InteractiveFly: GeneBrief

DP transcription factor: Biological Overview | References

BIOLOGICAL OVERVIEW

There are two E2F (dE2F1 and dE2F2), one DP (dDP), and two Rb family (RBF and RBF2) genes in the Drosophila genome. All the E2F and DP proteins, with the exception of the newly identified E2Fs 7 and 8, have both a conserved DNA-binding domain (DBD) and a dimerization domain (Dim, see Figure 2 of Du, 2006). Interestingly, the two Drosophila E2F proteins behave like the first two subgroups of the mammalian E2F proteins: dE2F1 mainly functions as a transcription activator, comparable to the mammalian-activating E2Fs 1–3, while dE2F2 primarily mediates active repression, similar to the mammalian-repressive E2Fs 4 and 5. Furthermore, similar to the mammalian Rb protein that can bind to both the activating and the repressive E2F proteins, RBF can bind to both dE2F1 and dE2F2 proteins in Drosophila. In contrast, RBF2 can only bind dE2F2 analogous to the mammalian p107/p130 proteins that bind preferentially to the repressive E2F proteins. Thus, the Rb-E2F pathway is well conserved and is much simpler in Drosophila than in the mammalian systems (Du, 2006 and references therein; full text of article).

To gain insight into the essential functions of E2F, the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila have been studied. The results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G₂/M-phase progression. Thus, dDP is not essential for developmental control of the G₁-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G₂/M progression (Frolov, 2005; full text of article).

The E2F transcription factor plays an important role in the regulation of cell cycle progression. Changes that activate E2F-dependent transcription, such as the overexpression of E2F genes or the inactivation of pRB family members (see Drosophila Retinoblastoma-family protein), promote the progression from G₁ to S phase. Conversely, changes that augment the formation of E2F repressor complexes, such as the overexpression of pRB family members or the inhibition of G₁ cyclin-dependent kinases, arrest cells in G₁ (Frolov, 2005).

E2F has been studied primarily in mammalian cells. Mammalian E2F refers to the net activity provided by a large number of proteins. The basic component of E2F is typically a heterodimer of DP and E2F subunits. Mammalian cells contain at least seven E2F genes and two DP genes, and the products of these can be combined in many different permutations. Functional overlap between different forms of E2F has made it difficult to identify the precise roles played by individual components. Moreover, the large number of E2F and DP genes has undermined attempts to assess the overall role of E2F in either cell cycle control or animal development (Frolov, 2005).

The Drosophila genome contains two genes with clear homology to the mammalian E2F genes (de2f1 and de2f2) and one gene that is highly homologous to the mammalian DP genes (dDP). dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F target genes. E2F regulation allows responsive genes to be coordinately regulated at the G₁-to-S transition. In de2f1 mutant embryos, the expression of E2F target genes, PCNA and RNR2, as revealed by in situ hybridization, is lost (Duronio, 1995; Royzman, 1997). Mutation of de2f1 severely reduces cell proliferation and DNA synthesis (Duronio, 1995), and de2f1 mutant embryos hatch to produce extremely slow-growing larvae that fail to develop, and die (Royzman, 1997). de2f2 mutants have reduced viability and fertility, but de2f2 is not an essential gene. However, mutation of de2f2 rescues the strong larval phenotype of de2f1, suggesting that de2f1 and de2f2 act antagonistically during larval development (Frolov, 2005).

One of the curious features of de2f2 de2f1 double-mutant animals is that they develop normally until late pupal stages. Imaginal disks taken from these animals display relatively normal patterns of DNA synthesis. This is remarkable, since the two de2fs are the only E2F genes found in a finished sequence of the Drosophila genome and cell proliferation occurs in de2f2 de2f1 double-mutant cells without E2F control. In a similar way, dDP mutant embryos have defects in the spatiotemporal pattern of E2F-dependent transcription, but these animals develop to late pupal stages with no clear larval defects. dDP mutant embryos appear to have fairly normal timing and levels of DNA synthesis at stage 13 (Royzman, 1997), but cells in the central midgut initiate S phase later than in wild-type embryos, during stage 14, raising the possibility that dDP may contribute to the correct timing of S phase (Duronio, 1998). It is uncertain precisely when maternal supplies of dDP are fully depleted, and this complicates the interpretation of the homozygous mutant phenotypes (Frolov, 2005).

To date, studies of dDP or de2f2 de2f1 mutants have been restricted to comparisons between wild-type and mutant animals. The properties of these mutant cells have not yet been compared side by side with wild-type cells in vivo. In addition, although dDP and the dE2Fs are heterodimeric partners, the issue of whether mutating dDP is equivalent to mutating de2f1 and de2f2 has not been carefully examined. Previous studies have stressed that the E2F-regulated patterns of RNR2 and PCNA expression are lost in dDP mutants (Duronio, 1998, Royzman, 1997), whereas analysis of de2f1 de2f2 double mutants has shown that the overall levels of expression of these and other E2F-regulated genes are similar to those of wild-type animals. To reconcile these discrepancies, and to better understand the roles of E2F and DP proteins in the regulation of cell proliferation, the consequences of inactivating dDP, dE2F1, and dE2F2 were compared. Somatic clones of dDP mutant cells were generated and used to study the effects of eliminating E2F regulation. This study showsthat the somatic mutation of dDP causes a strong reduction in cell proliferation and that dDP mutant clones display defects both in 5-bromo-2-deoxyuridine (BrdU) incorporation and in the number of mitotic cells. It is concluded that dDP is needed for efficient progression through both S phase and G₂/M and that the development of trans-heterozygous dDP mutant animals to late pupal stages disguises the fact that dDP mutant cells are at a severe disadvantage when compared directly with wild-type neighbors (Frolov, 2005).

The surprising finding that has emerged from the study of Drosophila de2f1 de2f2 double mutants is that these animals develop until late pupal stages with relatively normal patterns of cell proliferation (Frolov, 2001). This begs the question, which cellular functions require E2F regulation? Or, put another way, how 'normal' are E2F-deficient cells (Frolov, 2005)?

