Gene name - E2F transcription factor
Cytological map position - 93E8--93E9
Function - transcription factor
Keywords - cell cycle
Symbol - E2f
Genetic map position -
Classification - multifunctional
Cellular location - nuclear
The E2F protein is a critical component for normal cell cycle regulation. As a transcription factor, it positively regulates many of the genes required for initiation of S phase (the DNA synthetic phase). The complexity of E2F regulation is fairly well understood in human biology, while the availability of mutations makes it a prime target for investigation in Drosophila.
Drosophila E2F mutants complete early cell cycles, using maternal gene products, but DNA synthesis fails in cycle 17. Instead of undergoing their normal proliferation during a rapid cycle lasting about 40 minutes, cell cycles in the CNS of E2F mutants gradually decrease DNA synthesis after stage 12. Abdominal histoblasts also cease DNA replication in E2F mutants. Messenger RNAs coding for proteins required for DNA synthesis disappear in E2F mutants (Duronio, 1995a).
Interactions with cyclin E are especially complex. Cyclin E, which is the regulatory subunit of the cyclin E/cdc2c kinase heterodimer, regulates entry in S by phosphorylation of target substrates. Cyclin E expression at G1-S requires E2F. Activation of E2F without cyclin E is not sufficient for S phase. Early in G1, ectopic expression of cyclin E alone can bypass E2F and induce S phase. Thus cyclin E is a downstream target of E2F, coupling E2F activity to G1 control (Duronio, 1995b).
How is E2F regulated? In mammalian systems E2F is held inactive by the retinoblastoma (Rb) family of pocket proteins (See Drosophila Retinoblastoma-family protein). Hypophosphorylated Rb can interact with E2F during G1. This complex can bind to DNA and repress transcription of E2F target genes. A chain of events leads first to sequestration and ultimately the release of E2F. The sequestering of E2F is actively carried out by Rb family members until Rb is inactivated by hyperphosphorylation, carried out by cyclin dependent kinases. At this point, E2F is released allowing it to activate genes required for DNA synthesis and entry into S (Zhu, 1995 a and b).
An additional factor conspires to hold E2F inactive in G1 phase: the association of E2F with p53. As a consequence of its ability to physically associate with E2F, the expression of wild-type p53 can inhibit transcriptional activation by E2F. The expression of both E2F1 and DP1 (E2F's dimerization partner) can also downregulate p53-dependent transcriptional activation (O'Conner, 1995).
Equally important as the G1 phase regulation of E2F is the inactivation of E2F during S phase, when it is no longer required for gene activation. In mammalian systems the inactivation of E2F is again carried out by cyclin A dependent kinase activity. Just as there is a physical association of E2F with the Retinoblastoma protein in G1, there is an association of cyclin A with E2F in S phase (Kitagawa, 1995). These G1 and S phase interactions of E2B have yet to be documented in Drosophila. In Drosophila cyclin A is not synthesized until G2, too late to inactivate E2F in S phase.
E2F is capable of driving the G1-S transition of the cell cycle. However, mice in which the E2F-1 gene has been disrupted develop tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that disrupt G1-S transcription late in Drosophila embryogenesis and five mutations in the dDP gene (DP transcription factor) were identified. Sequencing of dDP reveals the presence of several important motifs, including the DNA-binding region, the DEF box that is predicted to be required for DP/E2F heterodimerization, and three other highly homologous regions named DP-conserved box 1 (DCB1), DCB2, and negatively charged box (NCB). Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The mutant phenotypes reveal that both genes promote progression of the cell cycle (Royzman, 1997).
