Activation of MMPs by EZH2 Figure 5. Transcriptional silencing of TIMP3 by EZH2. (A) Schematic representation of the promoter region of the TIMP3 gene. The bent arrow represents the transcription start sites (+1). The lines below the TIMP3 locus represent the regions amplified by PCR. (B–C) Immunoprecipitated DNA was analyzed by PCR with specific primer sets (also see Table S3). Chromatin obtained from PC3 (B) and DU145 (C) cells were immunoprecipitated using antibodies to EZH2, trimethyl-histone H3K27 (3meH3K27), RNA Polymerase II (Pol II), and IgG. The immunoprecipitated DNA was analyzed by PCR with the primer sets to amplify the region 4 in Figure 5A. The GAPDH promoter was used as a negative control. Each ChIP experiment was repeated at least three times and a representative experiment is shown. (D) Epigenetic regulation of TIMP3 expression in PC3 cells. ChIP assay was carried out using the antibodies to EZH2, 3meH3K27, MeCP2, RNAP II (Pol II), and IgG control after treatment with DZNep (1 mM), 5-Aza (10 mM), DMSO (control), or EZH2-specific shRNA (shRNA1). The immunoprecipitated DNA was analyzed by PCR with the primer sets to amplify the region 4 in Figure 5A. (E) qRT–PCR analysis of expression of TIMP3 in PC3 cells after treatment with DZNep (1 mM), 5-Aza (10 mM), or DMSO (control) for 2 days. The experiments were repeated at least twice and results were expressed as a ratio of TIMP2 or TIMP3 mRNA to GAPDH mRNA control. * P,0.05, ** p,0.005 (as compared with control (DMSO)). doi:10.1371/journal.pone.0030393.g005 subcloning corresponding cDNAs into pLenti4/V5-DEST vector (Invitrogen). We obtained lentiviral short hairpin RNAs (shRNAs) specific for EZH2 from Sigma-Aldrich (MISSIONH shRNA). Biologically active shRNAs were generated from the pLKO.1-puro vector, utilizing the Polymerase III U6-RNA promoter. The shRNA sequences for EZH2 knockdown were 59CCGGTAT- GATGGTTAACG GTGATCACTGAGTGATCACCGTTAA- CCATCATATTTTTG-39 (TRCN18365, sh1) and 59-CCGGG- CTAGGTTAATTGGGACCAAACTCGAGTTTGGTCCCAA- TTAACCTAGCTTTTTG-39 (TRCN40074, sh2). Because these two shRNAs are equally effective in suppression of EZH2 mRNA expression, we used EZH2-specific shRNA1 (sh1) in some of our experiments. For viral production, 293T cells were cotransfected with pLKO.1, pLenti4-EZH2-WT, pLenti4-EZH2-H689A or pLKO.1-EZH2-specific shRNA, and packaging plasmids (psPAX2 and pCMV-VSV-G) using CalPhos Mammalian Transfection Kit (Clontech). The virus-containing supernatants were collected 48– 72 h after transfection and aliquoted to be stored at 280uC. Quantitative real-time PCR Expression of TIMP and GAPDH mRNA was measured by real- time quantitative PCR using SYBR Green PCR Mastermix (Quanta biosciences). Total RNA was isolated from prostate cancer cells using Trizol Reagent (Invitrogen). 2 mg of total RNA was used for reverse transcription reactions using qScript cDNA SuperMix Kit following the manufacturer’s instructions (Quanta biosciences). Results shown were a representative of three independent experiments. The primers used in this study are listed in Table S3. Western blot analysis Cells were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche Applied Science) or lysed directly in 26 SDS-PAGE sample buffer, and subjected to Western blot analysis using antibodies against EZH2 (BD Biosciences, 612666), TIMP2 (Millipore Inc., MAB3310), TIMP3 (Abcam, ab39184), 3meH3K27 (Millipore Inc., 07-449), MeCP2 (Millipore Inc., 07-013), RNA polyersase II (Millipore Inc., 05-623), and Actin (Sigma-Aldrich, A2066). Protein bands were visualized using West Pico chemiluminescent detection system (Pierce) and subjected to densitometry analysis using the ChemiDoc XRS system with Quantity One software (Bio-Rad Laboratories). Immunostaining for EZH2 and TIMP3 4–5-mm-thick, formalin-fixed, paraffin-embedded human pros- tate cancer and normal sections were purchased from Cybrd and PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30393