E2F transcription factor 2


Targets of Activity

One possible mechanism by which E2F2 could inhibit DNA replication is to act as a transcription factor to modulate the expression of at least one crucial replication factor. For example, if E2F2 was part of a repressor complex, loss of E2f2 function could lead to increases in replication gene expression that might trigger widespread DNA synthesis. In order to test this idea, an examination was made of the abundance of several mRNAs encoded by genes either known to be (e.g. Orc1, RNR2, PCNA) or possibly (e.g. Orc2 and Orc5) regulated by E2F. RNA was extracted from total follicle cell preparations and subjected to RT-PCR. Relative to rp49 controls, more Orc5 mRNA was reproducibly (n=4) detected in Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f1-188 mutant samples, compared with wild type. An increased Orc2 mRNA level was also detected in some experiments (two out of four). For Origin recognition complex subunit 1 (Orc1), RNR2 and PCNA there was no substantial difference in the amount of mRNA detected between wild type and E2f2 mutants. These data suggest (1) that E2F target genes are expressed at or above wild-type levels after loss of E2f2 function and (2) that de-repression of specific target genes, such as those encoding members of the ORC complex, could contribute to the inappropriate DNA synthesis seen in E2f2 mutant follicle cells (Cayirlioglu, 2001).

E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species, but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, the control region of the Drosophila PCNA gene has been characterized. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins. The data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development (Thacker, 2003).

Two sequences upstream of the PCNA transcription start site are capable of binding E2F in vitro, and chromatin IP experiments have demonstrated that this region is occupied by both dE2F1 and dE2F2 in cultured SL2 cells. The two E2F binding sites have been reported to be necessary for PCNA expression in vivo, as determined by measuring the amount of β-Gal activity in whole-animal extracts from PCNA-lacZ transgenes. However, this approach does not assess the contribution E2F binding sites make to endogenous patterns of gene expression that represent the complex spatiotemporal program of PCNA expression during development. To determine this, transgenic flies were engineered carrying PCNA-GFP fusion constructs and in situ hybridization of embryos carrying these transgenes was performed with a GFP probe. The results indicate that a 100-bp sequence containing the two E2F binding sites reproduces the entire complex embryonic profile of cell cycle-correlated PCNA expression (Thacker, 2003).

PCNA-GFP fusion protein expression was analyzed in several larval tissues by confocal microscopy. The final synchronous mitotic cycle prior to cell differentiation (called the 'second mitotic wave') in third instar eye imaginal discs can be easily visualized as a stripe of cells that incorporates BrdU and that also expresses PCNA in an E2F-dependent fashion. The accumulation of PCNA-GFP fusion protein from the reporter transgene faithfully reproduces this pattern of cell cycle control. In the optic lobe of the larval brain, PCNA-GFP expression correlates with the inner and outer proliferative zones, which are separated by a field of quiescent cells. PCNA-GFP expression also accurately reports patterns of proliferation in the wing imaginal disc. Expression is absent in the zone of nonproliferating cells (ZNC) located at the juxtaposition of dorsal and ventral compartments in which E2F is thought to be inactive, and it is present in the surrounding cells in a pattern consistent with known proliferation patterns in this part of the disc. Thus, the 100-bp enhancer element containing two E2F binding sites accurately reproduces S phase-associated, E2F-dependent PCNA expression at many stages of Drosophila development. These transgenes should prove useful as tools to mark replicating cells in situ by using methods other than BrdU labeling (Thacker, 2003).

To determine the binding sites through which dE2F2 acts, the activity of each PCNA reporter construct was observed in dE2F2 mutant eye discs by in situ hybridization. The expression of PCNA-GFP and PCNA-Δ site I-GFP in dE2F2 mutant eye discs was very similar to wild-type, suggesting that dE2F1 is sufficient to provide both activating and repressing activities necessary to generate the pattern of PCNA expression. PCNA-Δ site II-GFP was expressed within the morphogenetic furrow of dE2F2 mutant eye discs, which is in contrast to the lack of expression of this reporter in wild-type eye discs. This ectopic expression appears to require dE2F1, because PCNA-Δ site I&II-GFP was not expressed in dE2F2 mutant eye discs. Thus, dE2F1 and dE2F2 can compete for site I when site II is absent, suggesting that activating and repressing influences on the PCNA gene can act through the same E2F binding site. However, the relevance of this observation to endogenous PCNA regulation is unclear, since E2F binding site II alone is sufficient to drive the spatiotemporal pattern of PCNA expression. Perhaps dE2F2 modulates the overall level of output of PCNA transcription by binding to site I, rather than whether the gene is fully repressed or not (Thacker, 2003).

