Accumulation of E2F-4.DP-1 DNA binding complexes correlates with induction of dhfr gene expression during the G1 to S phase transition.

Previously genomic DNase I footprinting showed changes in protein binding to two overlapping E2F sites correlates with activation of dhfr gene expression at the G1/S boundary of the Chinese hamster cell cycle (Wells, J., Held, P., Illenye, S., and Heintz, N. H. (1996) Mol. Cell. Biol. 16, 634-647). Here gel mobility and antibody supershift assays were used to relate changes in the components of E2F DNA binding complexes in cell extracts to repression and induction of dhfr gene expression. In extracts from log phase cells, E2F complexes contained predominantly E2F-4 and E2F-2 in association with DP-1, and DNA binding assays showed complexes containing E2F-2 preferentially interact with only one of the two overlapping E2F sites. In serum starvation-stimulation experiments, arrest in G1 by low serum was accompanied by decreased levels of dhfr mRNA and the appearance of an E2F-4.DP-1.p130 complex. After serum stimulation, induction of dhfr gene expression was preceded by loss of the p130 complex in mid G1 and coincided with marked increases in two free E2F.DP-1 complexes in late G1, one of which contained E2F-4 and a second which contained an unidentified E2F. We suggest activation of dhfr gene expression after serum stimulation requires at least two temporally distinct processes, relief of p130-mediated repression and subsequent activation of transcription by free E2F.