Activation of the murine dihydrofolate reductase promoter by E2F1 - A requirement for CBP recruitment

The E2F family of heterodimeric transcription factors plays an important ro le in the regulation of gene expression at the G(1)/S phase transition of t he mammalian cell cycle. Previously, we have demonstrated that cell cycle r egulation of murine dihydrofolate reductase (dhfr) expression requires E2F- mediated activation of the dhfr promoter in S phase. To investigate the mec hanism by which E2F activates an authentic E2F-regulated promoter, we preci sely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2 F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the d hfr promoter and protein-protein interactions with CBP, transcription facto r (TF) IIH, and TATA-binding protein (TBP), However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation betwe en activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP , Finally, transactivator bypass experiments indicated that direct recruitm ent of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates th e dhfr promoter.