Figure 4.
EZH2 is a cofactor for transcriptional activation together with MYCN. (A) Schematic representation of the top 100 MYCN direct target genes. (B) Normalized expression levels of Ezh2 in CD4 T cells and in MYCN-driven lymphoma cells. (C) Western blot showing expression levels of MYCN, Ezh2, and Suz12 in normal CD4 T cells and in MYCN-driven TCL spleen cells. (D) Representative ChIP-seq tracks for the Ezh2 promoter showing binding of MYCN, Ezh2, Suz12, p300, and histone marks H3K4me3, H3K27ac, and H3K27me3 in MYCN-driven TCL. (E) Normalized counts for EZH2 from the PTCL-NOS and FTCL cases from the Leuven cohort and the Kyoto cohort. Patients with overexpression of MYCN are indicated with stars. (F) Centered read-density heat maps showing binding locations of Ezh2, Suz12, MYCN, p300, H3K27ac, H3K4me3, and H3K27me3 in MYCN-driven TCL. Heat maps centered and ranked on Ezh2 signal strength in active promoters (top) or in regions enriched for H3K27me3 (bottom). (G) Venn diagram of EZH2 peaks in direct MYCN target genes and their overlap with H3K27ac or H3K27me3 peaks. (H) Normalized enrichment scores for different hallmark gene sets in the differentially expressed direct EZH2+MYCN target genes. (I) GSEA showing enrichment of different MYC target gene sets and stemness-associated gene sets in the differentially expressed direct EZH2+MYCN target genes. (J) Luciferase assay in HEK293T cells showing transcriptional activation of Firefly luciferase by MYCN and/or EZH2 (wild type or methyltransferase-inactivating mutants). (K) Forty-eight–hour ex vivo treatment of MYCN-driven TCL spleen cells with 2 μM of the EZH2 methyltransferase inhibitors tazemetostat and valemetostat, or the EZH2 degrader MS1943. n.s., not significant. (L) Fluorescence-activated cell sorter (FACS) analysis of EZH2 and MYCN in MYCN-driven TCL spleen cells treated ex vivo with DMSO or MS1943 for 12 hours. (M) In vitro treatment of RPMI-8402 cells with 5 μM MS1943, tazemetostat (Taz), or valemetostat (Val). (N) FACS analysis of EZH2 and MYCN in RPMI-8402 cells after treatment with 7 μM MS1943 or DMSO. (O) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of MYCN and EZH2 target gene expression in RPMI-8402 cells treated for 36 hours with 2.5 μM MS1943 or DMSO. See also supplemental Figures 4 and 5. ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05.

EZH2 is a cofactor for transcriptional activation together with MYCN. (A) Schematic representation of the top 100 MYCN direct target genes. (B) Normalized expression levels of Ezh2 in CD4 T cells and in MYCN-driven lymphoma cells. (C) Western blot showing expression levels of MYCN, Ezh2, and Suz12 in normal CD4 T cells and in MYCN-driven TCL spleen cells. (D) Representative ChIP-seq tracks for the Ezh2 promoter showing binding of MYCN, Ezh2, Suz12, p300, and histone marks H3K4me3, H3K27ac, and H3K27me3 in MYCN-driven TCL. (E) Normalized counts for EZH2 from the PTCL-NOS and FTCL cases from the Leuven cohort and the Kyoto cohort. Patients with overexpression of MYCN are indicated with stars. (F) Centered read-density heat maps showing binding locations of Ezh2, Suz12, MYCN, p300, H3K27ac, H3K4me3, and H3K27me3 in MYCN-driven TCL. Heat maps centered and ranked on Ezh2 signal strength in active promoters (top) or in regions enriched for H3K27me3 (bottom). (G) Venn diagram of EZH2 peaks in direct MYCN target genes and their overlap with H3K27ac or H3K27me3 peaks. (H) Normalized enrichment scores for different hallmark gene sets in the differentially expressed direct EZH2+MYCN target genes. (I) GSEA showing enrichment of different MYC target gene sets and stemness-associated gene sets in the differentially expressed direct EZH2+MYCN target genes. (J) Luciferase assay in HEK293T cells showing transcriptional activation of Firefly luciferase by MYCN and/or EZH2 (wild type or methyltransferase-inactivating mutants). (K) Forty-eight–hour ex vivo treatment of MYCN-driven TCL spleen cells with 2 μM of the EZH2 methyltransferase inhibitors tazemetostat and valemetostat, or the EZH2 degrader MS1943. n.s., not significant. (L) Fluorescence-activated cell sorter (FACS) analysis of EZH2 and MYCN in MYCN-driven TCL spleen cells treated ex vivo with DMSO or MS1943 for 12 hours. (M) In vitro treatment of RPMI-8402 cells with 5 μM MS1943, tazemetostat (Taz), or valemetostat (Val). (N) FACS analysis of EZH2 and MYCN in RPMI-8402 cells after treatment with 7 μM MS1943 or DMSO. (O) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of MYCN and EZH2 target gene expression in RPMI-8402 cells treated for 36 hours with 2.5 μM MS1943 or DMSO. See also supplemental Figures 4 and 5. ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05.

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