Although the dE2F mutant animals survive through larval life, a dramatic delay is observed in larval growth. It takes between 288 and 432 hr for the dE2F mutant larvae to pupate, compared to 120 hr for heterozygous sibling controls. Five days after egg laying (AEL) the dE2F mutant larvae are very sluggish and much smaller in size than their wild-type counterparts. The polytene salivary gland and diploid imaginal discs can not be identified in the 5-day-old dE2F mutant larvae, presumably because they are so small. The brains are also greatly reduced in size as compared to wild type. The size of the dE2F mutant larvae increases over time, and the internal tissues approached wild-type size. Therefore, DNA replication can occur during this larval period, but it is slow. Replication in the absence of dE2F is further evidenced by the formation of banded polytene salivary gland chromosomes in some of the 12- to 18-day larvae. Although the polytene chromosomes from the dE2F mutant larvae are smaller and more fragile than normal they are clearly visible. Thus, it is concluded that S phase occurs in the absence of dE2F, but dE2F is necessary for timely replication and growth. In addition to the growth delay, the dE2F mutant larvae had another striking phenotype: melanotic pseudotumors are formed. Melanotic tumors are groups of cells within the larvae that are recognized by the immune system and encapsulated in melanized cuticle. They are referred to as pseudotumors to emphasize that they are not necessarily the consequence of hyperproliferation but can be abnormal cells recognized by the immune cells. Small pseudotumors were first observed in the dE2F mutants 7 days AEL, and these early pseudotumors grow and darken as the larvae age. In the dE2F mutants that initiate pupation, numerous additional small pseudotumors form (Royzman, 1997).
Approximately half of the dDP mutant pupae reach adulthood in the pupal case. These adults struggled to eclose but ultimately die. Organisms dissected from the pupal case have essentially normal heads and thoraxes. However, their abdominal defects are severe. This is informative as the head and thorax are derived from imaginal discs, whereas the abdomen arises from the abdominal histoblast nests. The imaginal discs proliferate during larval stages, but the abdominal histoblast nests proliferate during pupal development. Thus, pupal lethality may result from a defect in abdomen formation that occurs during pupal development. Having shown that heat shock dDP rescues the dDP mutants, the developmental period during which ectopic dDP expression is capable of rescuing the lethality of the dDP mutants was defined. Ectopic expression of dDP results in 100% rescue of dDP mutant animals. Thus, the late lethality of dDP mutants is not a manifestation of a defect in the early development of the organism, but rather it stems from defects in larval/pupal life (Royzman, 1997).
The striking observation from the Drosophila dDP and dE2F mutants is that although cyclic transcription of cyclin E, PCNA, and ribonucleotide reductase 2 (RNR2) is not detectable, S phase still occurs. Although the possibility that cyclic transcription of these genes occurs at a low level driven by maternal pools of dDP and dE2F cannot be excluded, the bursts of transcription that normally precede S phase are not essential for the G1-S transition. In these mutants the cell cycle may be driven by basal levels of transcripts and post-transcriptional regulation. The maternal pools of components of the replication machinery can persist until late in development, as evidenced by the fact that mutations in PCNA and MCM2 cause late larval lethality (Royzman, 1997 and references).
E2F positively regulates many of the genes required for initiation of S phase (the DNA synthetic phase). In mammals, the tumor suppressor RB interacts with, and negatively regulates, E2F, but it is not clear whether the function of pRB is solely mediated by E2F. In addition, E2F has been shown to mediate both transcription activation and repression; it remains to be tested which function of E2F is critical for normal development. Drosophila homologs of the RB and E2F family of proteins Rbf and E2f have been identified. The genetic interactions between Rbf and E2f were analyzed during Drosophila development, and the results show that Rbf is required at multiple stages of development. Unexpectedly, Rbf null mutants can develop until late pupae stage when the activity of E2f is experimentally reduced, and can develop into viable adults with normal adult appendages in the presence of an E2f mutation that retains the DNA binding domain but lacks the transactivation domain. These results indicate that most, if not all, of the function of Rbf during development is mediated through E2f. In turn, the genetic interactions shown here also suggest that E2f functions primarily as a transcription activator rather than a co-repressor of Rbf during Drosophila development. Analysis of the expression of an E2F target gene Pcna in eye discs shows that the expression of PCNA is activated by E2f in the second mitotic wave and repressed in the morphogenetic furrow and posterior to the second mitotic wave by Rbf. Interestingly, reducing the level of Rbf restores the normal pattern of cell proliferation in E2f mutant eye discs but not the expression of E2f target genes, suggesting that the coordinated transcription of E2f target genes does not significantly affect the pattern of cell proliferation (Du, 2000).
Given that E2f is just one of many potential targets of Rbf, the dramatic suppression of the rbf mutant phenotypes by E2f mutants is very unexpected. (1) Lowering the activity of E2f can suppress the early larval lethality of the rbf mutants as well as the developmental phenotypes observed in the adult eyes and bristles. (2) An allele of E2f with an intact DNA binding domain but with no transactivation domain or Rbf binding domain can suppress the lethality of rbf null mutants, allowing the double mutant flies to develop into viable adults. Furthermore, these suppressed rbf null adults show normal adult structures. Thus the uninhibited E2f in rbf mutants mediates both the lethality as well as the observed eye and bristle phenotypes in adults. These observations provide strong evidence that E2F mediates most, if not all, of the phenotypes of rbf during development (Du, 2000).
