Figure 3
Figure 3. Phosphorylation of EZH2 by JAK3 is inhibitory for PRC2-mediated gene repression but required for gene activation toward proliferation. (A) GSEA plots show gene sets enriched among downregulated genes upon JAK3 knockdown. The 4 gene sets are described in Molecular Signatures Database: JAK-STAT signaling pathway; Genes bearing the H3K27me3 mark; Putative targets of EZH2 as an epigenetic silencer; Genes upregulated upon knockdown of SUZ12. (B) Correlation plot between the JAK-STAT gene signature and PRC2 canonical target gene signature indices in a dataset of NKTL patient samples (GSE80632). Each dot is a patient sample. Correlation coefficient and correlation test P value are also shown. (C) Hierarchical clustering of gene expression profiles of EZH2 KD, EZH2 WT, and it mutants (Y244A, Y244D, and SET∆) based on global profiling (GSE75680). Heat map shows expression profiles of noncanonical targets cross EZH2 KD, EZH2 WT, and its mutants (Y244A, Y244D, and SET∆) compared with their corresponding controls. (D) ChIP-qPCR assay in NKYS cells for endogenous EZH2 binding. RT-PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-EZH2 antibody or an irrelevant antibody (immunoglobulin G, IgG) as a control. (E) qRT-PCR analysis showing that JAK3 inhibitor PF decreased expression of noncanonical targets of EZH2. n = 4 independent experiments, means ± SD. *P < .05; **P < .01 (Student t test). (F) qRT-PCR analysis showing that gene expression of EZH2 noncanonical targets was not affected by PRC2 inhibitor EPZ-6438 (EPZ). The RNA harvested from NKYS cells exposed to EPZ at 200 nM was isolated, reverse-transcribed, subjected to qPCR by using primers as indicated, and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n = 5 independent experiments. Results are means ± SD. *P < .05; **P < .01 (Student t test). (G) Correlation plot between the JAK-STAT gene signature and EZH2 noncanonical target gene signature indices in a dataset of NKTL patient samples (GSE80632). Each dot is a patient sample, and no cell line is presented. Correlation coefficient and correlation test P value are also shown. (H) GSEA plots of significantly enriched gene sets show the regulation of oncogenesis-related genes and pathways by EZH2 noncanonical function. KD, EZH2 knockdown; NES, normalized enrichment score.

Phosphorylation of EZH2 by JAK3 is inhibitory for PRC2-mediated gene repression but required for gene activation toward proliferation. (A) GSEA plots show gene sets enriched among downregulated genes upon JAK3 knockdown. The 4 gene sets are described in Molecular Signatures Database: JAK-STAT signaling pathway; Genes bearing the H3K27me3 mark; Putative targets of EZH2 as an epigenetic silencer; Genes upregulated upon knockdown of SUZ12. (B) Correlation plot between the JAK-STAT gene signature and PRC2 canonical target gene signature indices in a dataset of NKTL patient samples (GSE80632). Each dot is a patient sample. Correlation coefficient and correlation test P value are also shown. (C) Hierarchical clustering of gene expression profiles of EZH2 KD, EZH2 WT, and it mutants (Y244A, Y244D, and SET∆) based on global profiling (GSE75680). Heat map shows expression profiles of noncanonical targets cross EZH2 KD, EZH2 WT, and its mutants (Y244A, Y244D, and SET∆) compared with their corresponding controls. (D) ChIP-qPCR assay in NKYS cells for endogenous EZH2 binding. RT-PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-EZH2 antibody or an irrelevant antibody (immunoglobulin G, IgG) as a control. (E) qRT-PCR analysis showing that JAK3 inhibitor PF decreased expression of noncanonical targets of EZH2. n = 4 independent experiments, means ± SD. *P < .05; **P < .01 (Student t test). (F) qRT-PCR analysis showing that gene expression of EZH2 noncanonical targets was not affected by PRC2 inhibitor EPZ-6438 (EPZ). The RNA harvested from NKYS cells exposed to EPZ at 200 nM was isolated, reverse-transcribed, subjected to qPCR by using primers as indicated, and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n = 5 independent experiments. Results are means ± SD. *P < .05; **P < .01 (Student t test). (G) Correlation plot between the JAK-STAT gene signature and EZH2 noncanonical target gene signature indices in a dataset of NKTL patient samples (GSE80632). Each dot is a patient sample, and no cell line is presented. Correlation coefficient and correlation test P value are also shown. (H) GSEA plots of significantly enriched gene sets show the regulation of oncogenesis-related genes and pathways by EZH2 noncanonical function. KD, EZH2 knockdown; NES, normalized enrichment score.

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