To gain an understanding of the role the E2F1 transcription factor plays in cell physiology, the full length protein (E2F1) and an amino terminal deletion of 87 amino acids (E2F1d87) were constitutively expressed in NIH3T3 fibroblasts. Multiple cell lines were generated for each construct. These cells do not proliferate in media containing low serum and do not proliferate in soft agar, indicating that they are likely not transformed. However, both sets of cell lines show increased DNA synthesis and increased numbers of cells in S phase when cultured in media containing low serum, compared to the control cell lines. Cells expressing E2F1d87 (but not E2F1) have an extremely rounded morphology when cultured in 10% serum-containing media. These rounded cells lack detectable microfilaments, microtubules, and focal contacts. However, when these cells are cultured in low serum-containing media (0.5%), they attain the flattened morphology and cytoskeletal structure of normal NIH3T3 cells.

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The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.

The E2F1 transcription factor was constitutively expressed in NIH3T3 fibroblasts to determine its effect on the cell cycle. These E2F1 cell lines were not tightly synchronized in G0 phase of the cell cycle following serum starvation, as are normal fibroblasts. Instead, the cells are spread throughout G0 and G1 phase with a portion of the population initiating DNA synthesis. Upon serum stimulation, the remaining cells in G0/G1 begin to enter S phase immediately but with a reduced rate. Constitutive expression of E2F1 appears to primarily affect the G0 phase, since transit of proliferating E2F1 cell lines through G1 phase is the same as control cells. Consistent with a shortened G0 phase, the E2F1 cell lines have a significantly reduced cellular volume. Additionally, the first S phase after serum stimulation, but not subsequent S phases, is nearly doubled in the E2F1 cell lines compared with control cells. Cell lines expressing a deletion mutant of E2F1 (termed E2F1d87), known to significantly affect cell shape, have cell cycle and volume characteristics similar to the E2F1 expressing cells. However, all S phase durations are considerably lengthened and the cells demonstrate delayed growth after plating.

The E2F1 transcription factor or an amino terminal deletion mutant termed E2F1d87 was constitutively expressed in NIH3T3 fibroblasts. Cells expressing wild-type E2F1 display a morphology indistinguishable from that of normal fibroblasts. However, the E2F1d87-expressing cells exhibited a distinct rounding during culture in media containing 10% calf serum. The morphology change was most pronounced during S phase, which was considerably lengthened in the E2F1d87-expressing cells. Consistent with this rounded shape, the E2F1d87-expressing cells have significantly increased levels of both p34cdc2 mRNA and protein. Also observed was an increase in active p34cdc2 in immunoprecipitates from extracts of the E2F1d87 cell line, as assayed by histone H1 kinase assay. The upregulation of p34cdc2 expression occurs at the transcriptional level and requires ectopic E2F1d87 along with serum growth factor stimulation, since culture of these cells in low serum media results in a flattened shape and a drop in p34cdc2 expression compared to that of the control cells.