An intergenic AML1-ETO binding non-coding element is essential for PLCG1 expression. (A) Schematic model of the 500-bp intergenic element characterized by p300 and AML1-ETO binding sites (green) in Kasumi-1 cells. sgRNAs targeting this region are shown with arrows. (B) PLCG1 mRNA expression in Kasumi_Cas9-EGFP cells (left) and SKNO-1_Cas9-Blast cells (right) following CRISPR/Cas9-induced knockout of the 500-bp intergenic region using specific gRNAs or a nontargeting control (NT). n = 3 independent experiments, in duplicate; paired Student t test. (C) Colony-forming assay of Kasumi-1_Cas9_EGFP cells (left) and SKNO-1_Cas9-Blast cells (right) (day 14) following genetic inactivation of the 500-bp enhancer region using specific sgRNAs compared with nontargeting control (NT); n = 2 independent experiments. (D-F) mRNA expression of PLCG1 (normalized to Beta2-microglobulin) in Kasumi-1 and SKNO-1 cells after treatment with (D) JQ1 (1 μM, 24 hours), (E) dBET6 (1 μM, 24 hours), and (F) Lys-CoA (1 μM, 24 hours) compared with diluent control (DMSO). n = 4 independent experiments, in duplicate; paired t test. (G) mRNA expression of PLCG1 in Kasumi-1 cells after knockout of JUN using CRISPR/Cas9 (gJUN #1 and #2) or a nontargeting control (NT). n = 3 independent experiments, in triplicate; paired t test. (H) Screenshot displaying binding patterns of AML1-ETO, JUN, and CEBPa at the PLCG1 locus in Kasumi-1 cells based on ChIP-seq. All data following inactivation of AML1-ETO (siAE) compared with nontargeting control (siMM). Relevant peaks are highlighted with boxes.