Activation of MMPs by EZH2 Figure 1. High levels of EZH2 promote invasion and migration of prostate cancer cells. (A) Western blot analysis of EZH2 expression in the benign prostate cell line RWPE-1 and the malignant prostate cancer cell lines LNCaP, DU145, and PC3 (top). Actin was used as an internal control. The protein bands were quantified using Quantity One software (Bio-Rad) (bottom). (B) Invasion assay of RWPE-1, PC3 and DU145 cells. Cell invasiveness was assessed by the invasion of cells through BME-coated inserts in the Transwell Boyden chamber (top). The invasion rate was determined by counting the cells that migrated through the inserts and expressed as the percentage relative to control (RWPE-1, set to 100%) (bottom). Each bar represents the mean 6 s.e of five fields counted. (C) Western blot analysis of EZH2 expression in PC3 and DU145 cells after an infection with EZH2- specific shRNA lentivirus (sh1 or sh2; two different EZH2-specific shRNAs) or with non-treated shRNA (NT, control) lentivirus (top). Invasion assay of PC3 or DU145 cells after an infection with EZH2-specific shRNA (sh1 or sh2) or control (NT) lentivirus (middle). Representative fields of invaded and stained cells are shown (middle). Each bar represents the mean 6 s.e of five fields counted (bottom). (D) Western blot analysis of overexpression of wild-type (WT) and mutant (H689A, enzymatically inactive) EZH2 proteins in RWPE-1 cells infected with EZH2 (WT)-GFP, EZH2 (H689A)-GFP, or control (GFP vector) lentivirus. EZH2 proteins were expressed from the CMV promoter (left). Invasion assay of RWPE-1 cells infected with EZH2 (WT)-GFP, EZH2 (H689A)-GFP, or control (GFP vector) lentivirus (right). (E) Wound healing assay of DU145 cells infected with EZH2-specific shRNA (sh1) or control shRNA (NT) lentivirus. Images were taken before (0 h) and after wound (24 h) (top). The results were expressed as the percentage of the remaining area determined by normalizing the area of wound after 24 h to the initial wound area at 0 h (set to 100%). Each bar represents the mean 6 s.e of five fields measured (bottom).* P,0.05, ** p,0.005 *** p,0.001 (as compared with control values). doi:10.1371/journal.pone.0030393.g001 ChIP assays in DU145 and PC3 cells using anti-EZH2 and anti- 3meH3K27 antibodies. Because PcG proteins are recruited to DNA via the DNA-binding protein YY1 [45], the immunoprecipitated DNA was analyzed by PCR with the primer sets designed to amplify regions containing the YY1-binding sites in the TIMP3 promoter (Figure 5A). The results indicate that: 1) EZH2 binds to the TIMP3 promoter and catalyzes trimethylation of H3K27 (3meH3K27) (Figures 5B and 5C), but does not bind to the GAPDH promoter, served as a negative control (Figure 5C); 2) RNA polymerase II (RNAP II) binds strongly to the GAPDH promoter but has no appreciable binding to the TIMP3 promoter (Figure 5C). These results suggest that EZH2-mediated trimethylation of H3K27 prevents RNAP II from binding to the TIMP3 promoter and thereby results in transcriptional silencing of the gene. We next determined whether H3K27 trimethylation is coupled with promoter DNA methylation for the repression of TIMP3, we treated PC3 cells with DZNep or the DNA methylation inhibitor 5-Aza followed by ChIP analysis. EZH2 knockdown or DZNep treatment markedly inhibited both H3K27 trimethylation and DNA methylation (MeCP2, methyl CpG binding protein 2) of the TIMP3 promoter; in contrast, 5-Aza treatment inhibited only DNA methylation but had no significant effect on H3K27 trimethylation of the promoter (Figure 5D). Furthermore, treatment of PC3 cells with DZNep or 5-Aza resulted in derepression of TIMP3 expression to almost the same degree (Figure 5E). Thus, these results suggest that transcriptional repression of the TIMP3 gene occurs through the EZH2-mediated H3K27 trimethylation and subsequent DNA methylation. Epigenetic silencing of TIMP2 expression by EZH2 We next investigated whether, like TIMP3, other three TIMPs (TIMP1, TIMP2 and TIMP4) are transcriptionally repressed by PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30393