Fig 2: Regulation of the endogenous RIP140 gene.(A) The levels of E2F1, cyclin D1 and RIP140 mRNAs were measured in MCF-7 cells 2 days after transient transfection with plasmids allowing the overexpression of the E2F1/DP1 heterodimer. Statistical analysis was performed using the Student t test (***p<0.0001). (B) HeLa cells were synchronized by 2 mM hydroxyurea (HU) and, after block release the distribution of cells in G1, S and G2/M was analyzed by flow cytometry (upper panel). Cyclin B1 and E, and RIP140 mRNA levels were quantified by real-time quantitative RT-PCR as described in Materials and Methods (lower panel). The results are expressed in arbitrary units after normalization by RS9 mRNA levels. Values are the means ± S.D. of three independent experiments.

Fig 3: Repression of E2F transactivation by RIP140 and pocket proteins.(A) The human WT RIP900-luc reporter plasmid (25 ng) and E2F1+/- DP1 (25 ng each) were transiently transfected in MCF-7 cells with a dose response of RIP140 expression plasmids (0, 50, 100 or 200 ng). (B) ChIP experiments using immunoprecipitation (IP) of E2F1 and RIP140 on the E2Fa site of the human RIP140 promoter. The cyclin A2 promoter was used as a positive control. Data are expressed as percent of the PCR signal obtained with the amount of chromatin used for IP (input). Statistical analysis performed using the Student t test (***p<0.0001 and *p<0.05). (C) MCF-7 cells were transfected with human RIP900-luc or cyclin E-luc reporter plasmid (25 ng), with E2F1+/- DP1 (25 ng) and pocket proteins Rb or RIP140 expression vectors (200 ng or 250 ng) in 96 well plates in the indicated combinations. Results were expressed as in Figure 2D (n = 4).

Fig 4: Effect of DP1 and Sp1 on the transactivation by E2F1.(A) MCF-7 cells were cotransfected respectively with the human RIP140 and cyclin E promoter reporter plasmids (25 ng) together with E2F1 (25 ng) and increasing dose of DP1 factors (0, 5, 25, 50 ng) (left). Results were expressed as in Figure 2D (n = 4). (B) The human RIP140, ARF and DHFR promoter reporter plasmids were tested with E2F1+/- DP1. The luciferase activity with E2F1 alone overexpressed was normalized at 100% (n = 3). (C) Different mutants of the RIP140 promoter (hRIP900wt, ?PScwt, mE2Fde) were tested for the response to overexpression of E2F1+/- DP1. Results were expressed as in Figure 5B (n = 4). (D) The effect of two deletion mutants of DP1 protein (DP1 ?107–126 that abolishes DNA binding and ?205–277 for E2F binding) on E2F1 activity was measured on hRIP140 and cyclin E promoters. Results were expressed as in figure 2D (n = 3). (E) MCF-7 cells were transiently transfected as indicated in Materials and Methods , with the point mutants for Sp1 binding sites (#6/7 or #8 shown in Figure 3A) of the proximal sequence ?PSc reporter plasmid (25 ng) together with expression vectors for E2F1+/- DP1 (25 ng each). Results were expressed as in Figure 2D (n = 3). (F) E2F1 mutated for Sp1 interaction (E2F1?Sp1) was used +/- DP1 on the proximal promoter region (?PSc reporter plasmid), in the same conditions as above. Results were expressed as in Figure 2D (n = 3).

Fig 5: Importance of the proximal region of the RIP140 promoter.(A) Schematic representation of the RIP140 promoter sequence (RIP900 plasmid) showing the E2Fs binding sites (a, b, c, d and e, indicated with grey ovals), the Sp1 binding sites (open circles) and the different 5' deletion mutants (?PAc, ?PPc and ?PSc). (B) MCF-7 cells were transiently transfected with the human RIP140 promoter reporter plasmids (25 ng) containing mutations of the E2Fs binding sites together with expression vectors for E2F1 and DP1 (25 ng each). In the left panel, each mutant has one E2F site mutated (mE2Fa, b, c, d and e) or multiple mutations which abolish all sites (E2Fnull). In the right panel, only the indicated site remains intact (E2Fa, b, c, d and e). (C and D) The same experiments as above were repeated with deletion mutants (respectively ?PAc, ?PPc, ?PSc) of the RIP140 promoter (C) or with the proximal promoter region (mutant ?PSc) with point mutations of the E2F binding sites (mE2Fd and e) (D). All results were expressed as described in Figure 2D and representative experiments are shown (n = 3).