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. 2022 Oct 28;12(12):2880–2905. doi: 10.1158/2159-8290.CD-21-1491

Figure 3.

Figure 3. Genetic knockout of SMARCA4 reduces chromatin accessibility and gene expression of OPC-like markers and enriches for the AC-like subpopulation in H3K27M-glioma cells. A, Schematic showing biological assessment of SMARCA4 knockout in H3.3K27M-glioma neurospheres by ChIP-seq for histone modifications (H3K27ac and H3K27me3), assay for transposase-accessible chromatin using sequencing (ATAC-seq) for chromatin accessibility, bulk RNA sequencing (RNA-seq), and scRNA-seq for gene expression. B, Heat map depicting z-scores for chromatin accessibility of OPC-like and AC-like marker genes in AAVS1-negative sgRNA control and SMARCA4-knockout (KO) BT869 neurospheres. C, Profile plot depicting the average H3K27ac ChIP-seq signal at BRG1 binding sites (n = 1,335) in BT869 4 days after SMARCA4 knockout. The plot shows 6.4-kb regions, centered on BRG1 peaks. D, Promoter-associated H3K27ac and H3K27me3 ChIP-seq signal for genes differentially expressed [determined by bulk RNA-seq analysis, log2(fold change [FC]) ≥ |1|] in SMARCA4-knockout cells (4 days after nucleofections). E, Profile plots (left) depicting the average H3K27ac signal at H3K27ac locations divided into positive (UP) or negative (DOWN) signal changes 4 days after SMARCA4 knockout. The plots show 6.4-kb regions, centered on H3K27ac peaks. Gene ontology (GO) analyses (right) performed on H3K27ac peaks with increased (n = 104, K27acUP) or decreased (n = 2,832, K27acDOWN) acetylation levels upon SMARC4 knockout for 4 days. The top five enriched GO terms are displayed. Pos. reg. of epidermal cell diff., positive regulation of epidermal cell differentiation.F, Heat map (left) depicting fold change (as log2) of H3K27ac levels after SMARCA4 knockout for 4 days. Displayed are log2 fold changes > |1|. Genes indicated on the heat map are OPC-like markers associated with decreased H3K27ac levels after SMARCA4 depletion. Gene tracks (right) displaying BRG1 ChIP-seq signal in BT869 neurospheres, as well as H3K27ac and ATAC-seq, at two OPC-like marker genes (TNR and EPN2). G, Changes in the percentage of cycling versus noncycling (left) and OPC- and AC-like cancer cell subpopulations (right) in SMARCA4-knockout and AAVS1-negative sgRNA control cells 4 days after knockout in BT869 neurospheres as determined by scRNA-seq analysis. H, Single-cell expression scores of upregulated genes in BT869 SMARCA4-knockout or AAVS1-negative sgRNA control knockout cells projected onto scRNA-seq data of pediatric H3K27M-glioma primary tumors (n = 6).

Genetic knockout of SMARCA4 reduces chromatin accessibility and gene expression of OPC-like markers and enriches for the AC-like subpopulation in H3K27M-glioma cells. A, Schematic showing biological assessment of SMARCA4 knockout in H3.3K27M-glioma neurospheres by ChIP-seq for histone modifications (H3K27ac and H3K27me3), assay for transposase-accessible chromatin using sequencing (ATAC-seq) for chromatin accessibility, bulk RNA sequencing (RNA-seq), and scRNA-seq for gene expression. B, Heat map depicting z-scores for chromatin accessibility of OPC-like and AC-like marker genes in AAVS1-negative sgRNA control and SMARCA4-knockout (KO) BT869 neurospheres. C, Profile plot depicting the average H3K27ac ChIP-seq signal at BRG1 binding sites (n = 1,335) in BT869 4 days after SMARCA4 knockout. The plot shows 6.4-kb regions, centered on BRG1 peaks. D, Promoter-associated H3K27ac and H3K27me3 ChIP-seq signal for genes differentially expressed [determined by bulk RNA-seq analysis, log2(fold change [FC]) ≥ |1|] in SMARCA4-knockout cells (4 days after nucleofections). E, Profile plots (left) depicting the average H3K27ac signal at H3K27ac locations divided into positive (UP) or negative (DOWN) signal changes 4 days after SMARCA4 knockout. The plots show 6.4-kb regions, centered on H3K27ac peaks. Gene ontology (GO) analyses (right) performed on H3K27ac peaks with increased (n = 104, K27acUP) or decreased (n = 2,832, K27acDOWN) acetylation levels upon SMARC4 knockout for 4 days. The top five enriched GO terms are displayed. Pos. reg. of epidermal cell diff., positive regulation of epidermal cell differentiation.F, Heat map (left) depicting fold change (as log2) of H3K27ac levels after SMARCA4 knockout for 4 days. Displayed are log2 fold changes > |1|. Genes indicated on the heat map are OPC-like markers associated with decreased H3K27ac levels after SMARCA4 depletion. Gene tracks (right) displaying BRG1 ChIP-seq signal in BT869 neurospheres, as well as H3K27ac and ATAC-seq, at two OPC-like marker genes (TNR and EPN2). G, Changes in the percentage of cycling versus noncycling (left) and OPC- and AC-like cancer cell subpopulations (right) in SMARCA4-knockout and AAVS1-negative sgRNA control cells 4 days after knockout in BT869 neurospheres as determined by scRNA-seq analysis. H, Single-cell expression scores of upregulated genes in BT869 SMARCA4-knockout or AAVS1-negative sgRNA control knockout cells projected onto scRNA-seq data of pediatric H3K27M-glioma primary tumors (n = 6).