AN INVERTED REPEAT MOTIF STABILIZES BINDING OF E2F AND ENHANCES TRANSCRIPTION OF THE DIHYDROFOLATE-REDUCTASE GENE

An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular and viral genes. This element, 5' TTTCGCGCCAAA-3', is comprised of two overlapping, oppositely oriented sites which match the consensus E2F site (5'-TTT(C/G) (C/G)CGC-3'). Recent work has shown that E2F binding activity is composed of at least six related cellular polypeptides which are capable of forming DNA-binding home- and heterodimers. We have investigated the binding of cellular E2F activity and of home- and heterodimers of cloned E2F proteins to the inverted repeat E2F element. We have demonstrated that mutations in this element that abolish its inverted repeat nature, while preserving a single consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes, Furthermore, the mutations decrease in vitro transcription and transient reporter gene expression 2-5-fold, an effect equivalent to that of abolishing E2F binding altogether. These data suggest a functional role that may explain the conservation of inverted repeat E2F elements among the DHFR promoters and several other cellular and viral promoters.