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E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1-3), and those that do not (E2Fs 4-5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology with E2Fs 1-5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1-5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-I and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly different to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of coexpression of E2F-1 and DP-1. The inhibitory effect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.