Two main conclusions are drawn from the experiments described in this study. The first is that in Drosophila the effects of inactivating dDP appear indistinguishable from the effects of inactivating both dE2F1 and dE2F2. This finding is of practical value because it validates the use of dDP mutant alleles to eliminate E2F activity. It is also an important conceptual point. E2F is generally considered to be a heterodimeric factor, but the idea that E2F and DP proteins function only in partnership with one another has not been rigorously examined in any experimental system. Indeed, there are several indications that this assumption is unlikely to be true in all species. Mammalian cells contain many different E2F and DP proteins, and several of them have been reported to interact with additional proteins (de la Luna, 1999; Hsien, 2002; Martin, 1995; Trimarchi, 2001). More significantly, the recent characterization of E2F7, an E2F family member with a duplicated DNA-binding domain that appears to bind to E2F-regulated promoters without a DP partner (Di Stefano, 2003), has added a new level of complexity. Similar genes are found in plants, suggesting that this DP-independent mode of action is conserved. Although the functions of most of these novel E2Fs are not yet known, they can, at least in the case of E2F7, bind to classic cell cycle E2F targets. Thus, in these species, it is expected that eliminating all E2F proteins will most likely have consequences different from those of removing all DP proteins (Frolov, 2005).

The Drosophila genome lacks any clear ortholog of E2F7, and the results described in this study strongly suggest that both of the known Drosophila E2F proteins require dDP to function, and vice versa. This conclusion is based on both biochemical and genetic data. In EMSAs, all of the complexes that bound to an E2F probe in a sequence-specific manner contained both dE2F and dDP proteins and were eliminated when dDP or dE2F1 and dE2F2 were removed by RNAi. Immunostaining experiments on polytene chromosomes show that E2F and DP proteins colocalize in vivo and that the presence of dDP at its natural targets requires dE2F1 and dE2F2; conversely, dE2F2 requires dDP. In addition, the removal of dDP (either by RNAi or in trans-heterozygous mutant animals) has effects on the expression of E2F target genes and on S-phase entry that are indistinguishable from the changes seen when dE2F1 and dE2F2 are removed. The direct targeting of dDP, dE2F1, and dE2F2 gave no further changes over cells in which either dDP or dE2F1 and dE2F2 were targeted (Frolov, 2005).

These results strongly suggest that dDP and the two dE2F proteins are exclusive and interdependent partners. However, there is a caveat to this interpretation. The levels of dDP and dE2F1/dE2F2 are strongly reduced in the absence of their binding partners. This may help to explain why dDP and de2f1 de2f2 mutant cells have similar properties: these cells may, in essence, lack the same set of proteins. However, this change in levels also means that there may not be sufficient dDP protein in de2f1 de2f2 mutants to perform any functions that are normally independent of dE2F1 or dE2F2. The converse could also be true for dDP-independent functions of dE2F1 and dE2F2. The idea that the levels of partner proteins can change dramatically in mutant animals adds an interesting complication to the interpretation of E2F/DP knockout phenotypes. Potentially, an indirect function of repressor E2F/DP complexes may be to maintain a reservoir of DP components that can be used to partner newly synthesized activator E2Fs. According to the Fly GRID database of two-hybrid interactions, dE2F1, dE2F2, and dDP have the potential to associate individually with >30 other proteins. At present, there is no evidence that most of these interactions occur in vivo, but the possibility cannot be formally excluded. Nevertheless, the results described in this study provide compelling evidence that E2F function can be eliminated in Drosophila by removing either dDP or dE2F1 and dE2F2, and hence, it can be safely assumed that dDP mutants are deficient for E2F regulation, once inherited products are exhausted (Frolov, 2005).

The second major conclusion that drawn from these experiments is that dDP mutant cells are at a strong proliferative disadvantage compared to wild-type cells. A requirement for E2F was seen in the slow proliferation of dDP mutant clones in vivo and the slow proliferation of dE2F1/dE2F2- or dDP-depleted cells in tissue culture. Cells in dDP mutant clones progress slowly through S phase and exhibit G₂/M defects. Since transient overexpression of cyclin E restores the normal rate of DNA synthesis but does not restore the normal number of M-phase cells, it is unlikely that abnormal G₂/M progression is a consequence of slow S phase. In agreement with this, no evidence is found of a significant extension of S phase in dDP mutant cells or that the cells enter mitosis with partially replicated DNA or suffer DNA damage and activate a checkpoint-mediated cell cycle arrest. This finding is consistent with the observation that many E2F target genes are expressed at abnormal levels in dDP-depleted cells. The reduced efficiency of DNA synthesis and G₂/M phase progression fits with the idea that proteins needed for the control of DNA synthesis and G₂/M progression are present at suboptimal levels when E2F regulation is absent. Indeed, E2F has been implicated in the regulation of expression of a set of G₂/M-specific genes both in Drosophila and in mammalian cells. The fact that dDP and de2f1 de2f2 mutant animals progress to such a late stage of development is a testament to the remarkable resilience of animal development, and in many respects, this development disguises the fact that cell proliferation is severely compromised in the absence of E2F regulation. This reduced rate of cell proliferation presumably contributes to the developmental delay that has been noted for dDP or de2f1 de2f2 mutants (Frolov, 2005).

It has been proposed from studies of embryos carrying a hypomorphic dDP allele that as maternally supplied dDP products run out, reduced E2F activity would result in delayed S-phase entry (Duronio, 1998). Such an effect may be transient, or cell type specific, because it is clear from the somatic clones described in this study that dDP mutant cells enter S phase of the second mitotic wave in the eye imaginal disk at precisely the same time as their wild-type neighbors. Although dDP mutant cells have been shown to be unable to control cell cycle phasing when challenged by the ectopic expression of cell cycle regulators (Reis, 2004), the relatively normal timing of S-phase entry in dDP mutants clones and in imaginal disks of trans-heterozygous dDP mutants shows that dDP mutant cells do respond fairly normally to the physiological signals that pattern S-phase entry during development. Clearly, further studies are needed to determine the precise settings in which normal cell cycle control requires E2F (Frolov, 2005).

Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program

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 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).

ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP

Drosophila oogenesis makes it possible to examine aspects of DNA replication that are not readily apparent during embryogenesis. Ovarian follicle cells undergo a set of mitotic divisions before switching to an endo cycle (a cycle consisting of only S phase and a gap phase) and becoming polyploid. Genomic replication ceases after four endo cycles, but two genomic regions that contain clusters of chorion genes continue to replicate so that the chorion genes are amplified as much as 80-fold relative to genomic DNA. The chorion genes encode the eggshell proteins. Amplification of the chorion genes is needed to produce sufficient chorion protein for a normal eggshell, and amplification occurs by repeated rounds of initiation of DNA replication and fork movement to produce a gradient of amplified DNA extending ~100 kb. Mutants with reduced amplification have a phenotype of thin eggshells and female sterility. Chorion gene amplification appears to use components that are required normally for initiating DNA replication. Origin recognition complex (ORC) is a complex of six subunits and is required for initiation of replication. Mutations in the Drosophila orc2 gene disrupt amplification. Overexpression and inhibition studies indicate that cyclin E is needed for amplification also. Because the levels of Cyclin E protein oscillate with genomic replication but remain constant in follicle cells undergoing amplification, it has been postulated that the high Cyclin E activity blocks genomic replication and that some mechanism permits the amplicons to escape this block to rereplication (Royzman, 1999 and references).

