Figure 5.
Figure 5. PRC1.1 and PRC2 antagonize NOTCH1 in the development of T-ALL. (A) Summary of H2AK119ub1 enrichment detected by the ChIP-seq analysis. The fold enrichment values of H2AK119ub1 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of RefSeq genes in WT and ΔCxxC DP thymocytes 8 weeks after the tamoxifen treatment and ΔCxxC DP T-ALL cells with a Notch1 active mutation in exon 34. Left, all RefSeq genes; right, KDM2B target genes (cluster 1 genes) that were defined by the K-means clustering of 3xFLAG-KDM2B ChIP-seq data in Figure 4B. (B) Scatter diagram of the H2AK119ub1 enrichment (ChIP/Input, RPKM+0.1 in log2) in ΔCxxC DP thymocytes (left) and T-ALL cells (right) vs WT DP cells. Representative direct target genes of NOTCH1 are shown as red dots. (C) A snapshot of 3xFLAG-KDM2B ChIP signals in DP thymocytes overexpressing 3xFLAG-KDM2B and H2AK119ub1 and H3K27me3 ChIP signals in WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells at the Myc gene locus. The structures of the Myc gene are indicated at the bottom. (D) ChIP quantitative PCR assays of KDM2B (left) and H2AK119ub1 levels (right) around Myc TSS. The cells used were WT and 3xFLAG-KDM2B-overexpressing DP thymocytes for KDM2B and WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells for H2AK119ub1. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. Data are shown as means ± SEM (n = 3). (E) Venn diagram of KDM2B and BCOR target genes in DP thymocytes and NOTCH1 target genes in T-ALL cells at the promoter region. Target genes were defined by a peak-calling analysis around TSS. Peaks were called using MACS2 v2.1.1 with a q value <0.05 for KDM2B, <0.2 for BCOR, and <10−10 for NOTCH1. All P values for the pairwise comparisons were <.0001 by Fisher's exact test. (F-G) Summary of H3K27me3 and EZH2 enrichment detected by the ChIP-seq analysis. The fold enrichment values of the H3K27me3 and EZH2 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of RefSeq genes in WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells. (H) Venn diagram of KDM2B and EZH2 target genes in DP thymocytes and NOTCH1 target genes in T-ALL cells around TSS (±2 kb). Peaks were called using MACS2 v2.1.1 with a q value <0.05 for KDM2B and EZH2 and <10−10 for NOTCH1. All P values for the pairwise comparisons were <.0001 by Fisher's exact test. (I) KEGG pathway analysis of the 1276 genes that have all peaks (KDM2B, EZH2, and NOTCH1) in Figure 6H (designated as overlapping genes). (J) H3K27me3 and H3K4me3 levels in EZH2 targets. The fold enrichment values of H3K27me3 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of 1276 overlapping genes and 1182 EZH2 target genes without KDM2B and NOTCH1 binding in Figure 6H (designated as EZH2 only) in WT DP thymocytes. *P < .05; **P < .01; ***P < .001 by Student t test.

PRC1.1 and PRC2 antagonize NOTCH1 in the development of T-ALL. (A) Summary of H2AK119ub1 enrichment detected by the ChIP-seq analysis. The fold enrichment values of H2AK119ub1 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of RefSeq genes in WT and ΔCxxC DP thymocytes 8 weeks after the tamoxifen treatment and ΔCxxC DP T-ALL cells with a Notch1 active mutation in exon 34. Left, all RefSeq genes; right, KDM2B target genes (cluster 1 genes) that were defined by the K-means clustering of 3xFLAG-KDM2B ChIP-seq data in Figure 4B. (B) Scatter diagram of the H2AK119ub1 enrichment (ChIP/Input, RPKM+0.1 in log2) in ΔCxxC DP thymocytes (left) and T-ALL cells (right) vs WT DP cells. Representative direct target genes of NOTCH1 are shown as red dots. (C) A snapshot of 3xFLAG-KDM2B ChIP signals in DP thymocytes overexpressing 3xFLAG-KDM2B and H2AK119ub1 and H3K27me3 ChIP signals in WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells at the Myc gene locus. The structures of the Myc gene are indicated at the bottom. (D) ChIP quantitative PCR assays of KDM2B (left) and H2AK119ub1 levels (right) around Myc TSS. The cells used were WT and 3xFLAG-KDM2B-overexpressing DP thymocytes for KDM2B and WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells for H2AK119ub1. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. Data are shown as means ± SEM (n = 3). (E) Venn diagram of KDM2B and BCOR target genes in DP thymocytes and NOTCH1 target genes in T-ALL cells at the promoter region. Target genes were defined by a peak-calling analysis around TSS. Peaks were called using MACS2 v2.1.1 with a q value <0.05 for KDM2B, <0.2 for BCOR, and <10−10 for NOTCH1. All P values for the pairwise comparisons were <.0001 by Fisher's exact test. (F-G) Summary of H3K27me3 and EZH2 enrichment detected by the ChIP-seq analysis. The fold enrichment values of the H3K27me3 and EZH2 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of RefSeq genes in WT and ΔCxxC DP thymocytes and ΔCxxC DP T-ALL cells. (H) Venn diagram of KDM2B and EZH2 target genes in DP thymocytes and NOTCH1 target genes in T-ALL cells around TSS (±2 kb). Peaks were called using MACS2 v2.1.1 with a q value <0.05 for KDM2B and EZH2 and <10−10 for NOTCH1. All P values for the pairwise comparisons were <.0001 by Fisher's exact test. (I) KEGG pathway analysis of the 1276 genes that have all peaks (KDM2B, EZH2, and NOTCH1) in Figure 6H (designated as overlapping genes). (J) H3K27me3 and H3K4me3 levels in EZH2 targets. The fold enrichment values of H3K27me3 signals were calculated against the input signals (ChIP/input, RPKM+0.1) around the TSS (±2 kb) of 1276 overlapping genes and 1182 EZH2 target genes without KDM2B and NOTCH1 binding in Figure 6H (designated as EZH2 only) in WT DP thymocytes. *P < .05; **P < .01; ***P < .001 by Student t test.

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