Fig 2: PRC2 facilitates PARP1 silencing by associating with RB1-E2F1-HDAC1-BRM/BRG1 repressor complex in G1 arrested cells. Epigenetic modifications (a,b and d), recruitment of HDAC1 (c), EZH2 (e), BRM (g), BRG1 (h) to PARP1 promoter and the density of histone H3 (f) were compared by ChIP between proliferating cells and cells arrested in G1 phase by their treatment with mimosine (Mim, 200 µM) for 48 h. Composition of PARP1 repressive complex in G1-arrested cells was determined by pulling down HDAC1 and followed by WB detection of RB1, E2F1, EZH2, BRM and BRG1 (j). The expression of PARP1 in HSPC supplemented with mimosine (50 µ) and additionally with 5 mM iHDAC, 125 nM iEZH2 and the mixture of 5 mM iHDAC and 125 nM and iEZH2 for 24 h was analyzed with western blot (k) and real-time PCR (l). Expression of PARP1 in G1-arrested cells treated with BRM/BRG1 inhibitor (iBRM/iBRG1 - PFI-3 – 1 µM) was determined with real-time PCR (n). Changes in epigenetic marks and in protein composition in PARP1 promoter upon THP-1 cells treatment with iHDAC/iEZH2/Mim versus Mim alone were monitored with ChIP (n–t).

Fig 3: E2F1 and CCND1 are two direct targets of miR-93.a 210 targets were predicted for miR-93 in four online databases. b The mRNA expression levels of 12 candidate target genes were measured in Bads-200 cells transfected with NC/miR-93. c, d E2F1 and CCND1 were upregulated in tumor samples of breast cancer, by comparing their mRNA levels in clinical tissues (c) and TCGA samples (d). e The dual luciferase assays were performed in Bads-200 cells. f MiR-93 notably downregulated the protein levels of E2F1 and CCND1 in Bads-200 cells, with or without PTX. g Representative images of Bads-200 xenografts with IHC staining of E2F1 and CCND1. Scale bars: (main) 200 µm, (inset) 50 µm. h, i The mRNA and protein levels of E2F1 and CCND1 were measured in BCap37, Bats-72, and Bads-200 cells. Bars indicate the mean ± SD of three independent replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Genome-wide effects of PRMT5 inhibition dependent on E2F1.a Sub-G1 fraction analysis of WT E2F1 p53+/+ or p53-/- HCT116 cells and E2F1 Cr p53+/+ or p53-/- HCT116 cells, treated for 6 days with 1 µM PRMT5 inhibitor (T1–44) or less active compound (T1–68) (i). (ii) A representative example of an immunoblot is included to demonstrate the input protein levels of E2F1, p53 and symmetric arginine methylation (SDMe); actin included as a loading control. n = 3. b An immunoblot displaying E2F1, p53 and SDMe protein levels in WT E2F1 and E2F1 Cr p53-/- HCT116 cells after 48 h of 1 µM PRMT5 inhibitor treatment (T1–44). c Venn diagrams showing the overlap of genes up- or downregulated with over 30% change (adjusted P-value threshold < 0.01) in each treatment condition with respect to DMSO-treated p53-/- HCT116 cells, filtered for genes containing an E2F1 motif in their proximal promoter region (-900 to +100) based on ENCODE. T1–44, cells treated with PRMT5 inhibitor; E2F1Cr, E2F1 CRISPR cells; E2F1Cr T1–44, E2F1 CRISPR cells treated with PRMT5 inhibitor, n = 3 (see also Supplementary Data S1–S3). d Heatmap of differentially expressed E2F1 target genes (adjusted P-value = 0.01, 30% change) in each treatment condition with respect to DMSO-treated p53-/- HCT116 cells. 2Log fold-change expression values were converted to the Z-score. Increased expression levels are indicated with darker red colouring, whereas decreased expression levels are indicated with darker blue colouring. Ivory colour represents no significant change (see also Supplementary Data S1–S3). e Cluster analysis of genes co-expressed with cortactin/CTTN identified with weighted correlation network analysis (WGCNA) from triplicate experiments. Genes connected with focal adhesions are highlighted in red. WT E2F1, wild-type E2F1 p53-/- HCT116 cells treated with DMSO; WT E2F1 T1–44, wild-type E2F1 p53-/- HCT116 cells treated with T1–44.

Fig 5: Combined E2F overexpression increases Sgo1 protein stability but not its transcription levels(A) Semi-quantitative PCR was performed for the Sgo1 transcript with cDNA synthesized from RNA extracted from cells expressing vector control (pBH) or cells overexpressing E2F1, E2F2, and E2F3a. Actin was used an internal control. (B) Real-time PCR was performed to quantify levels of Sgo1 mRNA. (C) Cells were treated with cycloheximide (2.5 µg/mL), cell protein lysates were prepared at the indicated time points, and Western blots were probed with antibodies recognizing cyclin B1, BubR1, and Sgo1. Actin was used as an internal control.