New mutations in Drosophila dE2F that cause cell-cycle defects in oogenesis have been identified and analyzed. These mutations, in addition to a female-sterile allele of dDP isolated previously (Royzman, 1997), allowed for an analysis of the role of E2F in DNA replication in follicle cells. There are multiple observations indicating that the dDP mutant stage-10B egg chambers undergo inappropriate genomic replication and are not delayed with respect to development or cell-cycle timing. (1) It is striking that the mutant stage 10B genomic replication is synchronous in all the follicle cells, a property associated with amplication but not genomic replication. Thus the genomic replication is not likely to be the consequence of follicle-cell genomic replication being delayed relative to egg chamber development and persisting into stage 10B. (2) It is unlikely that this genomic replication results from a slower S phase, with replicating follicle cells accumulating until replicating cells are continuous across the cell layer. Stage-10B egg chambers are seen with no replicating follicle cells or all of the follicle cells replicating: there is no evidence of a gradual increase in follicle cells in S phase. (3) These egg chambers are at stage 10B by morphological criteria, the oocyte and nurse cell size, and centripetal migration of the follicle cells. (4) The stage-appropriate change in Cyclin E protein distribution is seen in the mutant stage-10B nurse cells. It is postulated that the difference between the phenotypes is from variation in the levels of active dDP among egg chambers. Taken together, the phenotypes suggest that dDP plays a dual role in the regulation of replication in follicle cells. It is needed to activate chorion gene amplification, but it also may be required to inhibit follicle-cell genomic replication. Importantly, the results of the BrdU analysis correlate directly with the severe eggshell defects observed for the dDP mutants (Royzman, 1999).

Mutations in Drosophila E2F and DP affect chorion gene amplification and ORC2 localization in the follicle cells. In the follicle cells of the ovary, the ORC2 protein is localized throughout the follicle cell nuclei when they are undergoing polyploid genomic replication, and its levels appear constant in both S and G phases. In contrast, when genomic replication ceases and specific regions amplify, ORC2 is present solely at the amplifying loci. Mutations in the DNA-binding domains of dE2F or dDP reduce amplification, and in these mutants specific localization of ORC2 to amplification loci is lost. Interestingly, a dE2F mutant predicted to lack the carboxy-terminal transcriptional activation and RB-binding domain does not abolish ORC2 localization and shows premature chorion amplification. The effect of the mutations in the heterodimer subunits suggests that E2F controls not only the onset of S phase but also origin activity within S phase (Royzman, 1999).

Because the MCMs associate with chromatin in an ORC-dependent manner, the localization of the MCMs were examined in follicle cells. MCM2 is located throughout the nucleus and shows staining similar to that seen with MCM proteins in human and Xenopus replication systems. Similar staining was observed with Drosophila MCMs 4 and 5. During the mitotic divisions and subsequent follicle-cell genomic polyploidization, MCM staining appears bright in some follicle cells and faint in others. This staining pattern has been reported previously for embryonic and larval tissues: the bright MCM signal may correlate with binding of MCM to chromatin prior to replication. In contrast to ORC, MCM protein remains nuclear at stage 10 and discrete subnuclear foci are not observed. MCM staining is faint and diffuse in all the follicle cell nuclei of stage-10 egg chambers and all subsequent stages (Royzman, 1999).

Given that cyclin E is an important target of the E2F transcription factor, an attempt was made to determine whether the effects of the dDP and dE2F mutants on chorion gene amplification result from a change in either the levels or the activity of Cyclin E. No change is observed in the levels of cyclin E transcripts in stage 10A or 10B follicle cells from either wild-type or the dDP and dE2F mutant ovaries. A further test was made for an effect of E2F on Cyclin E in follicle cells by monitoring Cyclin E protein in wild-type and mutant ovaries with a monoclonal antibody against Cyclin E. Surprisingly, in dDP and dE2F mutants the levels of Cyclin E protein are normal in follicle cells at all stages, including stage 10B. In the dDP and dE2F mutants there was no effect on Cyclin E staining in follicle cells at any stage. Therefore, the dDP and dE2F mutant effects on chorion gene amplification appear not to occur via Cyclin E. Two other expected transcriptional targets of E2F, PCNA and RNR2, are not induced in the follicle cells of either wild-type or the dDP and dE2F mutants. This suggests that a G1-S transcriptional program is not driving amplification in the follicle cells (Royzman, 1999).

The fact that the levels and activity of Cyclin E are not affected in dDP or dE2F mutant follicle cells suggests that the role of Cyclin E in amplification is either parallel to or upstream of E2F. In evaluating the mechanism by which dDP and dE2F affect ORC localization and DNA replication it is useful to consider each of the three alleles and the distinct effects separately. There are two aspects of ORC localization: clearing of ORC uniformly present in the follicle cell nuclei and subsequent specific localization of ORC to the amplicons. The dDP^a1 mutation has the most severe effect in reducing BrdU incorporation and produces eggs with the thinnest shells. In addition, in some egg chambers continued follicle cell polyploidization occurs in place of amplification. The fact that in all the dDP mutant egg chambers nuclear localization of ORC2 persists, and ORC2 is not detectable specifically at amplifying foci could indicate that amplification requires that ORC be cleared from genomic chromatin and assembled at amplification origins. There are two outcomes from persistence of genomic ORC localization. It either blocks amplification or in a minority of egg chambers permits continued genomic replication. The clearing of ORC from genomic origins may be linked to a global change that permits rereplication and amplification of those loci that retain ORC binding (Royzman, 1999).

The dE2F^fi1 mutants have less severe phenotypic effects. ORC is cleared from genomic origins but is not localized to amplification origins. The outcome of this appears to be that genomic polyploidization appropriately stops, but amplification is reduced. These effects also support the idea that ORC concentration at amplifying foci is needed for rereplication. It is proposed that the dE2F^fi1 defect is less severe than that of dDP^a1 because a second dE2F gene exists that is able to compensate partially for the dE2F mutant protein (Royzman, 1999).

The absence of an effect of the dE2F^fi2 mutation on ORC localization is consistent with the fact that in this mutant, genomic replication ceases and amplification occurs. It is striking that amplification occurs earlier and has increased levels in mutant flies with a predicted truncated form of dE2F lacking the RB-binding domain. Thus restriction of the onset and extent of origin amplification may be regulated by E2F complexed with RB. It has been demonstrated that RB, when complexed to E2F, is capable of recruiting histone deacetylase and thereby converting chromatin to a compacted state. This state is correlated with inaccessibility to transcription factors, and it is reasonable to propose that it would also hinder binding of replication factors. Thus in this model, E2F in complex with RB would cause histone deacetylation in the vicinity of replication origins, leading to inhibition of amplification until stage 10B. The inability of dE2Fi2 protein to bind RB would prevent inhibition and result in premature amplification (Royzman, 1999).

