Figure 4
Figure 4. EZH2 positively regulates CCND1 transcription by binding to its promoter in NKTL cells. (A) Overexpression of EZH2 induces the expression of CCND1. Vectors expressing EZH2 (pcDNA-EZH2 or pcDNA-EZH2 SETΔ) were transiently transfected into NKYS cells. The RNA harvested from the cells at 24 hours after transfection was isolated, reverse-transcribed, subjected to qPCR by using primers specific for CCND1 mRNA, and normalized with GAPDH. (B) Luciferase promoter assay showing that EZH2 activates CCND1 transcription. NKYS cells were transfected with the luciferase reporter construct pGL4 containing the CCND1 promoter and various amounts of EZH2 WT/SETΔ plasmid or a control vector. Luciferase activities were measured after 24 hours. Luciferase readings were further normalized to the internal control pRL null. Results are presented as averages of triplicate experiments. Error bars represent standard deviation. * denotes P < .01 with respect to cells transfected with the same amount of control vector. (C) Genomic structure of human CCND1. The locations of the 6 pairs of primer sets used to detect the ChIP-enriched DNA fragments are indicated. (D) ChIP-qPCR for endogenous EZH2 binding to the CCND1 gene. ChIP assays were performed by using NKYS. Real-time PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-EZH2 antibody or an irrelevant antibody (IgG) as a control. A known EZH2 binding site in the promoter region of the MYT-1 gene was amplified as a positive control for the ChIP assays. (E) ChIP-qPCR for ectopically expressed EZH2 WT and EZH2 SETΔ binding to the CCND1 gene. ChIP assays were performed by using NKYS transfected by a pcDNA4.1/Myc-His vector or a plasmid expressing EZH2 WT and EZH2-SETΔ. Real-time PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-His antibody. Primer set 3, which amplifies a region representing EZH2 binding, was used to detect the ChIP-enriched DNA fragments. (F) Luciferase promoter assay showing the inability of EZH2 SANT domain deletion mutants to activate CCND1 transcription. NKYS were transfected with 3µg of EZH2 WT/SETΔ/SANTΔ plasmids or a control vector. Luciferase activities were measured after 24 hours. (G) Correlation between mRNA levels for EZH2 and CCND1 determined by qRT-PCR in NKTL clinical samples. Spearman correlation coefficient = 0.8608; P < .0001. (H) Western blot analysis of protein levels of CCND1 and EZH2 in NK cell lines and normal NK cells.

EZH2 positively regulates CCND1 transcription by binding to its promoter in NKTL cells. (A) Overexpression of EZH2 induces the expression of CCND1. Vectors expressing EZH2 (pcDNA-EZH2 or pcDNA-EZH2 SETΔ) were transiently transfected into NKYS cells. The RNA harvested from the cells at 24 hours after transfection was isolated, reverse-transcribed, subjected to qPCR by using primers specific for CCND1 mRNA, and normalized with GAPDH. (B) Luciferase promoter assay showing that EZH2 activates CCND1 transcription. NKYS cells were transfected with the luciferase reporter construct pGL4 containing the CCND1 promoter and various amounts of EZH2 WT/SETΔ plasmid or a control vector. Luciferase activities were measured after 24 hours. Luciferase readings were further normalized to the internal control pRL null. Results are presented as averages of triplicate experiments. Error bars represent standard deviation. * denotes P < .01 with respect to cells transfected with the same amount of control vector. (C) Genomic structure of human CCND1. The locations of the 6 pairs of primer sets used to detect the ChIP-enriched DNA fragments are indicated. (D) ChIP-qPCR for endogenous EZH2 binding to the CCND1 gene. ChIP assays were performed by using NKYS. Real-time PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-EZH2 antibody or an irrelevant antibody (IgG) as a control. A known EZH2 binding site in the promoter region of the MYT-1 gene was amplified as a positive control for the ChIP assays. (E) ChIP-qPCR for ectopically expressed EZH2 WT and EZH2 SETΔ binding to the CCND1 gene. ChIP assays were performed by using NKYS transfected by a pcDNA4.1/Myc-His vector or a plasmid expressing EZH2 WT and EZH2-SETΔ. Real-time PCR was performed with immunoprecipitated chromatin fragments obtained by using an anti-His antibody. Primer set 3, which amplifies a region representing EZH2 binding, was used to detect the ChIP-enriched DNA fragments. (F) Luciferase promoter assay showing the inability of EZH2 SANT domain deletion mutants to activate CCND1 transcription. NKYS were transfected with 3µg of EZH2 WT/SETΔ/SANTΔ plasmids or a control vector. Luciferase activities were measured after 24 hours. (G) Correlation between mRNA levels for EZH2 and CCND1 determined by qRT-PCR in NKTL clinical samples. Spearman correlation coefficient = 0.8608; P < .0001. (H) Western blot analysis of protein levels of CCND1 and EZH2 in NK cell lines and normal NK cells.

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