DNA methylation is an epigenetic barrier to enhancer hijacking. (A) Experimental strategy for 5-azaD–mediated DNA demethylation. (B) Expression of TLX3 mRNA following 5-azaD treatment in Jurkat^WT and Jurkat^SV cells normalized to GAPDH. Results are mean ± SD (N = 3 biological replicates) and analyzed by two-way ANOVA with Dunnett multiple comparison test. Fold changes and P values represent comparisons between dimethyl sulfoxide (DMSO) and 5-azaD treatment in Jurkat^SV cells. ∗P < .05, ∗∗P < .01. (C) Volcano plot of gene expression changes upon 5-azaD treatment in Jurkat^WT and Jurkat^SV cells (N = 3 biological replicates). Red dots indicate genes displaying log₂ (fold change) ≥1.5 and an adjusted P value <1 × 10⁻⁶. (D) CpG methylation changes at the TLX3 promoter upon 5-azaD treatment in Jurkat^WT and Jurkat^SV cells. Average methylation levels were derived from 2 independent biological replicates. Differential methylation analysis was performed by the Fisher exact test. (E) Scatterplot of global methylation changes for all gene promoters in Jurkat^WT and Jurkat^SV cells and analyzed by Pearson correlation. Results are averaged methylation differences between 5-azaD and DMSO treatment from 2 replicate RRBS experiments. (F) Boxplots showing the mRNA expression changes of SV-associated genes (N) in SV-containing sample (yes) vs all other samples that do not contain the corresponding SVs (No) for 5 patients with CMML. The overview of treatment and experimental scheme is shown on the left. Boxes show median of the data and quartiles, and whiskers extend up to 1.5× of the interquartile range. P values were calculated by a one-sided paired t test. (G) Model for the cooperation between 3D genome organization and the epigenetic state of target genes as the molecular determinant of enhancer hijacking–mediated oncogenic transcription.