The differences between the three mutations in the E2F subunits provides insights into the mechanism by which E2F may influence ORC localization. This effect could be direct or indirect. Both the dDP^a1 and dE2Fⁱ¹ mutations are predicted to weaken E2F DNA binding. Thus the known E2F activities should be present but at reduced levels. For example, these two mutant proteins should retain transactivation activity and the ability to bind RB, repress transcription, and alter chromatin structure. Despite these activities, ORC foci are not detected, implying that the ability of E2F to bind DNA is crucial for its ability to influence ORC localization. This conclusion is supported by the fact that ORC is localized properly in the dE2Fⁱ² mutant in which the protein has a normal DNA-binding motif and is predicted to lack the transactivation and RB-binding domains (Royzman, 1999).

The suggestion that the critical activity of E2F in controlling ORC localization is DNA binding makes it possible that E2F has a direct interaction with ORC to localize it to amplification origins. There are candidate E2F-binding sites within the amplification control region for the third chromosome cluster. dE2F could not be detected at discrete nuclear foci when amplification is occurring (I. Royzman and T. Orr-Weaver, unpubl. cited in Royzman, 1999); however, dE2F may be more difficult to visualize in situ than ORC. Another alternative is that E2F influences ORC by one of its transcriptional targets. There may be an E2F transcriptional target whose gene product affects ORC localization. Alternatively, the key target might be another subunit of ORC. In human cells ORC1, but not ORC2, is transcriptionally regulated by E2F. The observation that the truncated form of dE2F (dE2Fi2) is sufficient for ORC2 localization would then suggest that dE2F normally activates the transcription of the critical target gene by recruiting another positive regulator to the promoter or by displacing a negative regulator (Royzman, 1999).

The mutations in dDP and dE2F reveal a previously unrecognized role for E2F in controlling replication origin activity within S phase by affecting ORC localization. These results both define a new cell cycle function for E2F and suggest that it affects replication complex assembly directly or via one of its targets. Defining this mechanism will greatly enhance understanding of the regulation and developmental control of replication initiation (Royzman, 1999).

A role for the DP subunit of the E2F transcription factor in axis determination during Drosophila oogenesis

The E2F family of transcription factors contributes to cell cycle control by regulating the transcription of DNA replication factors. Functional 'E2F' is a DNA-binding heterodimer composed of E2F and DP proteins. Drosophila contains two E2F genes (E2F and E2F2) and one DP gene. Mutation of either E2F or DP eliminates G1-S transcription of known replication factors during embryogenesis and compromises DNA replication. However, the analysis of these mutant phenotypes is complicated by the perdurance of maternally supplied gene function. To address this and to further analyze the role of E2F transcription factors in development mitotic clones of DP, mutant cells in the female germline have been phenotypically characterized. DP has been shown to be required for several essential processes during oogenesis. In a fraction of the mutant egg chambers the germ cells execute one extra round of mitosis, suggesting that in this tissue DP is uniquely utilized for cell cycle arrest rather than cell cycle progression. Mutation of DP in the germline also prevents nurse cell cytoplasm transfer to the oocyte, resulting in a 'dumpless' phenotype that blocks oocyte development. This phenotype likely results from both disruption of the actin cytoskeleton and a failure of nurse cell apoptosis, each of which is required for normal cytoplasmic transfer. DP is required for the establishment of the dorsal-ventral axis, since loss of DP function prevents the localized expression of the Egfr ligand Gurken in the oocyte: Gurken initiates dorsal-ventral polarity in the egg chamber. Thus new functions for E2F transcription factors during development have been uncovered, including an unexpected role in pattern formation (Myster, 2000).

Drosophila oogenesis relies on communication between the nurse cells and the oocyte; this includes transfer of nurse cell cytoplasm to the oocyte. There are two successive phases of cytoplasmic transport during oogenesis. Early transport is slow and highly selective. Later in oogenesis a rapid phase of cytoplasmic transfer occurs as nurse cells contract to squeeze their contents into the oocyte. DP mutant egg chambers are defective in this rapid transport. They are also defective in nurse cell apoptosis. How might dDP affect both nurse cell cytoplasmic transport and apoptosis? Recent evidence reveals that entry into apoptosis may be coupled to rapid cytoplasm transport. In wild-type egg chambers several events indicate an apparent initiation of a nurse cell apoptotic pathway just prior to rapid cytoplasm transfer. The nurse cells undergo actin cytoskeleton rearrangements and their nuclear membranes become permeabilized. Delays in apoptosis seen in dumpless mutants that disrupt the actin cytoskeleton, such as chickadee (profilin) and kelch (a ring canal component), suggest that an apoptotic pathway and a rapid cytoplasmic transport pathway are interconnected. Germline mutant clones of Drosophila dcp-1, a CED-3-related caspase, display defects in actin bundle assembly, nuclear envelope permeabilization and cytoplasmic transport. Thus, it has been suggested that activation of an apoptotic pathway in nurse cells may lead to the formation of the actin bundles and subsequent nurse cell to oocyte cytoplasmic transfer. This model places a signal to enter apoptosis at the start of the pathway, followed by dcp-1 function, followed by chickadee, quail (villin) and singed (fascin) functions in bundling actin, and ending with cytoplasm transfer. In the context of this model, DP could act either by directly regulating genes involved in actin cytoskeleton function (e.g., chickadee) or an apoptotic pathway (e.g., dcp-1). The well-documented ability of both mammalian and Drosophila E2F to induce apoptosis suggests the latter hypothesis, i.e., that dDP is required for the initiation of nurse cell apoptosis, which triggers subsequent cytoplasmic dumping (Myster, 2000).

