DZNep treatment removes the binding of PRC2 to their target genes and increases the binding of RNA Pol II. (A) Schematic representations of 5′-flanking regions of candidate PRC2 target genes. Arrows point to the transcription start sites. Vertical bars indicate CpG sites. Regions analyzed by PCR are shown by black bars at the bottom. ChIP primers are located on the core promoter regions of analyzed genes. (B) ChIPs show that DZNep treatment reduces the binding of SUZ12 to the promoter of target genes but increases binding of RNA Pol II to these gene promoters. Nonspecific IgG was used as a control. Input represents the genomic DNA.

DZNep preferentially induces apoptosis in cancer cells. (A) Chemical structure of DZNep. (B) MCF-7 and HCT116 cells were treated with 5 μM DZNep for 48 and 72 h, followed by PI staining and FACS analysis. (C) MCF-7 and HCT116 cells were treated with DZNep for 72 h, followed by JC-1 staining and FACS analysis. MTP was quantified by the cells with lower membrane potential (ΔΨm). (D) MCF-7 and HCT16 cells were treated with 5 μM DZNep for 48 and 72 h, and whole-cell extracts were analyzed by Western blotting. Cleavage of PARP was detected after DZNep treatment. β-Actin was used as a loading control. (E) Cell death response of a variety of cancer cells and normal cells to DZNep. Indicated cells were treated with 5 μM DZNep for up to 120 h and the cell death was measured by PI staining and FACS analysis. Data represent ±SD from three independent experiments.

Effects of DZNep on PRC2 proteins and histone methylations. (A) MCF-7 and HCT116 cells were treated with 5 μM DZNep for 48 and 72 h and cells were harvested for Western blot analysis using antibodies detecting the indicated proteins. (B) MCF-7 cells were treated with DZNep for 48 h and the levels of indicated histone methylations were examined by Western blot analysis. (C) MCF-7 cells were treated with DZNep for the indicated times and cells were harvested for Western blot analysis of the indicated proteins. (D) MCF-7 and HCT116 cells were treated as in A and total RNA was isolated for RT–PCR analysis of EZH2, EED, and SUZ12 mRNA levels. (E) MCF-7 cells were treated with DZNep for 18 h, followed by the addition of proteosome inhibitors MG132 (5 μM), LLNL (50 μM), or MG115 (20 μM) for 8 h. Cells were harvested for Western blot analysis of EZH2 and SUZ12. (F) MCF-7 cells were treated with siRNA targeting EZH2, EED, or SUZ12 individually. After 72 h, cells were harvested and the levels of PRC2 proteins and H3-K27me3, H3-K9me3, and total H3 were examined by Western blot analysis as indicated. (G) MCF-7 and MCF-10A cells were treated as in F and cell death was measured by PI staining and FACS analysis. Data represent ±SD from three independent experiments.

Effects of DZNep and 5-AzaC on PRC2 target gene expression and DNA methylation status. (A) MCF-7 cells were untreated or treated with DZNep, 5-AzaC, or 5-AzaC plus TSA, and RNA was isolated for gene array expression analysis. The averaged expression levels of 140 PRC2 targets are shown on the left. Inductions of p18INK4C expression by 5-AzaC or 5-AzaC plus TSA as a positive control are shown on the right. (B) Methylation status of six representative PRC2 target gene promoters in MCF-7 cells either untreated or treated with DZNep or 5-AzaC. Each color-coded ball represents one CpG site. Red indicates low level of methylation and yellow indicates high level of methylation. Gray indicates the missing data. The corresponding changes in gene expression upon treatment with DZNep, 5-AzaC, or 5-AzaC plus TSA were evaluated by quantitative RT–PCR and the results are shown on the right. The detailed information about the genomic locations of each amplicon and the percentage of methylation at each CpG site in each of the amplicons can be found in Supplementary Table S3.

Identification of PRC2 target genes associated with DZNep sensitivity. (A) Indicated cell lines were treated with 5 μM DZNep for 72, 96, and 120 h and cell death was measured by FACS analysis. The data presented are the average of three independent experiments. (B) Unsupervised clustering of 44 PRC2 targets in indicated breast cancer cell lines. Genes highlighted in green are highly expressed in resistant cell lines relative to sensitive cell lines. (C) Cluster program showing genes up-regulated by DZNep in at least three sensitive cell lines. (D) Indicated cell lines were treated with or without DZNep for 72 h and cells were harvested to examine the mRNA levels of FBXO32, LAMB3, PLAU, and PPP1R15A by quantitative real-time RT–PCR analysis. Shown are the folds of induction after 72-h treatment of DZNep. (E) Indicated breast cancer cell lines were treated with DZNep for 48 h and EZH2 and SUZ12 protein levels were determined by Western blotting.

FBXO32 is functionally required for DZNep-induced apoptosis. (A) Each of indicated Dharmacon SMARTpool siRNA was transfected into MCF-7 cells for 24 h, followed by 5 μM DZNep treatment for 72 h. Apoptosis was measured by FACS analysis. Only FBXO32 siRNA was capable of reducing DZNep-induced apoptosis. (B) A FBXO32 siRNA targeting a different region of FBXO32 also inhibited DNZep-induced apoptosis in MCF-7 cells compared with a nontargeting siRNA control (NC). Shown are the results of three independent experiments. The knockdown of FBXO32 mRNA expression is shown by RT–PCR at the bottom. (C) The levels of FBXO32 mRNA in two MCF-7 stable clones expressing FBXO32 shRNA or nontargeting control shRNA (NC shRNA) were determined by real-time RT–PCR. The graph shows the fold of induction of FBXO32 mRNA following DZNep treatment in each stable clone as indicated. (D) MCF-7 cells expressing FBXO32 shRNA or NC shRNA were treated with DZNep for 72 h and the cell death was measured by PI staining and FACS analysis. (E) Cells were treated as in D and JC-1 staining was performed. (F) Cells were treated as in D and Western blot analysis was performed to detect the PARP cleavage.

Identification of DZNep-activated PRC2 target genes in breast cancer cells. (A) Vent diagram showing the overlapping genes up-regulated in DNZep-treated and PRC2 siRNA-treated MCF-7 cells. Cells were treated with DZNep or individual EZH2, EED, or SUZ12 siRNA for 72 h and gene expression was performed using the Illumina 24K BeadArray system. Genes (751) were up-regulated for twofold or greater upon DZNep treatment and 1402 genes were up-regulated for twofold or greater in at least one out three PRC2 knockdown conditions. (B, left panel) Cluster diagram showing the PRC2 targets expressed differentially in MCF-7 and MCF-10A cells. Genes are rank-ordered by levels of high expression (red) and low expression (green) in MCF-7 cells relative to MCF-10A cells. Clustering of 44 target genes selectively repressed in MCF-7 cells and their reactivation by DZNep or siRNAs targeting individual PRC2 proteins is shown in the right panel. (C) RT–PCR validation of 10 PRC2 target genes. (D) Averaged fold of induction of 44 PRC2 targets by DZNep in MCF-10A versus MCF-7 cells. (E) GO assignments of the gene functions. (F) Hierarchical clustering of PRC2 target genes in primary breast tumors (T) and normal breast tissues (N). Cluster I represents the PRC2 target genes whose expression is down-regulated in primary tumors compared with the normal tissues.