Research Article Fabrice Sircoulomb et al. Figure 5. ZNF703 overexpression regulates ER and E2F1 transcriptional activity. A. MCF7 GFP-ZNF703 cells were fixed and immunostained with anti-ER. ZNF703 (green) and ER (red) do not co-localize and are expressed in different cells (white arrowheads). Nuclei were counterstained with Hoechst (blue). B. Oestrogen receptor-dependent transcriptional activity was evaluated using a luciferase reporter system (p17M-ERE-Luc) in the pre- sence and absence of 17-b estradiol (E2) in MCF7 GFP and GFP-ZNF703 breast cancer cell lines. ER-dependent transcriptional activity decreases in MCF7 GFP-ZNF703 cells as com- pared to GFP expressing cells, independently of E2 induction. C. Analysis of ER, FOXA1, and GATA3 protein expression by immunoblotting in MCF7 GFP- ZNF703 and MCF7 GFP breast cancer cell lines. MCF7 GFP-ZNF703 cells show a decrease in ER- associated proteins. D. E2F1-dependent transcriptional activity was evaluated using two different luciferase reporter systems. Both vectors code for the luciferase protein under the control of either three E2F1 responsive elements (pGL2- 3XE2F1-Luc) or CCNE1 promoter (pGL3- CCNE1-Luc), which is a direct E2F1 target gene. E2F1-dependent transcriptional activity decreases with ZNF703 overexpression. E. Analysis of RB1 and P27Kip1 protein expression by immunoblotting in MCF7 GFP-ZNF703 and MCF7 GFP cells. ZNF703 overexpression induces a hyperphosphorylation of RB1 protein and a decrease in P27Kip1 protein expression. a- Tubulin protein expression was used as a control. F, G. To test E2F1 or ER transcriptional activity in luminal B primary tumours compared to luminal A, we performed a GSEA to screen the activated transcription factors from the Broad Institute (MSigDB C3: motif gene sets TFT). We represented the different gene sets that share a transcription factor binding site defined in the TRANSFAC database enriched in luminal B tumours, compared to luminal A tumours. Each gene sets is plotted according to its association to either luminal A or B tumours ( log(diff GSEA p-value)); E2F1-related gene sets are in red, ER- related gene sets are in blue and other gene sets are in grey (F). A total of 117 gene sets were differentially regulated between luminal B and luminal A primary tumours (FDR q- value < 0.25; Nominal p-value < 0.05). E2F1- related gene sets are significantly enriched in luminal B tumours, whereas luminal A tumours, gene expression programme is enriched in ER-related gene sets (G). to luminal A tumours. A total of 117 gene sets were differentially regulated between luminal B and luminal A tumours (FDR q- value < 0.25; Nominal p-value < 0.05) (Fig 5F, Supplementary Table 11). Among these gene sets, 10 were associated with luminal B tumours and 107 with luminal A tumours. Eight out of the ten gene sets (80%) associated with luminal B tumours were related to genes containing E2F1 cis-regulatory motifs. Among the 107 gene sets associated with luminal A tumours, 38 www.embomolmed.org EMBO Mol Med 3, 153–166 2011 EMBO Molecular Medicine 159