Surprisingly, loss of dDP function significantly inhibits the establishment of dorsal-ventral polarity during oogenesis. Germline cells mutant for either of two alleles of dDP prevent the normal accumulation of Grk protein. In DP oocytes, Grk protein fails to accumulate to wild-type levels. Grk protein is detected at higher levels in DP mutants than DP mutants, but fails to properly localize to the oocyte plasma membrane. These data suggest that both Grk accumulation and intracellular localization are disrupted by mutation of DP. Because Grk is required for an oocyte-follicle cell signaling system that initiates dorsal follicle cell fate, the DP mutants displayed a ventralized eggshell. Defects in Gurken accumulation may result from disruption of the cell cycle caused by loss of DP. There is already evidence that Gurken expression is linked to cell cycle control. Mutation of the 'spindle' class genes spnB and okra prevent normal Grk accumulation and establishment of the dorsal-ventral axis. spnB and okra encode proteins with sequence similarity to yeast genes required for repair of double strand breaks (DSBs). The failure to accumulate Grk in spnB and okra mutants is a downstream developmental consequence of a meiotic checkpoint activated in response to the persistence of DSB generated during meiotic recombination. Perhaps loss of DP function alters replication sufficiently to activate a checkpoint pathway. Alternatively, the dorsal-ventral defect in DP mutants may reflect a DP requirement for expression of spnB and okra during meiosis (Myster, 2000).

Drosophila E2F1 has context-specific pro- and antiapoptotic properties during development

E2F transcription factors are generally believed to be positive regulators of apoptosis. This study shows that dE2F1 and dDP are important for the normal pattern of DNA damage-induced apoptosis in Drosophila wing discs. Unexpectedly, the role played by E2F varies depending on the position of the cells within the disc. In irradiated wild-type discs, intervein cells show a high level of DNA damage-induced apoptosis, while cells within the D/V boundary are protected. In irradiated discs lacking E2F regulation, intervein cells are largely protected, but apoptotic cells are found at the D/V boundary. The protective effect of E2F at the D/V boundary is due to a spatially restricted role in the repression of hid. These loss-of-function experiments demonstrate that E2F cannot be classified simply as a pro- or anti-apoptotic factor. Instead, the overall role of E2F in the damage response varies greatly and depends on the cellular context (Moon, 2005).

The proapoptotic potential of E2F is well documented. Overexpression of E2F1 induces apoptosis, and a significant number of the proposed targets for E2Fs are genes with proapoptotic functions. Moreover, mammalian E2F1 is activated, specifically, in response to DNA damage. It is a less well-publicized fact that the lists of E2F-regulated genes discovered by microarray studies include many genes with antiapoptotic properties. For example, the overexpression of mammalian E2F1 increases the expression of Bcl-2, TopBP1, and Grb2 -- genes that have been shown to suppress apoptosis in other studies (Moon, 2005).

This study takes advantage of the substantial development of dDP and de2f1;de2f2 mutant animals to examine the net contribution of E2F regulation to the DNA damage response. Because of the size of the mammalian E2F family, this type of genetic analysis would be very difficult to carry out in mammalian cells, and the results reveal how the complete elimination of E2F function influences the cellular response to DNA damage response in vivo. The results demonstrate that E2F/DP proteins are, indeed, critical determinants of the cellular response to DNA damage. Surprisingly, however, the role played by E2F/DP is completely context dependent. In vivo, E2F/DP proteins vary from being strongly proapoptotic in some cells to being strongly antiapoptotic in others. One wonders whether the tissue culture systems that are often used to study E2F-induced apoptosis adequately reflect this complexity (Moon, 2005).

What determines whether E2F makes cells sensitive or resistant to DNA damage-induced apoptosis? The results suggest that a combination of factors are involved. Interestingly, the cells that are most sensitive to DNA damage-induced apoptosis in wild-type discs, and in which dE2F1/dDP is strongly proapoptotic, are actively proliferating. Conversely, the apoptotic cells seen in irradiated dDP and de2f1ⁱ² mutant wing discs are centered on a region of nondividing cells, indicating that the antiapoptotic function of E2F occurs largely in cells that are under cell cycle arrest. However, these differential sensitivities are not simply an indirect effect of cell cycle position, because they are changed in dDP mutant discs, even though the distribution of cycling/noncycling cells is the same as in wild-type discs (Moon, 2005).

At first glance, these differences would seem to fit a model in which dE2F1/dDP complexes promote the expression of proapoptotic genes in proliferating cells, but combine with RBF1 to repress the same targets in arrested cells. Although this model is appealing, it cannot be the full explanation. Cells are actively proliferating throughout the wing disc, but dE2F1/dDP only sensitizes a spatially restricted subset of cells to DNA damage-induced apoptosis. Moreover, the zone of nonproliferating cells (ZNC) that separates the cells of the disc that will form the dorsal and ventral surfaces of the adult wing, does not completely correspond to the pattern of cells with activated caspases in dDP mutant discs. In addition to cell cycle-dependent fluctuations in E2F activity, clearly there must be additional, developmentally regulated signals that heighten or lessen the cellular sensitivity to dE2F1/dDP-induced apoptosis. Notch and Ras signaling pathways appear to be likely candidates for this. The cells with the highest sensitivity to DNA damage-induced apoptosis in the wild-type wing disc are situated in a region in which Notch signaling is high and Ras signaling is low. Clonal experiments show that Ras signaling protects cells against DNA damage-induced apoptosis and that Notch signaling promotes apoptosis. It has previously been shown that Ras signals can suppress HID activity by both transcriptional inhibition and at the level of posttranslational modification, but precisely how Notch signals affect E2F-dependent apoptosis is unclear. Future studies are needed to discover whether the Notch- and Ras-mediated signals alter the program of E2F transcription, or whether these pathways converge with E2F on the apoptotic machinery (Moon, 2005).

One of the major difficulties in studying the biological functions of E2F is that E2F complexes affect the expression of a large number of genes and can act in a variety of different ways. It is difficult to assess the overall role of E2F regulation in a given process by studying an individual E2F gene, or a single E2F target. The rate-limiting targets for E2F function most likely vary from context to context, and they may not always be the usual suspects. In the D/V boundary of the developing wing disc, in which E2F/DP complexes protect from DNA damage-induced apoptosis, E2F/DP proteins are needed specifically to limit the expression of hid. Remarkably, the loss of E2F/DP leads to an upregulation of hid in this one part of the disc. This change occurs prior to irradiation, and it alters the cellular sensitivity to DNA damage. No apoptosis was found in unirradiated dDP mutant wing discs, implying that the change in hid expression in the dDP mutant wing disc is not, by itself, sufficient to induce apoptosis. The elevated hid expression is clearly important, because reducing the gene dosage of hid almost completely eliminated DNA damage-induced apoptosis in dDP mutant discs, but not in wild-type discs (Moon, 2005).

