Multiomic profiling of MECOM-depleted cells reveals a predominantly repressive role at target sites. (A) Schematic representation of experimental protocol for multiomic characterization of dTAG^V-1–treated MUTZ-3 MECOM-FKBP12^F36V cells. The CD34⁺, GFP⁺ MECOM-expressing population was preenriched via MACS before treatment with 500 nM dTAG^V-1 or DMSO. Cells were then harvested and processed for bulk RNA-seq, ATAC-seq, and PRO-seq to profile transcriptional and epigenetic changes. (B-C) Volcano plots representing changes in nascent gene expression assessed via PRO-seq in MUTZ-3 MECOM-FKBP12^F36V cells treated with dTAG^V-1 vs DMSO for 1 and 4 hours. n = 3 independent replicates (supplemental Table 4). (D) Volcano plot representing changes in gene expression assessed via bulk RNA-seq in MUTZ-3 MECOM-FKBP12^F36V cells treated with dTAG^V-1 vs DMSO for 6 hours. n = 3 independent replicates (supplemental Table 3). (E) Volcano plot representing changes in chromatin accessibility as assessed by ATAC-seq in MUTZ-3 MECOM-FKBP12^F36V cells treated with dTAG^V-1 vs DMSO for 6 hours. n = 3 independent replicates. Red data points represent chromatin peaks that are also bound by MECOM as assessed by MECOM-HA ChIP-seq. There are 837 of these sites that are schematically highlighted in the top right corner of the plot (supplemental Table 5). (F-G) Assessment of eRNA transcription levels at 837 MECOM-bound differentially accessible peaks measured from PRO-seq data. (F) Average PRO-seq read density across all MECOM-regulated cisREs with ±2000 bp on each side of the peak summit in dTAG^V-1–treated vs DMSO-treated samples. (G) Box plot showing average PRO-seq read density in aggregate for each MECOM-regulated cisRE ±500 bp on each side of the peak summit in dTAG^V-1–treated vs DMSO-treated samples. Two-sided Student t test was used for comparisons. n = 3 independent replicates. (H) Unbiased motif enrichment analysis of ATAC-seq differentially accessible peaks between dTAG^V-1–treated and DMSO-treated samples. (I) Venn diagram comparing gene expression and chromatin accessibility changes across sequencing modalities. Bulk RNA-seq DEGs from 6 hours and 24 hours dTAG^V-1 treatment, PRO-seq DEGS from 4 hours dTAG^V-1 treatment, and genes in proximity (within 1 MB) to at least 1 MECOM-bound, differentially accessible ATAC-seq peak were overlapped to yield a consensus MECOM gene network consisting of 122 genes. Cutoffs for bulk RNA-seq and PRO-seq were P < .05 (supplemental Tables 6 and 7). Peak-to-gene proximity was determined using the GREAT.³¹ (J) Schematic depiction of MECOM’s interaction with transcriptional corepressor CtBP2 via MECOM’s PLDLS motif. This protein-protein interaction can be inhibited by a genetically encoded 4x-PLDLS peptide inhibitor³² (top) or if MECOM’s PLDLS motif were mutated to PLASS (bottom). (K) H3K27ac and CtBP2 ChIP-seq analysis. Heat map (left) displays CtBP2 ChIP-seq signal at MECOM-regulated cisREs in MUTZ-3 cells expressing a 4x-PLDLS peptide inhibitor of the MECOM-CtBP2 interaction compared with cells expressing 4x-PLASS control.³² Heat map (right) showing H3K27ac ChIP-seq signal at MECOM-regulated cisREs in MUTZ-3 MECOM-FKBP12^F36V cells treated with 500 nM dTAG^V-1 or DMSO for 6 hours. (L-M) Experimental overview for lentiviral MECOM add-back rescue experiment. (L) MUTZ-3 MECOM-FKBP12^F36V cells were transduced with lentiviruses constitutively expressing either WT MECOM (EVI1 isoform) or MECOM PLDLS>PLASS along with a TagRFP transduction reporter at high MOI. (M) CD34 expression assessed by flow cytometry as a function of treatment duration (500 nM DMSO vs dTAG^V-1) (bottom). Histogram of CD34 expression at day 15 (top). Samples were transduced 48 hours before treatment. n = 3 independent technical replicates. Mean and standard deviation are shown, but many are hidden due to low variation between replicates. DEGs, differentially expressed genes; DAPs, differentially accessible peaks; eRNA, enhancer RNA; GFP, green fluorescent protein; MACS, magnetic-activated cell sorting; MB, megabase; MOI, multiplicity of infection; ns, not significant; nt, nucleotide; TF, transcription factor; WT, wild-type.