiation of blood cells” [P = 8.39 × 10^–43], “Lymphohematopoietic cancer” [P = 3.2 × 10^–12], “AML” [P = 2.47 × 10^–6], and “Cell transformation” [P = 1.41 × 10^–12]) are depicted.