What is the connection between dE2F1 and hid? Since dE2F1 binds to sequences upstream of the hid transcription start site, the transcription of hid is most likely reduced by the direct action of E2F complexes. Previous studies in mammalian cells have shown that E2F1 can directly repress transcription of some E2F1-specific targets, although the mechanisms underlying these effects are not well understood. The dE2F1 binding site upstream of hid has two interesting features that may be significant. (1) Unlike most dE2F-regulated promoters that have been examined to date, this binding site is bound specifically by dE2F1, but not by dE2F2. This specificity fits with the genetic evidence that de2f1, rather than de2f2, is important for protection from DNA damage-induced apoptosis, and it may explain why hid is not generally repressed by dE2F2 complexes. (2) Another curious feature is that the dE2F1 binding site upstream of hid is surprisingly distal from the transcription start site. In most E2F-induced promoters, E2F binding sites are typically within 500 bp of the transcriptional start site. The position of the E2F1 binding site in the hid promoter, at -1.4 kb, may be part of the reason why hid differs from other dE2F1 targets and is not activated in a cell cycle-dependent manner. It is noted that the pattern of hid expression that sensitizes cells to apoptosis in dDP mutants occurs in the absence of E2F regulation; therefore, dE2F1 does not directly contribute to the pattern itself, but it presumably serves to interfere with another transcription factor (Factor X) that is specifically active within this region. Since the hid promoter is largely uncharacterized, the possibility that dE2F1 may act through additional sites or that it may also repress hid expression via an indirect mechanism cannot be excluded. In order to test this model, it will be necessary to identify the factors that control hid expression in vivo (Moon, 2005).

A simple model is presented for the context-specific effects of dE2F1. In the intervein region, dE2F1 increases the expression of proapoptotic genes. In doing so, dE2F1 helps set a level of sensitivity for DNA damage-induced apoptosis, and this threshold is reduced when dE2F1 or dDP are removed. At the D/V boundary, dE2F1/dDP complexes are also needed, most likely in conjunction with RBF, to limit the expression of hid. When E2F regulation is removed, the increase in hid expression outweighs the changes in expression of other E2F targets, making cells more sensitive to apoptosis (Moon, 2005).

If E2F's contribution to the DNA damage response varies in mammalian cells as much as it does in Drosophila, then this would have implications for the use of general E2F inhibitors that are currently under development. These results suggest that a global inhibitor of E2F activity, or even a specific inhibitor of activator E2Fs, may have the unintended consequence of making some normal cell types very sensitive to DNA damage-induced apoptosis (Moon, 2005).

E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila

The ability of ionizing radiation (IR) to induce apoptosis independent of p53 is crucial for successful therapy of cancers bearing p53 mutations. p53-independent apoptosis, however, remains poorly understood relative to p53-dependent apoptosis. IR induces both p53-dependent and p53-independent apoptoses in Drosophila, making studies of both modes of cell death possible in a genetically tractable model. Previous studies have found that Drosophila E2F proteins are generally pro-death or neutral with regard to p53-dependent apoptosis. This study reports that dE2F1 promotes IR-induced p53-independent apoptosis in larval imaginal discs. Using transcriptional reporters, evidence is provided that, when p53 is mutated, dE2F1 becomes necessary for the transcriptional induction of the pro-apoptotic gene hid after irradiation. In contrast, the second E2F homolog, dE2F2, as well as the net E2F activity, which can be depleted by mutating the common cofactor, dDp, is inhibitory for p53-independent apoptosis. It is concluded that p53-dependent and p53-independent apoptoses show differential reliance on E2F activity in Drosophila (Wichmann, 2010).

This study has taken advantage of the relative simplicity of Drosophila E2F and p53 families to study the role of E2Fs in p53-independent apoptosis. The results indicate that Drosophila E2F homologs play opposing roles in regulating p53-independent apoptosis in response to IR. dE2F1, a homolog of the mammalian 'activator' E2Fs, is required for Chk2-/p53-independent apoptosis, while dE2F2, a homolog of the mammalian 'repressor' E2Fs, limits p53-independent apoptosis. The net E2F activity in the cell, reduced by mutations in dDP, is inhibitory towards p53-independent apoptosis (Wichmann, 2010).

One surprising finding from these studies is that 2 kb of hid promoter confers IR-induced transcriptional activation in a p53-dependent manner. This is surprising because in embryos, transcriptional activation of hid by IR in a p53-dependent manner requires the IRER (irradiation responsive enhancer region) that lies next to rpr, ~ 200 kb away from hid, and is regulated epigenetically by histone modification (Zhang, 2008). Yet, as shown previously, 2 kb of hid promoter is enough to allow IR-induced GFP expression in eye and wing imaginal discs (Tanaka-Matakatsu, 2009). This study shows that this induction is p53-dependent. Clearly, regulation of hid by IR is different between embryos and larval discs (Wichmann, 2010).

Mammalian p53 consensus is a tandem repeat of 10 nucleotides with the sequence RRRCWWGYYY where R = G/A, W = A/T and Y = T/C and invariant C and G are shown in bold. Drosophila p53 binds to a DNA damage response element at the rpr locus that differs from the mammalian consensus at one position shown in lower case; tGACATGTTT GAACAAGTCg. Manual examination of 2 kb of hid promoter fragment that responds to p53 status shows a potential binding sequence at −2006 from the start of hid transcription that deviates from the mammalian consensus at two positions, and another at −1667 that deviates at three positions. These are ttGCATGCTC GctCATGTTC and GtGCAAGagT GtGCTTGaat respectively. Since the consensus for Drosophila p53 has not been determined, it is possible that either or both of these are responsible for the effect of p53 on hid-driven GFP reporter (Wichmann, 2010).

The 2 kb hid enhancer includes E2F consensus sequences. Rb has been shown to repress the expression of hid-driven GFP reporter when E2F binding sequences are intact but not when these are mutated (Tanaka-Matakatsu, 2009). That is, E2F binding sites allow for repression of hid by Rb although which E2F mediates this repression is not known. In any case, the finding that net E2F activity is inhibitory towards apoptosis would be consistent with the published result that Rb inhibits hid expression via E2F consensus sites. It is not known if dE2F1 plays a permissive role (e.g. by allowing elevated basal expression of pro-apoptotic genes) or an instructive role (e.g. by allowing for induction of pro-apoptotic genes by IR), or both. The results with hid-driven GFP reporter are consistent with an instructive role but do not rule out a permissive role (Wichmann, 2010).

In the absence of p53, dE2F1 is needed for the transcriptional induction of hid> GFP reporter by IR. This can explain two published results: that hid is necessary for IR-induced p53-independent apoptosis (McNamee, 2009), and that hid is transcriptionally induced in p53 mutants after a time delay. Human E2F1 can bind to the promoter of a hid ortholog, Smac/DIABLO, and can, when ectopically expressed, activate the transcription of the latter in vivo. The role of p53 status in this process or the significance of this mode of regulation was not investigated. It is speculated that the role of E2F1 in IR-induced, p53-independent transcriptional activation of Smac/DIABLO genes may be conserved in mammals (Wichmann, 2010).

