Fig 2: DREAM, E2F, and FOXM1/B-MYB/MuvB complex components bind to the MKI67 promoter in vivo and in vitro.E2F4 and p130 were detected as representatives of DREAM. B-MYB is specific for B-MYB-MuvB. NF-YA is a subunit of NF-Y. E2F1/2/3 proteins are detected to represent E2F proteins independent of DREAM. FOXM1 detection indicates FOXM1-MuvB binding. LIN37 and LIN54 are representative components of all MuvB-based complexes. A In vivo protein binding to the MKI67 promoter in serum-starved (0 h, gray) and restimulated (22 h, blue) human T98G cells was analyzed via chromatin immunoprecipitation (ChIP) followed by real-time qPCR. Mean values ± SD are given of one representative experiment with two technical replicates for each precipitate. B–F Nuclear extracts from proliferating HeLa cells (B), density-arrested human T98G cells (C), restimulated T98G cells (D), serum-starved mouse NIH3T3 cells (E), as well as restimulated NIH3T3 cells (F) were subjected to DNA affinity purification, to assay in vitro binding of complex components by western blot analysis. Fragments of the MKI67 promoter containing wild-type (wt) or mutant promoter fragments with mutations in CHRprox, CHRdist, and CDE sites were used for DNA probes. 2CHRmut, CDE/CHRdist, and 3mut mutant probes carry combined mutations in CHRdist/CHRprox, CDE/CHRdist, and CHRdist/CHRprox/CDE, respectively. As a negative control of background binding (Ctrl.), a fragment of the mouse Gapdh2 promoter was employed. Input samples were taken from the nuclear extract before purification. For detection with one specific antibody, all samples were run on the same gel and analyzed via western blotting.

Fig 3: YAP1 knockdown inhibits the proliferation of bladder cancer cells in vitro. The expression of YAP1 subgroup target genes was partially restored by YAP1 expression. (a) UC3 cells were infected with lentivirus expressing shYAP1 or scrambled shRNA (scRNA). YAP1 mRNA and protein levels were detected using qRT–PCR and Western blotting, respectively. (b) Cell viability was analysed at 0, 24, and 48 h with the MTT assay to determine the proliferation of the UC3 cell line. The data are presented as the means ± standard deviations of three independent experiments. (c) A clonogenic assay was performed with control and YAP1 knockdown UC3 cells. Cells were stained with a crystal violet solution, and colonies were counted in three independent experiments. Invasion and migration assays of UC3 cells transfected with shNTS and shYAP1 for 24 h were performed using Boyden chamber assays. Data are presented as the means ± standard deviations of three experiments. (d) The YAP1 activation 1 (YA1) genes FOXM1, E2F1, CCNA2, AURKA, BIRC5, LMNB1, and TEAD2 were validated at the mRNA level after YAP1 knockdown in the UC3 cell line. The mRNA levels of CTGF, CYR61, and ZEB1 included in YAP1 activation 2 (YA2) group were verified using qRT–PCR. The YAP1 inactivation (YI) genes GATA2, SMAD3, and SMAD6 were validated in YAP1 knockdown cells using qRT–PCR. The data are presented as the means ± standard deviations of three independent experiments. (e) Levels of the YAP1, CTGF, CYR61, E2F1, and FOXM1 proteins were detected using Western blotting. (f) In qRT–PCR experiments, the levels of YA1 and YA2 subgroup genes were detected following YAP1 overexpression, and the levels of YA1 and YA2 subgroup genes were restored by YAP1 overexpression. This experiment was performed three times. ns, not significant; *, p < 0·05; **, p < 0·01; and ***, p < 0·001.

Fig 4: Regulation of E2F1, PRMT5 and cortactin/CTTN expression in human cancer.a Heatmap representation of expression levels for E2F1 and PRMT5 genes in human cancers (pancreatic, colon, and head and neck cancer) compared with normal tissue, generated using Xena Browser. Data from The Cancer Genome Atlas and Genotype-Tissue Expression projects were used to display expression levels from cancer tissue or healthy tissue, respectively. Each line on the heatmap represents a single patient sample and each column represents the expression level of a particular gene (E2F1, PRMT5). High expression levels are indicated with darker red colouring, whereas low expression levels are indicated with darker blue colouring (i); graphical representation of absolute expression values is also shown (ii). b Expression level of the cortactin/CTTN gene in different pancreatic cancer data sets (Ishikawa et al.50 (i), Pei et al.51 (ii) and Badea et al.52 (iii) data sets), generated using the Oncomine online tool, were plotted as log2 median intensity. c The expression level of the cortactin/CTTN gene in different colorectal cancer data sets (Kaiser et al.46 (i), Hong et al.47 (ii), Skrzypczak et al.48 (iii), and Sabates-Beliver et al.49 (iv) data sets), generated using the Oncomine online tool, were plotted as log2 median intensity. d The expression level of the cortactin/CTTN gene in different head and neck cancer data sets (Cromer et al.53 (i), Estilo et al.54 (ii), Talbot et al.55 (iii), and Ye et al.56 (iv) data sets), generated using the Oncomine online tool, were plotted as log2 median intensity. e Kaplan–Meier curves of overall survival of patients with pancreatic cancer for E2F1, PRMT5 and CTTN genes are presented; generated using Xena Browser. For each analysis, patients were divided into two groups: one with high expression (above or equal to the mean gene expression of all patients; red line) or one with low expression (below the mean gene expression of all patients; blue line). f The model describes the interplay between PRMT5, E2F1 and motility regulating genes in migration, invasion and adhesion. By regulating the methylation of E2F1, it is proposed that PRMT5 can influence the expression level of motility-related genes. In the absence of E2F1 methylation (under conditions of PRMT5 inhibition), the expression of these genes is decreased, which results in a loss of cell motility.

Fig 5: E2F1 also interacts with alternatively spliced transcripts from the MECOM gene.(A) Schematic representation of exon structure for the MECOM gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads spanning exons 1 and 3 (indicated by the red numbering), which occurs in MECOM transcript V7 (highlighted in red text). (B) U2OS (i), MCF7 (ii), or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor as indicated. An anti-E2F1 RIP was then performed, and coimmunoprecipitated MECOM transcript variant V7 was analyzed by qRT-PCR using specific primers. Input protein levels for the U2OS experiment are also included (iv), while the input protein levels for HCT116 and MCF7 cells are the same as those displayed in Fig. 3 (E and F). n = 2. (C) Examination of the promoter region of the MECOM gene identified an E2F1 DNA binding motif lying within the first intron of V7 or the second intron of V4 (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in HCT116 or HCT116 E2F1 CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in MECOM or against the known E2F motif in the promoter sequence of CDC6 (ii). Input protein levels are the same as those displayed in Fig. 4F. n = 3. (D) U2OS cells (i) or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor, where indicated. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific MECOM transcript variants or total MECOM RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS/HCT116 cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated cells is also displayed over each bar. Input protein levels for U2OS cells are also displayed (ii), while the input protein levels for HCT116 cells are the same as those displayed in Fig. 4E. n = 4.