Fig 1: Screening for Polycomb group proteins (PcGs) regulated by E2F1. (A) Gene expression heatmap of representative E2F1 targets and PcG genes that are differentially regulated in SK-Mel-147 upon E2F1 knockdown (11). Red and green shading indicates high and low gene expression relative to sh.control set to 1. (B and C) Analysis of PcGs at mRNA (B) and protein levels (C) following knockdown of E2F1 in SK-Mel-147 cell line. E2F1 target gene BIRC5 is shown as positive control. ß-actin served as loading control. (D and E) mRNA (D) and protein levels (E) of EPC1 and EZH2 in indicated cell lines after induction of ER.E2F1. Saos-2 or SK-Mel-29 cells stable expressing ER-E2F1 were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) or solvent for 24 hrs. Actin was used for loading control. (F) Expression of EPC1 (green) and E2F1 (red) in SK-Mel-29.ERE2F1 treated with 4-OHT for 24 h. Nuclei were counter-stained with DAPI (blue) and fluorescence was visualized by laser scanning microscopy.
Fig 2: Elevated EPC1 expression in invasive and chemoresistant tumor cells. (A) Endogenous EPC1 and E2F1 expression levels in different cancer cell lines as assessed by Western blot. Actin was used for loading control. (B) Protein levels of endogenous E2F1 and EPC1 in epirubicin-resistant MCF7 human breast cancer cells (MCF7-EPIR) and parental cells (left). Protein levels of E2F1 and EPC1 in cisplatin (cDDP, 30 µM) treated SK-Mel-147 cells expressing sh.E2F1 or sh.control compared with untreated cells (right). Actin was used for loading control.
Fig 3: E2F1 directly binds and activates EPC1 promoter. (A) Schematic representation of EPC1 gene promoter region (upper). Putative E2F1-binding sites were predicted using JASPAR database. Arrows indicate position of PCR amplicons in chromatin immunoprecipitation (ChIP) experiments relative to ATG (P1, -1292/-1148; P2, -825/-507; P3, -230/+42). ChIP assay shows the binding of E2F1 on EPC1 promoter in SK-Mel-29.ER-E2F1 treated with or without 4-OHT (1 µM) for 24 h (lower). Input represents 10% of sheared chromatin prior to immunoprecipitation. DNA was analyzed by semi-quantitative PCR with primers corresponding to the amplicons shown in upper panel. Primers amplifying the E2F1 sites in the EZH2 promoter were used as a positive control. (B) Schematic representation of EPC1 full-length promoter region (upper). Luciferase reporter assays in Saos-2, SK-Mel-29 and MCF7 cells upon E2F1 activation (lower). Relative light units (RLU) were measured after co-transfection of EPC1 promoter construct and E2F1. Bars indicate mean values ± SD of three independent experiments. (C) Dose-dependent regulation of EPC1 reporter by E2F1. SK-Mel-29 cells were co-transfected with EPC1 promoter construct and increasing amounts of E2F1 expression plasmid (0.25, 0.5, 0.75 and 1 µg). RLU were measured 24 h post-transfection. p73-prom-luc was used as positive control. (D) Luciferase reporter activities after co-transfection of SK-Mel-29 cells with EPC1 promoter construct and different E2F family members or DNA binding-deficient mutant E132 (1 µg). pcDNA was transfected as control in all reporter assays. E2F1, E2F2, E2F3 and E132 expression was verified by Western blot. Actin serves as loading control. Error bars indicate one SD of the mean. All experiments were performed three times independently. Statistical significance was determined by two-sided Student's t-test. **P = 0.01, ***P = 0.001.
Fig 4: EPC1 regulates E2F1 targets. (A) Equal amounts of RNA from sh.control and sh.EPC1 infected cells were subjected to microarray analysis. Affymetrix gene expression data were analyzed using hierarchical clustering. Gene expression levels for E2F target genes are displayed. Colors represent normalized expression levels. Red column represents high gene expression, while green column indicates low expression. (B and C) mRNA (B) and protein levels (C) of Bcl-2, BIRC5, p27 and Bim were regulated after knockdown of EPC1. SK-Mel-147 cells were infected with Lenti.shRNA against EPC1 or scrambled RNA and harvested 96 h later. GAPDH and actin were used as normalization as well as loading control for qPCR and Western blot, respectively. Bar graphs indicate the mean ± SD of two separate experiments. (D) SK-Mel-147 cells stably expressing sh.EPC1, sh.E2F1 or both were harvested for Western blot analysis of EPC1, E2F1, Bim and p27 expression (left). Actin was used as loading control. SK-Mel-147 stable cell lines expressing EPC1 shRNA or scrambled shRNA were transfected with E2F1 expression vector (right). 48 h after transfection cells were harvested and EPC1, E2F1, BIRC5 and Bcl-2 protein levels were analyzed by Western blot. Actin was used for loading control. (E) Binding of endogenous EPC1 to indicated E2F1-regulated promoters after 4-OHT treatment in SK-Mel-29.ER-E2F1 (left) and E2F1 knockdown in SK-Mel-147 cells (right) was determined by ChIP assays. (F) Recruitment of Tip60 and EZH2 to E2F target gene promoters and associated acetylation (Ac) or tri-methylation (Me3) levels in the presence and absence of EPC1. (E and F) Input represents 10% of sheared chromatin prior to IP. Statistical significance was determined by two-sided Student's t-test. *P = 0.05.
Fig 5: Modulation of E2F1 transcriptional activity by EPC1. (A) Regulation of E2F1 transcriptional activity by EPC1. SK-Mel-29 cells were transfected with 0.5 µg Bcl-2-luc (left) or BIRC5-luc reporter plasmids (right) together with expression vectors for E2F1 (0.5 µg) in the absence or presence of EPC1 expression vector (1, 2, 3 µg). E2F1 and EPC1 expression was confirmed by Western blot. Actin was used as loading control. (B) SK-Mel-29 cells were cotransfected with the p27-luc reporter plasmid (0.5 µg) and expression plasmids for E2F1 (0.5 µg) in the absence or presence of EPC1 expression vectors (3 µg). E2F1, EPC1 and actin expression was confirmed by Western blot. (C and D) H1299 cells were transfected with Bcl-2-luc (C) or p27-luc reporter plasmids (D) together with expression vectors for Flag-tagged full-length E2F1 or Flag-tagged E2F1 mutant in the presence or absence of EPC1 expression vector. Luciferase assays were performed after 24 h using pcDNA plasmid as control that was set to 1. Data are presented as the mean with standard deviation of technical replicates (duplicates or triplicates) in an experiment representative of several independent others. (E) Schematic representation of full-length and truncated E2F1 proteins. Western blot shows expression of Flag-tagged full-length E2F1 or Flag-tagged E2F1 mutant and EPC1 in H1299 cells. Actin was used as loading control. Statistical significance was determined by two-sided Student's t-test. *P = 0.05, **P = 0.01, ***P = 0.001.
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