Previous work has shown that dE2F1 and dE2F2 exhibit antagonistic functions, with dE2F1 activating and dE2F2 repressing the transcription of a reporter containing canonical E2F sites from the PCNA promoter. dE2F1 and dE2F2 occupy the PCNA promoter and the ratio of the two E2F proteins influenced the degree of transcriptional activation or repression. In wild type, PCNA expression is tightly coupled to the pattern of S phase, in eye imaginal discs for example. In de2f2 mutants, PCNA is no longer down-regulated outside pattern of the S phase. The loss of all E2F activities, either in de2f1, de2f2 double mutants or in dDP single mutants, results in de-repression of PCNA such that a low but significant level is expressed throughout the cell cycle. Thus the net result of opposing E2F activities is the cyclical expression of PCNA in concert with DNA replication (Wichmann, 2010).

The paradigm of E2F-dependent regulation of PCNA aids in understanding the role of E2F proteins in p53-independent apoptosis. dE2F1 and dE2F2 might similarly influence p53-independent apoptosis by regulating pro-apoptotic gene(s) such as hid. According to this model, dE2F2 (with dDp) provides a net repressive activity that inhibits IR-induced apoptosis. This activity must be operative only in the absence of p53;dE2F2 mutations that have no effect on apoptosis when p53 is present. In p53 mutants, dE2F1 counteracts dE2F2 after irradiation and thus promotes apoptosis. Disabling transcriptional activation by dE2F1, which is what the allelic combination de2f1^i2/de2f1⁷¹⁷² is predicted to cause, would result in the failure to overcome dE2F2/Dp. Removal of dE2F2 with null alleles, would result in increased gene expression and more apoptosis. Reducing the ability of dDP to interact with dE2Fs, which is what the allelic combination dDP^a1/dDP^a2 is predicted to cause, would reduce dE2F1 and dE2F2 activities simultaneously. Since this results in more apoptosis, the net E2F activity is inhibitory on apoptosis when p53 is absent (Wichmann, 2010).

This study and published studies in wing imaginal discs reveal significant differences in the effect of E2F/DP mutations on p53-dependent apoptosis (typically assayed at 4–6 h post irradiation) and p53-independent apoptosis (18–24 h after irradiation in p53 mutants). The clearest difference is that dE2F2 null mutations have little or no effect on p53-dependent apoptosis, but increase the level of p53-independent apoptosis. dDP loss-of-function mutations decrease p53-dependent apoptosis throughout eye imaginal discs and in most cells of the wing pouch, whereas they increase the level of p53-independent apoptosis. In contrast, loss-of-function mutations in dE2F1 reduced both p53-dependent apoptosis in most cells of the wing imaginal disc and p53-independent apoptosis in the wing imaginal disc. These differences raise the question ‘how does p53 status alter the role of dE2F2 and dDP in IR-induced apoptosis?’ In the presence of p53, dE2F2 has little effect and dDP is stimulatory. In the absence of p53, dE2F2 and dDP play inhibitory roles. Perhaps the occupancy of transcriptional factors at the target loci such as the hid promoter is sensitive to p53 status (Wichmann, 2010).

In the eye imaginal disc, mutations in ago, a ubiquitin ligase, result in elevated apoptosis. This mode of cell death occurs via elevated E2F1 activity, increased expression of hid and rpr and is independent of apoptosis. Thus, the role of dE2F1 in promoting p53-independent apoptosis is conserved in another tissue of the larvae (Wichmann, 2010).

Previous studies found that the role of Drosophila E2F transcription factors in apoptosis is context-dependent and is influenced by, for example, whether the cells are in the wing pouch or at the dorsal/ventral margin of the wing disc and whether apoptosis is induced by radiation or by the loss of a tumor suppressor homolog, Rb. The current study addresses the role of E2F family members in IR-induced p53-independent apoptosis. The most significant finding in this study is that reducing E2F activity, as in the case of dDP mutants, allows p53-null cells to die following IR exposure. This is in clear contrast to the finding that a similar reduction of E2F activity prevents p53 wild type cells from dying following IR exposure. Several E2F antagonists are being considered in cancer therapy. The current results from Drosophila studies would caution that p53 status must be considered when using such therapies in conjunction with radiation treatment. If the findings in Drosophila apply to human cancers, an E2F antagonist would help kill p53-deficient cancer cells following radiation treatment, but would help p53-wild type cancer cells survive. In addition, an E2F antagonist may be particularly suitable for combination therapy with radiation to eradicate p53-deficient tumors because it may sensitize p53-deficient cancer cells to radiation while protecting p53-wild type somatic cells from the cell-killing effects of IR (Wichmann, 2010).

Loss of dE2F compromises mitochondrial function

E2F/DP transcription factors regulate cell proliferation and apoptosis. This study investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. This defect is attributed to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, these results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation (Ambrus, 2013).

E2F transcription factors are best understood for their role in controlling the cell cycle, apoptosis, and differentiation. In this report, evidence is presented that E2F is also involved in the regulation of mitochondrial function and identify a specific biological context, DNA damage-induced apoptosis, in which this aspect of E2F control becomes critical. It is suggested that mitochondrial dysfunction, and not the failure to induce the apoptotic geneexpression program, makes E2F-deficient cells refractory to apoptosis (Ambrus, 2013).

In flies and mammals, the conserved mechanism by which E2F triggers apoptosis is transcriptional control of apoptotic targets. Therefore, it is believed that in irradiated cells, dE2f1, like dp53, contributes to the normal transcriptional induction of apoptotic genes. However, the current data do not support such a model since the apoptotic gene expression program was induced properly in irradiated dDP mutants. Thus, in the context of the DNA damage response, the contribution of dE2f1 to the normal transcriptional induction of apoptotic genes is negligible. It is emphasized that the data do not imply that dE2f1 is unimportant. For example, unrestrained dE2f1 activity in rbf mutants has been shown to markedly increase the induction of hid and rpr in response to DNA damage, and this increase determines the elevated sensitivity of rbf mutants to irradiation-induced apoptosis. Since ablation of dp53 completely blocks irradiation-induced apoptosis and induction of apoptotic genes, the irradiation-induced apoptotic program is governed primarily by dp53, and hyperactive dE2f1 can provide additional assistance to dp53 in activating apoptotic genes. This contribution of dE2f1 becomes evident in certain settings, such as in rbf mutants (Ambrus, 2013).

