Animal E2Fs

In the majority of eukaryotic organisms, the decision to enter or leave the cell division cycle is taken in the G1 phase. The E2F-RB pathway plays an important role in this regulatory process. RB was the first tumour suppressor gene cloned from mammalian cells, while E2F1 was identified through its ability to form a complex with the RB protein (Bagchi et al. 1991; Bandara and La Thangue 1991; Du and Pogoriler 2006).

More than 100 proteins are potentially able to interact with the animal RB, indicating that RB function is far more complex than it was initially suggested, but still the best studied binding partners of RB are the E2F transcription factors (Du and Pogoriler 2006). Structural relatives of this pathway have been identified in the unicellular green algae Chlamydomonas reinhardtii as well as in multicellular organisms, indicating that these regulators might play a role in an evolutionary conserved mechanism. The structural relatives of RB and E2Fs are missing from yeast, but the regulatory logic also applies; the G1 to S transition is regulated by the removal of a transcriptional repressor Whi5 of G1 regulation (Cooper 2006).

The widely accepted model for E2F function is the formation of het-erodimers between E2F and its dimerization partner (DP) that will activate the expression of genes required for entering the cell cycle. RB inhibits this event by physically binding to E2Fs at their carboxyl terminal RB binding motif and inhibiting its activity. Upon mitogen stimulation, this repression is suppressed by hyperphosphorylation of RB by specific CDK-cyclin D complexes (Sherr and Roberts 1999), leading to the activation of genes required for DNA synthesis. Further studies, however, revealed that the function of E2Fs is much more complex, since animal E2Fs can either activate or repress transcription (Du and Pogoriler, 2006; Rowland and Bernards 2006). For instance, the majority of the eight mammalian E2Fs (E2F1-8) are repressors (E2F4-8) that could either inhibit transcription in RB-dependent (E2F4 and E2F5) or RB-independent manner (E2F6, E2F7, E2F8).

The basis of the RB-dependent transcriptional repression through E2Fs is the ability of RB, and its pocket protein relatives, p107 and p130, to simultaneously bind to E2Fs and chromatin remodelling enzymes such as histone deacetylases (HDACs) (Rayman et al. 2002) or histone methyl transferases (e.g. SUV39H1) (Liu et al. 2005). In addition, there are examples that activator E2Fs are also able to repress transcription (Aslanian et al. 2004). Nevertheless, loss of activator E2Fs in mammalian cells reduces gene expression of E2F target genes and inhibits cell division (Wu et al. 2001). On the other hand, mutation of repressor E2Fs resulted in an increase in E2F-dependent gene expression in quiescent cells (Attwooll et al. 2004). However, whether the re-pressor/activator function of cell proliferation is the most important role of the mammalian E2Fs is still not clear, since blocking all E2F activities by overexpression of a dominant negative E2F mutant form lacking the C-terminal transactivation and RB binding domains, rather than inhibiting cell proliferation, results in blocking of cell cycle exit and differentiation (Rowland et al. 2002; Zhang et al. 1999).

In Drosophila, the interplay between E2Fs is simplified as there are only two E2F transcription factors, dE2F1 and dE2F2, that have antagonistic effects on cell division during larval development: dE2F1 is an activator while dE2F2 is a repressor (Frolov et al. 2001). Loss of dE2F1 function resulted in a serious proliferation defect in the mutant fly, which surprisingly was restored by the simultaneous loss of the repressing dE2F2. This observation indicates that dE2F1 activates transcription by replacing the repressor dE2F2 from promoter sequences of target genes containing E2F-binding sites. According to this model, E2F-mediated repression limits the rates of cell proliferation. Further studies also revealed that dE2F2 and the retinoblastoma family (RBF) of proteins provide a repressor activity that is uncoupled from cell cycle progression, and that loss of E2F-mediated repression results in the inappropriate expression of tissue-specific genes and markers for differentiation (Dimova et al. 2003) (Fig. 1). Similarly loss of repressor E2F4 in mouse embryonic fibroblasts allows cells to undergo spontaneous differentiation (Landsberg

Fig. 1 Model of E2F transcriptional regulation in Drosophila. dE2F1 and dE2F2 are the only E2Fs in Drosophila and both need to interact with the single dDP protein for efficient DNA-binding, but they show different preferences for the retinoblastoma protein. dE2F1 interacts only with RBF1, while dE2F2 associates both with RBF1 and RBF2. dE2F1 and dE2F2 are functionally antagonistic transcription factors. dE2F1 activates the expression of key cell cycle regulators for both G1-S and G2-M transitions such as cyclin E and Cdc25 genes, respectively (Neufeld and Edgar 1998), by preventing the recruitment of repressor dE2F2 to the DNA on cell cycle gene promoters. dE2F2, on the other hand, binds and inhibits the expression of cell cycle genes as well as of a variety of tissue-specific genes involved in differentiation that are not activated by dE2F1 (Dimova et al. 2003; Frolov et al. 2001)

Fig. 1 Model of E2F transcriptional regulation in Drosophila. dE2F1 and dE2F2 are the only E2Fs in Drosophila and both need to interact with the single dDP protein for efficient DNA-binding, but they show different preferences for the retinoblastoma protein. dE2F1 interacts only with RBF1, while dE2F2 associates both with RBF1 and RBF2. dE2F1 and dE2F2 are functionally antagonistic transcription factors. dE2F1 activates the expression of key cell cycle regulators for both G1-S and G2-M transitions such as cyclin E and Cdc25 genes, respectively (Neufeld and Edgar 1998), by preventing the recruitment of repressor dE2F2 to the DNA on cell cycle gene promoters. dE2F2, on the other hand, binds and inhibits the expression of cell cycle genes as well as of a variety of tissue-specific genes involved in differentiation that are not activated by dE2F1 (Dimova et al. 2003; Frolov et al. 2001)

et al. 2003). Although it has been suggested that dE2Fs are not essential for cell proliferation, since an E2F-independent mechanism is sufficient for high basal level of gene expression in the absence of dE2Fs, it is likely, however, that the balance of E2F-dependent activation and repression is involved in the coordination of cell proliferation and differentiation.

Even though initially the animal E2F function was associated mainly with the control of G1-S transition, further studies clearly show that they are involved in the regulation of G2-M transition as well. There are a number of mitotic target genes for mammalian E2Fs including cyclin B1 (Zhu et al. 2005) and Mad2, a spindle checkpoint gene (Hernando et al. 2004). In Drosophila, the key target gene of dE2F1 to control G2-M transition is the phosphatase, CDC25, an activator of the mitotic CDK1 (Neufeld and Edgar 1998).

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