Transcriptional silencing of TIMP2 by EZH2.
(A) Schematic representation of the promoter regions of the TIMP2 gene. The bent arrow represents the transcription start sites (+1). The lines below the TIMP2 locus represent the regions amplified by PCR using primer sets (set 1, set 2, and set 3 in (B); set 2 in (C) and (D)) designed to amplify regions containing the YY1-binding sites (see Table S3). (B–C) Chromatin immunoprecipitation (ChIP) experiments were performed with chromatin isolated from PC3 (B) and DC145(C) cells using antibodies to EZH2, 3meH3K27, and IgG. Results of our ChIP in both PC3 and DU145 cells are very similar to each other. Shown are ChIP results in DU145 cells. (D) Epigenetic regulation of TIMP2 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 µM), 5-Aza (10 µM), DMSO (control), or EZH2-specific shRNA (shRNA1). (E) qRT–PCR analysis of expression of TIMP2 in PC3 cells after treatment with DZNep (1 µM), 5-Aza (10 µM), or DMSO (control) for 2 days. The experiments were repeated at least twice and results were expressed as a ratio of TIMP2 mRNA to GAPDH mRNA control. * P<0.05, ** p<0.005 (as compared with control (DMSO)).