Given that irradiated dDP mutants have a properly induced apoptotic gene expression program that should trigger apoptosis in these cells, their failure to undergo apoptosis is puzzling. It is suggested that the resistance of dDP mutants to apoptosis is the consequence of a mitochondrial dysfunction. In dDP mutants, mitochondria exhibit an abnormal morphology and reduced mitochondrial membrane potential and ATP levels. The lower level of expression of dE2f/dDP mitochondria-associated target genes is a critical event in determining the response of dDP mutants to irradiation, since genetic attenuation of their expression mimics the dDP mutant mitochondrial phenotype and protection from irradiation-induced apoptosis. Significantly, the strongest protection was observed with the genes that exerted the most severe mitochondrial defects upon downregulation. Thus, the response to irradiation-induced apoptosis correlates with the extent of the mitochondrial defects. One possibility is that the mitochondrial dysfunction of dDP-deficient cells lowers their mitochondrial readiness for apoptosis, and therefore the irradiation-induced apoptotic transcriptional program is insufficient to trigger cell death. Intriguingly, 'mitochondrial readiness for apoptosis' is thought to be the molecular basis of a differential response to chemotherapy in cancer patients with acute myelogenous leukemia. Among the mitochondria-associated dE2F/dDP target genes investigated in this work, Mdh2 is particularly interesting. It was previously shown that an Mdh2 mutation prevented apoptosis in another context during ecdysone-induced cell death in salivary glands. Destruction of salivary glands is normally triggered by the induction of rpr and rpr, and both genes were induced in Mdh2 mutants to the level observed in the wild-type. In addition, Mdh2 mutants display a defect in energy production and reduced ATP levels, which is thought to compromise their ability to undergo apoptosis. This setting is highly reminiscent of dDP mutants, which are also remarkably resistant to cell death even in the face of a high level of induction of a DNA damage-dependent apoptotic transcription program (Ambrus, 2013).

The idea that mitochondrial defects could impact execution of apoptosis is consistent with the recently uncovered importance of mitochondria for cell death in Drosophila. Several studies demonstrated that Rpr, Grim, and Hid, the key apoptotic proteins in flies, are localized to mitochondria and that this localization is required for efficient activation of apoptosis. Thus, one possibility is that proapoptotic proteins are not efficiently localized to mitochondria in dDP mutants. It is also possible that the dysfunctional mitochondria of dDP mutants fail to remodel in response to irradiation, which has been shown to be necessary for execution of stress-induced apoptosis. It is noted that the current findings do not imply that dE2F/ dDP normally triggers apoptosis by modulating mitochondrial function, but rather that mitochondrial function is compromised in E2F-deficient cells, which in turn would result in less efficient apoptosis (Ambrus, 2013).

Another important conclusion of this study is that a mechanistic link between the Rb pathway and mitochondria is conserved in mammalian cells. Several Drosophila dE2F/dDP-regulated, mitochondria-associated genes are also E2F targets in mammalian cells, and their expression is similarly reduced in cells when E2F is inactivated. Significantly, this leads to strong mitochondrial defects that are highly reminiscent of the mitochondrial phenotype in Drosophila dDP mutant eye discs. The data are consistent with the recent finding that mammalian E2f1 and pRB regulate expression of oxidative metabolism genes during the adaptive metabolic response in mice. The Rb pathway has been also implicated in the regulation of the mitochondrial biogenesis transcriptional program in erythropoiesis. Intriguingly, a recent study demonstrated that a fraction of endogenous pRB is present at mitochondria, where it directly participates in mitochondrial apoptosis (Hilgendorf, 2013). Given the prominent role of mitochondrial pathways in apoptosis in mammalian cells, it is conceivable that the loss of E2F could impact the efficacy of apoptosis in mammals by a mechanism analogous to that observed in Drosophila. Such an idea is consistent with the finding that inactivation of E2F reduces DNA damage-induced apoptosis in mammalian cells (Ambrus, 2013).

Interestingly, Nicolay (2013) recently demonstrated that an rbf mutation alters cellular metabolism and an abnormal metabolism sensitizes Drosophila to various types of stress. This work, found that inactivation of E2F results in a severe mitochondrial dysfunction, which is the basis for the failure of dDP mutants to undergo DNA damage-induced apoptosis. Thus, a general emerging theme is that perturbation of the Rb pathway may exert profound metabolic changes within the cell that can have a major impact on cell survival (Ambrus, 2013).

Multicilin drives centriole biogenesis via E2f proteins

Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. This study shows that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, this study shows that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation (Ma, 2014)

Search PubMed for articles about Drosophila Dp

Ambrus, A. M., Islam, A. B., Holmes, K. B., Moon, N. S., Lopez-Bigas, N., Benevolenskaya, E. V. and Frolov, M. V. (2013). Loss of dE2F compromises mitochondrial function. Dev Cell 27: 438-451. PubMed ID: 24286825

de la Luna, S., et al. (1999). Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor. EMBO J. 18: 212-228. PubMed ID: 9878064

Di Stefano, L., Jensen, M. R. and Helin. K. (2003). E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes. EMBO J. 22: 6289-6298. PubMed ID: 14633988

Du, W. and Pogoriler, J. (2006). Retinoblastoma family genes. Oncogene 25(38): 5190-200. PubMed ID: 16936737

Duronio, R. J., et al. (1995). The transcription factor E2F is required for S phase during Drosophila embryogenesis. Genes Dev. 9: 1445-1455. PubMed ID: 7601349

Frolov, M. V., et al. (2001). Functional antagonism between E2F family members Genes Dev. 15: 2146-2160. PubMed ID: 11511545

Frolov, M. V., Moon, N. S. and Dyson, N. J. (2005). dDP is needed for normal cell proliferation. Mol. Cell. Biol. 25(8): 3027-39. PubMed ID: 15798191

Hilgendorf, K. I., Leshchiner, E. S., Nedelcu, S., Maynard, M. A., Calo, E., Ianari, A., Walensky, L. D. and Lees, J. A. (2013). The retinoblastoma protein induces apoptosis directly at the mitochondria. Genes Dev 27: 1003-1015. PubMed ID: 23618872

Hsien, J.-K., et al. (2002). Novel function of the cyclin A binding site of E2F in regulating p53-induced apoptosis in response to DNA damage. Mol. Cell. Biol. 22: 78-93. PubMed ID: 11739724

Ma, L., Quigley, I., Omran, H. and Kintner, C. (2014). Multicilin drives centriole biogenesis via E2f proteins. Genes Dev 28: 1461-1471. PubMed ID: 24934224

Martin, K., et al. (1995). Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein. Nature 375: 691-694. PubMed ID: 7791903

McNamee, L. M. and Brodsky, M. H. (2009). p53-independent apoptosis limits DNA damage-induced aneuploidy. Genetics 182: 423-435. PubMed ID: 19364807

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date revised: 25 October 2014

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