Fig. 3: Functional assessment of methylation differences between CIMP and other leukemias.

All statistical analyses presented here have been performed using MCIP-seq data from CIMP (n = 13), AML (n = 50), T-ALL (n = 14), and healthy CD34+ cells (n = 3), unless otherwise specified. a Box plot showing methylation levels at different genomic features. The lower and upper edges of the boxplots represent the first and third quartiles, respectively; the horizontal line inside the box indicates the median. The whiskers extend to the most extreme values within the range comprised between the median and 1.5 times the interquartile range. The lines between boxes indicate the effect size as Cohen’s d, defined as the number of standard deviation units between groups (all comparisons were significant in a two-tailed Welch’s t test). Typically, d values below 0.2 are considered small, and above 0.8 are considered large⁹⁶. b Average methylation levels of different leukemias and healthy cells at putative gene promoters, defined as 4-kb regions surrounding the center of H3K4me3 ChIP-seq peaks in CD34+ HSPCs. Each line depicts a smoothed average (LOESS function) for a group of patients, with the shaded band indicating the 95% confidence interval. c Tornado plots depicting methylation (MCIP-seq) at putative HSPC promoters (H3K4me1 peaks), sorted by chromatin accessibility in HSPCs (DNase). The color code distinguishes different types of leukemia and HSPCs, and the intensity reflects the degree of methylation. The HSPC tracks in purple were downloaded from ENCODE⁷⁸ and show chromatin accessibility (DNase) as well as histone marks for enhancers (H3K4me1), promoters (H3K4me3), activation (H3K27ac) and repression (H3K27me3). GC density was downloaded from the UCSC browser¹⁵³. d Volcano plot of differentially methylated regions (DMRs) annotated with the closest genes in the linear genome. The statistical significance of the comparisons between these groups was determined by the Wald test (two-sided) in the DESeq2 package and corrected for multiple testing with the Benjamini–Hochberg procedure. Regions with false discovery rate (FDR) < 0.05 and log2 fold change >2 are highlighted; the numbers at the top indicate the number of differentially expressed genes for each comparison. e Summary plot of the top 5 most significant results of pre-ranked GSEA conducted on genes in the vicinity of DMRs between CIMP and AML. The C2 (top) and C5 (bottom) MSigDB collections were used in the analysis. f Genomic tracks of MCIP-seq data for a few selected samples of each leukemia (CIMP, T-ALL, AML) at promoters of hematopoietic genes with significant changes in methylation.