Figure 4.
SETD1B inactivation results in dysregulation of gene expression and H3K4 trimethylation. (A) Heatmap illustrating the differential gene expression of significantly differentially expressed genes (Log2FC > 0.5, FDR < 0.05) between OCI-LY19 cells (KMT2Dmut) carrying sgLacZ (n = 3) or sgSETD1B (sg1 clone 1) (n = 3). (B) Bar graphs demonstrating the top enriched gene sets within the hallmark dataset from MsigDB. The x-axis represents the differential gene expression’s FDR between SETD1B knockout and control. (C) Enrichment plot for P53 pathway and apoptosis hallmark gene sets of GSEA comparing OCI-LY19;sgSETD1B and OCI-LY19;sgLacZ. NES, normalized enrichment score. (D) Histograms showing the average H3K4me3 read density plot of ChIP-seq from SETD1B mutant (sgSETD1B) and OCI-LY19 control (sgLacZ). (E) Doughnut chart demonstrating the genomic distribution of H3K4me3-bound peaks with at least 30% loss (P < 0.05, peak numbers n = 1,553) in OCI-LY19 sgSETD1B cells versus sgLacZ cells located at the promoter (defined as ± 2 kb windows centered on TSS), intragenic (inside gene body), and intergenic (up- or downstream of the closest gene but not overlapping with the promoter). (F) Bar graphs representing the GSEA in significantly differentially expressed genes, as compared to the hallmark dataset (MsigDB). (G) Normalized Integrative Genomics Viewer (IGV) read-density tracks of H3K4me3 ChIP-seq peaks at the loci of representative genes (BIK and CD40) from the ChIP-seq experiments. Signals are plotted on a normalized read per million (RPM) bases. (H) ChIP-qPCR analysis of H3K4me3 loss in the promoter of BIK in OCI-LY19; sgSETD1B clones compared with control cells (sgLacZ). BIK downstream locus with no detectable H3K4me3 peaks in either sgSETD1B clones or control cells (NTC). Values represent means and SD. P values were calculated by Student’s t test. ****P < 0.0001, n = 3. (I) Bar graph representing the relative expression of BIK in OCI-LY19 sgSETD1B vs. clones OCI-LY19;sgLacZ treated with either DMSO or JIB-04. Values represent means and SD. P values were calculated by two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001; n = 3.

SETD1B inactivation results in dysregulation of gene expression and H3K4 trimethylation. (A) Heatmap illustrating the differential gene expression of significantly differentially expressed genes (Log2FC > 0.5, FDR < 0.05) between OCI-LY19 cells (KMT2Dmut) carrying sgLacZ (n = 3) or sgSETD1B (sg1 clone 1) (n = 3). (B) Bar graphs demonstrating the top enriched gene sets within the hallmark dataset from MsigDB. The x-axis represents the differential gene expression’s FDR between SETD1B knockout and control. (C) Enrichment plot for P53 pathway and apoptosis hallmark gene sets of GSEA comparing OCI-LY19;sgSETD1B and OCI-LY19;sgLacZ. NES, normalized enrichment score. (D) Histograms showing the average H3K4me3 read density plot of ChIP-seq from SETD1B mutant (sgSETD1B) and OCI-LY19 control (sgLacZ). (E) Doughnut chart demonstrating the genomic distribution of H3K4me3-bound peaks with at least 30% loss (P < 0.05, peak numbers n = 1,553) in OCI-LY19 sgSETD1B cells versus sgLacZ cells located at the promoter (defined as ± 2 kb windows centered on TSS), intragenic (inside gene body), and intergenic (up- or downstream of the closest gene but not overlapping with the promoter). (F) Bar graphs representing the GSEA in significantly differentially expressed genes, as compared to the hallmark dataset (MsigDB). (G) Normalized Integrative Genomics Viewer (IGV) read-density tracks of H3K4me3 ChIP-seq peaks at the loci of representative genes (BIK and CD40) from the ChIP-seq experiments. Signals are plotted on a normalized read per million (RPM) bases. (H) ChIP-qPCR analysis of H3K4me3 loss in the promoter of BIK in OCI-LY19; sgSETD1B clones compared with control cells (sgLacZ). BIK downstream locus with no detectable H3K4me3 peaks in either sgSETD1B clones or control cells (NTC). Values represent means and SD. P values were calculated by Student’s t test. ****P < 0.0001, n = 3. (I) Bar graph representing the relative expression of BIK in OCI-LY19 sgSETD1B vs. clones OCI-LY19;sgLacZ treated with either DMSO or JIB-04. Values represent means and SD. P values were calculated by two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001; n = 3.

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