E2F Activity Is Regulated by Cell Cycle-Dependent Changes in Subcellular Localization

Abstract

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G 1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.E2F is a transcriptional regulator that plays a pivotal role in the regulation of cellular proliferation (reviewed in reference 59; 8). Many E2F-responsive genes have been identified, and their products are components of either cell cycle control (e.g., cyclin E, cyclin A, and cdc2) or DNA synthesis (e.g., dihydrofolate reductase, thymidine kinase, or DNA polymerase ␣) machinery. In each case, E2F is thought to restrict the expression of these genes to the point of the cell cycle at which their products act (38).E2F is regulated by the retinoblastoma protein (pRB) (3, 5, 12), a tumor suppressor that is functionally inactivated in a large proportion of all human tumors (reviewed in reference 67). Consistent with its antiproliferative role, pRB blocks the ability of E2F to activate transcription (32, 33). In addition, overexpression studies have indicated that the resultant pRB-E2F complex can act as a transcriptional repressor, in which E2F provides the sequence-specific DNA binding activity and pRB inhibits transcription by sequestering adjacent transcription factors (2,10,62,69,70). This idea suggests that E2F participates in both the activation and the inhibition of cellular proliferation. Consistent with this hypothesis, homozygous deletion of the murine E2F-1 gene causes atrophy in some tissues and tumors in others (26, 73).The growth-inhibitory properties of pRB are regulated by its cell cycle-dependent phosphorylation (reviewed in reference 7). Phosphorylation is catalyzed by one or more of the cell cycle-dependent kinases...

E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells

“…To test this possibility, we transfected into satellite-cell derived myotubes a plasmid encoding an E2F-4 chimeric protein carrying a nuclear localization signal (E2F-4-NLS) (Muller et al, 1997). The NLS ensures that the protein is carried into the nucleus, since wild type E2F-4 is cytoplasmic (Magae et al, 1996;de la Luna et al, 1996;Lindeman et al, 1997;Muller et al, 1997;Verona et al, 1997). Similar to the other E2Fs, E2F-4-NLS did not activate S phase in myotubes (data not shown).…”

Exogenous E2F expression is growth inhibitory before, during, and after cellular transformation

“…For example, E2F activates its negative regulators, such as Rb, p107, p16, and p19, at the transcriptional level. Also, several groups have observed that E2F4 is regulated by cellular localization, being shuttled between the cytoplasm and the nucleus in a cell cycle-dependent fashion (Magae et al, 1996;Lindeman et al, 1997;MuÈ ller et al, 1997;Verona et al, 1997). In addition, E2F1 and E2F3 proteins have short half lives due to ubiquitin-mediated proteolysis (Flores et al, 1998;Marti et al, 1999).…”

“…Using this criterion, E2F4 should be as e ective an inhibitor of Ras-mediated transformation as E2F1. It has been previously noted that during S phase, when E2F target genes are expressed, E2F1 is nuclear whereas E2F4 is mostly cytoplasmic (Magae et al, 1996;Lindeman et al, 1997;MuÈ ller et al, 1997;Verona et al, 1997). To determine if the di erence in cellular localization decreases the e ectiveness of E2F4 as a Ras inhibitor, we tested an E2F4 protein fused to a consensus nuclear localization signal from the SV40 large T antigen in the focus formation assay (Lindeman et al, 1997).…”

Retinoblastoma family genes

“…This subgroup of E2F proteins still contains the Rb-binding sequence at the C-terminus but does not have the cdk binding or the nuclear localization sequences in the N-terminus and has nuclear export sequences instead (Dynlacht et al, 1994b;Muller et al, 1997;Gaubatz et al, 2001; Apostolova et al, 2002). Consistent with these structural features, this subgroup of E2Fs functions mainly as repressors of E2F target gene expression, and they translocate into the nucleus when in complex with the Rb family of proteins during the G0/G1 phases of the cell cycle (Muller et al, 1997;Verona et al, 1997). E2Fs 6-8, representing the remaining two subgroups, also function as transcriptional repressors (Trimarchi et al, 1998(Trimarchi et al, , 2001de Bruin et al, 2003a;Di Stefano et al, 2003;Attwooll et al, 2004b;Maiti et al, 2005).…”