Fig. 4: DREAM and RB:E2F complexes bind to the BRCA1 and BRCA2 promoters.

A Chromatin immunoprecipitations (ChIPs) were performed with cross-linked chromatin from serum-starved (0 h) or restimulated (22 h) T98G cells. Antibodies targeted E2F4, E2F1, E2F3, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. B ChIPs were performed with cross-linked chromatin from starved (0 h) or restimulated (22 h) RPE-1 cells. Antibodies targeted E2F4, LIN9, LIN37, A-MYB, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. A, B Mean ± SD; two-way ANOVA; n = 3; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. C Nuclear extracts of density-arrested RPE-1 cells and cells restimulated for 20 h were employed for DNA affinity purification using biotinylated wt and mutant (E2F-A, E2F-B, and A/B) BRCA1 promoter probes and a promoter probe of the late cell cycle gene Cyclin B2 as well as a mouse promoter probe of the early cell cycle gene Dhfr. As a negative control, a fragment of the mouse Gapdhs promoter (neg. Ctrl.) was used. D DREAM components (p130, E2f4, Lin37, Lin54, and Lin9) were purified from nuclear extracts of density-arrested NIH3T3 mouse cells by DNA affinity purification and detected by Western blot. Binding to the human BRCA2 wild-type promoter probe (wt) was compared with binding to mutant probes (E2F-A, E2F-B, and A/B). Background protein binding was determined with a probe of the mouse cyclin B2 CHR mutant promoter (neg. Ctrl.). E p130, RB, E2F4, and LIN37 were purified from nuclear extracts of proliferating HCT116 human cells by DNA affinity purification and detected by Western blot. Binding to the wild-type promoter probe (wt) was compared with binding to E2F site mutant probes (E2F-A, E2F-B, and A/B) as well as binding to the promoter of the late cell cycle gene Cyclin B2 and the mouse promoter of the early cell cycle gene Dhfr. As a negative control, binding to a fragment of the mouse Gapdhs promoter was tested (neg. Ctrl.).