Fig 2: Interaction with p130 is essential for the recruitment of RBP2 onto the E2F-responsive promoters. (A) Mutation of LxCxE motif in RBP2 leads to loss of binding of RBP2 to the E2F-responsive promoters. Chromatin affinity purification (ChAP) assay was performed in HeLa cells stably expressing either wild type (wt.) SFB-RBP2, SFB-RBP2 (E1377K), SFB-RBP2 (H483G, E485Q) or SFB-RBP2 (W1625A) are shown. The amount of RBP2 pulled down in each sample is shown in Supplementary Figure S5A. The error bars represent S.D. Tukey's multiple comparison test *P=0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P>0.05. (B) ChIP assay was performed in cells expressing either RBP2 wt., RBP2 E1377K or empty SFB-pcDNA5-FRT vector. The H3K4me3 levels on different promoters are shown. H3 levels are shown in Supplementary Figure S6B. The error bars represent S.D. Tukey's multiple comparison test was employed to calculate the significance *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P > 0.05. (C) RBP2 affects transcription of E2F-responsive gene. G1/S synchronized cells were treated with either siRNA against 3'UTR region of RBP2 or control (luciferase) siRNA in cells stably expressing either empty vector, RBP2 wt., RBP2 E1377K or RBP2 H483G, E485Q. cDNA synthesized from total RNA was amplified using real-time PCR for indicated mRNAs. All signals were normalized to actin mRNA levels from respective samples. The expression level is relative to control siRNA-treated cells (which is arbitrarily set to 1) from respective cell line but for ease of understanding only one control bar is shown. The error bars represent S.D. Dunnett's multiple comparison test was employed to calculate the significance*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P > 0.05.

Fig 3: p130 interacts with RBP2. (A) The association of E2F4 with RBP2 is mediated by p130. HeLa cells were transfected with shRNA either targeting p130 (two shRNA: #3and #5,were used for efficient knockdown) or Scramble (non-targeting) shRNA (see also Supplementary Figure S3B and S3C). Nuclear extract was prepared from both types of cells and used for pull-down experiment using GST or GST-E2F4 TAD. The blot was probed with antibody against p130 (panel a) and RBP2 (panel b). Panel c shows bead-bound GST or GST-E2F4TAD proteins stained with CBB. (B) The interaction between p130 and RBP2. MEFs were used for making nuclear extract and pull-down experiment was performed using GST or GST-p130 T/E1A (small pocket). The blot was probed with antibodies against RBP2 (panel a) and E2F4 (panel b). Panel c shows the CBB staining of bead-bound protein used in each lane (see also Supplementary Figure S3D and S3E). (C) Endogenous RBP2 interacts with endogenous p130. Endogenous RBP2 was immunoprecipitated from Hela cell NE with anti-RBP2 or IgG antibody and immunoblotted with anti- RBP2 (panel a), anti-p130 (panel b) and anti-E2F4 (panel c) antibody. (A–C) The positions of the molecular weight markers are indicated on the left.

Fig 4: RBP2 associates with the Transactivation Domain of E2F4. (A) Association of GST-E2F4 with endogenous RBP2. Bacterially expressed GST, GST-E2F4 and GST-E2F1 were used for pull down experiment using nuclear extract (NE) produced from Mouse embryonic Fibroblasts (MEFs). The blot was probed with anti-RBP2 antibody (panel a). Panel b shows the bead-bound GST, GST-E2F1 or GST-E2F4 proteins stained with Coomassie Brilliant Blue (CBB). (B) Cell cycle-stage specific immunoprecipitation of E2F4 from mouse embryonic Fibroblasts. MEFs were synchronized by Nocadazole block and timed release in normal media to obtain cells populations in early G1 (G1E) or G1/S stage (see also Supplementary Figure S1 and S2). NE from these phases was immunoprecipitated by E2F4 or IgG antibody. The blots were probed with anti-RBP2 and anti-E2F4 antibodies as indicated. E2F4 antibody used for immunoblotting is described in Supplementary Figure S1B. (C) Schematic structure of E2F4 protein. Full-length and deletions of E2F4 used in this study are shown. All deletions were expressed as C-terminal fusion of GST protein. Functional domains are indicated on top. Numbers indicate amino acids. FL, full length; TAD, transactivation Domain; DD, DP protein dimerization domain. (D) Mapping of E2F4 domain which associates with RBP2. GST-tagged E2F4FL (lane 5) and its deletions: GST-E2F4?TAD (lane 4) and GST-E2F4?DD (lane 3), were used for pull down experiment from HeLa NE. The blots were probed with anti-RBP2 (panel a) and GST (panel b) antibody. (E) The Association between E2F4 and RBP2 is mediated by p130. GST-E2F4TAD (wild type, lane 5) and its mutant forms GST-E2F4TAD M1 (Y 392 H, lane 3) and GST-E2F4TAD M2 (D 404 G, L405 P, F 406 L, and D 407 G, lane 4) (see also Supplementary Figure S3A) were used for pull down experiment from HeLa cell NE. The blot was probed with anti-RBP2 (panel a), p130 (panel b) and GST (panel c) antibody. (A–B, D–E) The positions of the molecular weight markers are indicated on the left.

Fig 5: p130 and RBP2 bind to the E2F responsive promoters in a cell-cycle stage specific manner for demethylation of H3K4me3. (A, B) p130 and RBP2 bind to E2F-responsive promoters in early G1 phase. Chromatin immunoprecipitation (ChIP) assay with E2F4, E2F1, p130 and RBP2 (A) and, H3K4me3 and H3K4me2 (B) antibodies, performed on the HeLa cells synchronised in early G1 (G1E; light grey) and G1/S (black) phase using double thymidine block are shown. See Supplementary Figure S4A for H3 and IgG ChIP. All experiments shown were done at least three, usually more times with similar results. (C, D) ChIP assay with p130 and RBP2 (A) and, H3K4me3 (B) antibodies, performed on mouse embryonic fibroblasts synchronized in early G1 (G1E; grey) and G1/S (black) phase, using Nocodazole block, are shown. See Supplementary Figure S4B for E2F4 and S4C for H3 and IgG ChIP. The data shows average of two independent experiments. (A–D) The immunoprecipitated DNA was quantified with real-time PCR and the results are plotted as percent input enrichment. The antibody used is indicated in the top right corner of each box. The error bars represent S.D. Student's t-test, *P = 0.05, **P = 0.01, *** P = 0.001, ****P = 0.0001, ns: not significant, P > 0.05.