Epigenetic modulatory genes influence lineage-specific expression profiles. (A) Intersection between identified hits of clonal mutations (variant allelic frequency [VAF] >30%), differentially expressed genes and alternatively spliced, differentially used exon-exon junctions (adj. P < .01) in lineage-switched myeloid relapse/myeloid fraction of MPALs, present in the analyzed cohort. (B) Fold change in expression of NuRD complex members (CHD4, MTA1, RBBP4, and MBD3) and PHF3, after lineage-switched relapse (left) and in MPAL cases (right). (C) CHD4 structure; the R1068H mutation (red) is located in the critical helicase domain of CHD4 at a highly conserved residue. ∗Number of positions that have a single, fully conserved residue; colon, conservation between groups of strongly similar properties, scoring >.5 in the Gonnet PAM 250 matrix; period, conservation between groups of weakly similar properties, scoring ≤0.5 in the Gonnet PAM 250 matrix. (D) Identification of regions of differential chromatin accessibility before and after knockdown of CHD4 and PHF3 depicted in red in MLLr SEM cells (left) and non-MLLr 697 cells (right). For all reads, the fold change in ATAC-peak height was calculated relative to the control (shNTC) and ATAC peaks from knockdown cells were plotted according to their fold change vs the control signals. CHD4 ChIP density plots from SEM cells (depicted in blue) were plotted along with the corresponding DNA regions of the shNTC control. Differentially expressed genes associated with changing ATAC peaks (log₂FC analyzed vs shNTC) identified in each cellular variant are represented by heat maps included at the right side of each gene (for SEM and 697 cells). (E) UCSC genome browser screenshots representing differential chromatin accessibility (ATAC-seq) and gene expression level (RNA-seq) in the myeloid CEBPA and the lymphoid RAG2 loci after CHD4 and PHF3 knockdown in MLLr SEM cells and non-MLLr 697 cells. ChIP-seq traces representing normal CHD4 occupancy in non-MLLr B-ALL (REH cells), MLLr B-ALL (SEM cells) and MLLr AML cells (MV-4;11) are shown as a reference at the bottom of each screenshot. TSS, transcriptional start site is depicted for each gene. (F) Expression of the lineage-specific cell surface markers CD19 (lymphoid) and CD33 (myeloid) after culture of MLL/Af4-transformed hCD34⁺ cord blood progenitor cells in lymphoid-permissive conditions. Knockdown of PHF3, CHD4, or a combination disrupts the dominant lymphoid differentiation pattern in nontargeting control (shNTC). (G) PHF3 knockdown is capable of influencing the surface marker expression after a longer incubation period (33 days). CHD4 knockdown impaired cellular survival upon longer in vitro culture (data not shown). ChIP, chromatin immunoprecipitation.