Figure 5

H3K9ac and E2F1-mediated transcriptional regulation of DNMT1. Ai, RT-PCR showing GCN5, PCAF and E2F1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii, the results from three independent experiments are represented as mean ± SD. Bi, EdU labeling showing proliferation of GCN5, PCAF and E2F1-silenced and control cells. Blue, Hoechst 33342 labeling of cell nuclei; Red, EdU labeling of nuclei of proliferative cells. Bii, the EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst33342 positive cells. Ci and Cii, analysis of histone modification H3K9ac and transcription factor E2F1 enrichment around the E2F1 motif within the CpG islands after the deletion of GCN5, PCAF or E2F1. D, The interaction of E2F1 and GCN5 or PCAF were examined by the immunoprecipitation of cell extracts with an antibody to E2F1, and co-immunoprecipitation of E2F1, GCN5 and PCAF by western blot analysis. Results of Figure5A-D were obtained in BRCA1-mutated breast cancer cells, and the same results were also obtained in 293 T cells and non-BRCA1 mutated breast cancer cells. Ei-Ev, the DNMT1 expression levels after deletion of H3K9ac and E2F1 around the E2F1 motif in 293 T cells, and 15 primary non-mutated and BRCA1-mutated breast cancer and their normal breast cells. Each experiment was repeated four times for 293 T cells and primary breast cells of each patient. Bar graphs show mean ± SD. * P < 0.05 vs. Control.