Fig. 3.

The Dom and MBT proteins form complexes with E2F and are present on E2F target gene promoters. (A) The Dom and MBT proteins associate with E2F. Cellular extracts from regular S2* cells or S2* cells transiently transfected with a Flag-L3mbt expression vector were subjected to affinity purification with antibodies for Dom, Rbf1, dE2F1, and Flag and Western blot analysis with anti-dE2F1. No signal was observed in control precipitation in which cellular lysates from untransfected cells were incubated with the anti-Flag antibody. (B) The Dom, H2Av, and MBT proteins are present at the promoters of E2F target genes. Chromatin from cross-linked regular S2* cells was immunoprecipitated with anti-Dom (Dom), anti-H2Av (H2Av), and anti-Flag (control) antibodies. In a parallel experiment (for the right three columns), extracts from S2* cells expressing Flag-L3mbt were precipitated with anti-Flag (F-L3mbt) and nonspecific IgG (control) antibodies. The pelleted chromatin fragments were analyzed by PCR with primers specific for PCNA, DNA polα (Pol), CG14545, CG8399, CG3105 (belonging to the groups A to E, respectively), and RP49 promoters. (C) Recruitment of Dom and MBT proteins to E2F targets depends on E2F. Flag-L3mbt transfected S2* cells were treated with RNAi of dE2Fs (dE2F1, or combined dE2F1 and dE2F2), and then subjected to ChIP with Dom and Flag antibodies, respectively. Several E2F targets were selected for promoter-binding assay. (D) Deposition of H2Av at selected E2F target promoters depends on Dom and dE2F1. S2* cells were incubated with Dom or dE2F1 dsRNAs and subsequently subjected to ChIP assays with an anti-H2Av antibody.