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. 2022 Oct 20;13(1):146–169. doi: 10.1158/2159-8290.CD-22-0416

Figure 1.

Figure 1. CRISPR screens uncover the functional interplay between the mammalian MLL1 and MLL3/4 chromatin-modifying complexes. A, CRISPR-Cas9–based screening strategy to identify regulators of response to Menin-MLL inhibition. CPD, cell population doublings; sgRNA, single guide RNA; Tf, final time point; T0, initial time point. B, Chromatin-focused CRISPR screening data showing the top 20 most significantly enriched (red) and depleted (blue) genes in the Menin-MLL inhibitor (MI-503) treatment relative to vehicle (DMSO). Gene scores are shown as the mean log2 fold change in abundance of the 6 sgRNAs targeting each gene in each condition. C, Genome-wide CRISPR screening data showing gene-level ranking based on differential enrichment of sgRNAs in the Menin-MLL inhibitor treatment (VTP-50469) relative to vehicle (DMSO). Differential (Δ) beta-score between VTP-50469 and DMSO conditions was calculated using MaGeCK. A positive Δ beta-score denotes enrichment of specific gene-targeting sgRNAs. A negative Δ beta-score denotes depletion of specific gene-targeting sgRNAs. Red circles denote MLL3/4-UTX complex subunits. Yellow circles denote PRC1.1 complex subunits. D, Schematic representation of the top-scoring chromatin regulators in the chromatin-focused MI-503 screen and their corresponding protein complexes. Red denotes enriched subunits. Blue denotes depleted subunits. Color scale represents the log2 fold change in abundance of the 6 sgRNAs targeting each subunit in the Menin-MLL inhibitor (MI-503) treatment relative to vehicle (DMSO). E, Viability assay from cells treated with vehicle (DMSO, black) or Menin-MLL inhibitor (MI-503, red) for 96 hours (mean ± SEM, n = 3 infection replicates, P value calculated by Student t test). sgCtrl, control sgRNA targeting a nongenic region on chromosome 8. F, Relative cell proliferation is shown as the proliferation of double-positive cells (sgMen1-RFP + sgUtx-BFP or sgMen1-RFP + sgCtrl-BFP) relative to single-positive cells (sgMen1-RFP) 16 days after infection measured by flow cytometry (mean ± SEM, n = 3 infection replicates, P value calculated by Student t test). Representative FACS plots are shown for sgControl- and sgUtx-targeted cells.

CRISPR screens uncover the functional interplay between the mammalian MLL1 and MLL3/4 chromatin-modifying complexes. A, CRISPR–Cas9-based screening strategy to identify regulators of response to Menin–MLL inhibition. CPD, cell population doublings; sgRNA, single-guide RNA; Tf, final time point; T0, initial time point. B, Chromatin-focused CRISPR screening data showing the top 20 most significantly enriched (red) and depleted (blue) genes in the Menin–MLL inhibitor (MI-503) treatment relative to vehicle (DMSO). Gene scores are shown as the mean log2 fold change in abundance of the 6 sgRNAs targeting each gene in each condition. C, Genome-wide CRISPR screening data showing gene-level ranking based on differential enrichment of sgRNAs in the Menin–MLL inhibitor treatment (VTP-50469) relative to vehicle (DMSO). Differential (Δ) beta-score between VTP-50469 and DMSO conditions was calculated using MaGeCK. A positive Δ beta-score denotes enrichment of specific gene-targeting sgRNAs. A negative Δ beta-score denotes depletion of specific gene-targeting sgRNAs. Red circles denote MLL3/4–UTX complex subunits. Yellow circles denote PRC1.1 complex subunits. D, Schematic representation of the top-scoring chromatin regulators in the chromatin-focused MI-503 screen and their corresponding protein complexes. Red denotes enriched subunits. Blue denotes depleted subunits. Color scale represents the log2 fold change in abundance of the 6 sgRNAs targeting each subunit in the Menin–MLL inhibitor (MI-503) treatment relative to vehicle (DMSO).E, Viability assay from cells treated with vehicle (DMSO, black) or Menin–MLL inhibitor (MI-503, red) for 96 hours (mean ± SEM, n = 3 infection replicates, P values calculated by Student t test). sgCtrl, control sgRNA targeting a nongenic region on chromosome 8. F, Relative cell proliferation is shown as the proliferation of double-positive cells (sgMen1-RFP + sgUtx-BFP or sgMen1-RFP + sgCtrl-BFP) relative to single-positive cells (sgMen1-RFP) 16 days after infection measured by flow cytometry (mean ± SEM, n = 3 infection replicates, P values calculated by Student t test). Representative FACS plots are shown for sgControl- and sgUtx-targeted cells.