Interestingly, lowering the activity of Rbf can also partially suppress the E2f null phenotypes. There are at least two possible explanations for this observation. One possibility is that Rbf has a function downstream of E2f; the other possibility is that Rbf can affect the E2f mutant phenotypes through a parallel pathway (for example Rbf may be able to regulate the expression of E2f target genes through another target such as dE2F2). The second explanation seems to be more likely for the following reasons: (1) in the suppressed E2f null mutants, the expression of E2f target genes is not restored, and the larvae growth is still greatly retarded, suggesting that reducing the level of Rbf bypasses rather than restores the function lost by E2f mutation; (2) the rbf null mutant phenotype is fully suppressed by an E2f mutant that lacks transcription activation and an Rbf binding domain, demonstrating that Rbf functions upstream of E2f. In summary, these results do not point to a role for Rb downstream of E2f. In contrast, loss of Rbf indeed causes deregulation of the expression of PCNA even in the absence of transcription activation by E2f, supporting the notion that Rbf can regulate the expression of PCNA by targets other than E2f (Du, 2000).
E2F transcription factors can function both to activate transcription and to repress transcription by recruiting RB family proteins to specific promoters. Although analysis of E2f mutants shows that E2f is required for the coordinated expression of replication functions such as PCNA and Ribonucleoside diphosphate reductase small subunit (RnrS), it is not clear whether the lack of transcription of these set of genes is the cause of the larval lethality. It is formally possible that the lethality of E2f mutant is caused by the failure to repress certain critical E2f target genes. Depending on the function of E2f as a transcription activator or a co-repressor of Rbf, completely different predications are expected regarding the genetic interaction between Rbf and E2f. The observation that lowering the level of Rbf can suppress the larval lethality of E2f mutants and allow the E2f mutants to develop into pharate adults, suggests that during Drosophila development, the function of E2f is mainly to activate transcription and not to recruit Rbf to repress transcription (Du, 2000).
In addition to the first E2f to be identified in Drosophila, a second, termed E2f2, has been identified (Sawado, 1998). Interestingly, E2f2 can bind to E2F binding sites, but the function of E2f2 appears to be distinct from that of E2f. Cotransfection of E2f2 represses the expression from the PCNA gene promoter while cotransfection of E2f activates the expression (Sawado, 1998). Similar findings are also observed in transfection experiments in which E2f strongly activates transcription, while E2f2 fails to activate a reporter with E2F binding sites. These results suggest that E2f2 may function mainly to repress transcription while E2f functions mainly to activate transcription. Taken together, these data suggest a model for the function of Rbf, E2f and E2f2. In this model, E2f functions mainly to activate transcription of the E2f target genes. Rbf negatively regulates the activity of E2f to inhibit the expression of E2f target genes. In addition, Rbf can also repress the expression of E2F targets genes through other targets of Rbf such as E2f2. Thus the expression of E2F target genes will have three different states: activated, when there is free E2f/Dp to activate transcription; repressed, when there is E2f2/Dp/Rbf (and possibly E2f/Dp/Rbf) to repress transcription; and basal, when there is neither activation nor repression. At present, it is not clear whether E2f also has a function to repress transcription during development, nor is it is clear about the function of free E2f2/Dp (Du, 2000).
Bases 5' UTR -701
Bases in 3' UTR - 1261
Although Drosophila and human proteins share three regions with a high degree of homology, the fly protein is much larger, in part due to the insertion of a 300-aa block with a unique sequence between the two homologous blocks closest to the C-terminal. The Drosophila gene is equally related to each of the human family members. Amino acids 249-318 of the fly protein share a striking homology with DNA-binding domains of the human E2F genes. In addition, a region termed the "marked box" is highly conserved. This domain may function in protein dimerization. The C-terminal Rb-binding domain has also been largely conserved in the fly protein, sharing 56% similarity to the human proteins (Dynlacht, 1994 and Ohtani, 1994).
date revised: 11 August 97
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