Figure 5. HES4 enforces the expression... | American Society of Hematology

Figure 5.

$HES4 enforces the expression of the Δ133p53 isoform in CUTLL-1 human T-ALL cell line. (A) Heat map and hierarchical clustering of the gene expression RNA-seq data from the human CUTLL-1 cell line after transduction with the HES4/mTag2BFP, 2 shHES4/Cherry, or empty/Cherry (Ctrl) lentivectors as indicated. After 10 days from the transduction, mTag2BFP+ or Cherry+ cells were sorted using FACS for RNA isolation and sequencing. The top 2000 differentially expressed genes, scaled to mean = 0 and SD = 1, are represented (adjusted P value ≤.1). (B) Lollipop plots of the Hallmark GeneSets significantly enriched in CUTLL-1 cells by GSEA of genes identified in the analysis of the RNA-seq data. (C) GSEA showing the enrichment score for the Hallmark G2/M checkpoint gene signature obtained by analysis of the RNA-seq data in CUTLL-1 cells. (D) Volcano plot of up- and downregulated genes by HES4 expression in CUTLL-1 cells as determined by RNA-seq assay (adjusted P value <.05). (E) Heat map of the ChIP-seq reads for HES4 and input around the transcriptional starting site (TSS) in the CUTLL-1 cell line. Scales indicate the normalized counts in the ChIP-Seq signal. (F) Venn diagram showing the up- and downregulated genes following HES4 expression in CUTLL-1 cells, determined by RNA-seq, and the genes with HES4 peaks within 1 Kb around the TSS as determined by the ChIP-seq assay. (G) Plots of the Hallmark GeneSets significantly enriched in GSEA of upregulated genes (n.240) following HES4 expression in CUTLL-1 cells as identified by the integrated analysis of the RNA-seq and ChIP-seq data sets. (H) Location of predicted HES4 sites relative to the TSS, and the ChIP-seq peak score of up- and downregulated genes following HES4 expression in CUTLL-1 cells as determined by the RNA-Seq assay. (I-J) The Δ133p53 messenger RNA expression level in the CUTLL-1 and TALL-1 cell lines after transduction with the sh-scramble or shHES4 lentivectors as indicated (I) and in the DND-41 and ALL-SIL cell lines and 2 independent clones of PDXs after transduction with HES4 lentiviruses or the EV as control (J). Four days after the transduction, the cells were sorted using FACS for RNA isolation to perform the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The values in the plot indicate the ratio of Δ133p53 messenger RNA expression level over the full length FSp53α isoform, both normalized to B2M gene expression as control. In each data set, 3 biologic replicates for each condition are indicated. (K) The western blot analysis of the P53 isoforms in the DND-41 and ALL-SIL cell lines following transduction with the HES4 lentiviruses or EV as negative control. (L) ChIP-quantitative polymerase chain reaction (qPCR) analysis. Local ChIP was performed using antibodies against HES4 and RNA polymerase II in the DND-41 cell line after transduction with the HES4 lentiviruses or EV as negative control. In the schematic map of the TP53 human region, the primer pairs used for ChIP-qPCR assays are indicated by the small arrows. Values are expressed as a fraction of input DNA controls. Mean values are plotted for assays performed in triplicate. The error bars indicate the SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (Student t test). FDR, false discovery rate q-value; Min, minimum; Max, maximum; NES, normalized enrichment score; PolII, polymerase II.$

HES4 enforces the expression of the Δ133p53 isoform in CUTLL-1 human T-ALL cell line. (A) Heat map and hierarchical clustering of the gene expression RNA-seq data from the human CUTLL-1 cell line after transduction with the HES4/mTag2BFP, 2 shHES4/Cherry, or empty/Cherry (Ctrl) lentivectors as indicated. After 10 days from the transduction, mTag2BFP+ or Cherry+ cells were sorted using FACS for RNA isolation and sequencing. The top 2000 differentially expressed genes, scaled to mean = 0 and SD = 1, are represented (adjusted P value ≤.1). (B) Lollipop plots of the Hallmark GeneSets significantly enriched in CUTLL-1 cells by GSEA of genes identified in the analysis of the RNA-seq data. (C) GSEA showing the enrichment score for the Hallmark G2/M checkpoint gene signature obtained by analysis of the RNA-seq data in CUTLL-1 cells. (D) Volcano plot of up- and downregulated genes by HES4 expression in CUTLL-1 cells as determined by RNA-seq assay (adjusted P value <.05). (E) Heat map of the ChIP-seq reads for HES4 and input around the transcriptional starting site (TSS) in the CUTLL-1 cell line. Scales indicate the normalized counts in the ChIP-Seq signal. (F) Venn diagram showing the up- and downregulated genes following HES4 expression in CUTLL-1 cells, determined by RNA-seq, and the genes with HES4 peaks within 1 Kb around the TSS as determined by the ChIP-seq assay. (G) Plots of the Hallmark GeneSets significantly enriched in GSEA of upregulated genes (n.240) following HES4 expression in CUTLL-1 cells as identified by the integrated analysis of the RNA-seq and ChIP-seq data sets. (H) Location of predicted HES4 sites relative to the TSS, and the ChIP-seq peak score of up- and downregulated genes following HES4 expression in CUTLL-1 cells as determined by the RNA-Seq assay. (I-J) The Δ133p53 messenger RNA expression level in the CUTLL-1 and TALL-1 cell lines after transduction with the sh-scramble or shHES4 lentivectors as indicated (I) and in the DND-41 and ALL-SIL cell lines and 2 independent clones of PDXs after transduction with HES4 lentiviruses or the EV as control (J). Four days after the transduction, the cells were sorted using FACS for RNA isolation to perform the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The values in the plot indicate the ratio of Δ133p53 messenger RNA expression level over the full length FSp53α isoform, both normalized to B2M gene expression as control. In each data set, 3 biologic replicates for each condition are indicated. (K) The western blot analysis of the P53 isoforms in the DND-41 and ALL-SIL cell lines following transduction with the HES4 lentiviruses or EV as negative control. (L) ChIP-quantitative polymerase chain reaction (qPCR) analysis. Local ChIP was performed using antibodies against HES4 and RNA polymerase II in the DND-41 cell line after transduction with the HES4 lentiviruses or EV as negative control. In the schematic map of the TP53 human region, the primer pairs used for ChIP-qPCR assays are indicated by the small arrows. Values are expressed as a fraction of input DNA controls. Mean values are plotted for assays performed in triplicate. The error bars indicate the SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (Student t test). FDR, false discovery rate q-value; Min, minimum; Max, maximum; NES, normalized enrichment score; PolII, polymerase II.

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