Abstract
Previous work has provided evidence for E2F-dependent transcription control of both G1/S- and G2/M-regulated genes. Analysis of the G2-regulated cdc2 and cyclin B1 genes reveals the presence of both positive- and negative-acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. We find that E2F1, E2F2, and E2F3 bind to the positive-acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative-acting site. We also find that binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF-Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B-Myb interaction with the G2-regulated promoters coincides with the activation of the genes, and RNAi-mediated reduction of B-Myb inhibits expression of cyclin B1 and cdc2. The ability of B-Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B-Myb, which is an E2F-regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M.