RT-PCR was used to directly measure whether loss of E2F binding sites could support some expression below the level detectable by cytological methods. Whereas GFP transcripts were undetectable in wild-type embryos, they were detected at similar levels in both PCNA-GFP and PCNA-Δ site I-GFP lines. GFP transcripts were also reproducibly detected in Δ site II and Δ site I&II lines compared to controls. In the Δ site I and Δ site I&II lines, the amount of GFP mRNA detected was lower than with PCNA-GFP or PCNA-Δ site I-GFP lines, although there was enough line to line variability within a single construct that conclusive quantitative comparisons could not be made between constructs. Nevertheless, these data indicate that the PCNA enhancer can support transcription in the absence of functional E2F binding sites. Similarly, simultaneous loss of dE2F1 and dE2F2 reduces, but does not eliminate, endogenous PCNA expression in the eye disc (Thacker, 2003).

Chronic derepression in the absence of E2F proteins might provide sufficient expression to permit cell cycle progression. Thus, the essential function of the PCNA gene could possibly be provided in the absence of E2F-regulated transcription. To test this, PCNA minigenes were constructed that lack one or both of the E2F binding sites (by fusing the site mutants used above to the PCNA coding region instead of GFP) and it was asked if these transgenes could complement the lethality of PCNA null mutants. As expected, both a wild-type control transgene and two independent Δ site I-PCNA transgenes fully complement the lethality of PCNA null mutant animals. The two independent Δ site I&II-PCNA and one Δ site II-PCNA transgenes tested in this assay also complemented PCNA lethality, although less efficiently than wild-type or Δ site I transgenes. The Δ site II-PCNA rescue was the least efficient (7% of expected for each of two PCNA alleles), although, since only a single line was tested, an inhibitory effect from insertion position cannot be excluded. Nevertheless, it is possible that E2F2-mediated inhibition through site I further inhibits expression from this transgene relative to the Δ site I&II-PCNA construct. In sum, while E2F regulation is not absolutely essential for PCNA gene function, it appears to provide a level of PCNA expression necessary for normal Drosophila development (Thacker, 2003).

Loss of dE2F1 function is lethal, and replication in cells that lack dE2F1 is impaired relative to that in wild-type cells. Because dE2F1 is also required for the expression of genes encoding essential replication factors, dE2F1-mediated activation appears to be necessary for normal cell cycle progression. Nevertheless, sufficient PCNA function is provided in the absence of E2F regulation to support development. This expression may result from the absence of E2F/RBF-dependent repression and/or the presence of other factors (e.g., DREF) that bind within the 100-bp enhancer and contribute to PCNA activation. S. cerevisiae cells can proliferate in the absence of cell cycle-regulated activation of transcription of replication factors. Similarly, DNA synthesis in the second mitotic wave appears rather normal in eye imaginal discs lacking both dE2F1 and dE2F2. Here, direct control of cyclin E transcription by the Ci transcription factor in response to Hh signaling appears to control the onset of S phase (Thacker, 2003).

If sufficient biosynthesis of essential replication factors like PCNA can be achieved in the absence of E2F, then why is E2F essential? The data indicate that the absence of E2F-regulated PCNA expression is not tolerated very well. Thus, the optimal level of PCNA gene function requires E2F input. Since PCNA is not expected to provide a cell cycle regulatory role, per se, it is suspected that the lack of cell cycle regulation and the consequent low level of ubiquitous expression is not the problem. Rather, the lack of high-level expression achieved during S phase may attenuate DNA synthesis and inhibit normal development. This may be true for many E2F target genes, such that the coordinated loss of entire E2F-directed programs of gene expression is much more detrimental than the loss of E2F regulation of a single gene like PCNA (Thacker, 2003).

, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-related genes
Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Advantage was taken of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), potential corepressors were depleted and the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2 was sought. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, the results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. The chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulates the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but has no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins (Taylor-Harding, 2004).

Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. This study used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. Sites were also found where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants (Georlette, 2007).

MMB is ~700 kDa, and contains a unique ensemble of nine proteins, of which five are capable of binding to DNA: Myb, E2F2/DP, and Mip120 are site-specific DNA-binding factors, and a fifth protein, Mip130, contains an A-T hook domain capable of binding to AT-rich DNA. Although E2F2 and Myb have been widely studied individually, nothing has yet been explored as to how these DNA-binding proteins behave in an ensemble. Perhaps the more biologically relevant question is: How do the different site-specific DNA-binding proteins choose where to bind among the potentially large number of binding sites within the genome of a cell? It is likely that all or some combination of the DNA-binding activities may participate in MMB targeting to specific DNA sites. The other MMB factors (RBF1 or RBF2, Caf1/p55, Mip40, and Lin-52) are not known as DNA-binding proteins, but may contribute indirectly to DNA targeting through association with histones (Georlette, 2007).

The major findings reported here revealed the extraordinary diversity of use and combinatorial requirements for the factors of the MMB/dREAM complex in regulating gene expression. The Venn diagrams and subclasses for each set of genes regulated by MMB members illustrate this point for both the transcriptional repression and activation of gene expression by MMB. In the initial characterization of the complex, E2F2 and the RBFs but not Myb were found central for transcriptional repression. A more complete description of the network of genes regulated by MMB, and the genome-wide location of the individual MMB members analyzed in this study revealed a more complex picture of the MMB regulatory network than previously thought. Some of this complexity may be the result of complexes containing subsets of individual MMB members. However, this scenario would not significantly change the present prospective. This is so because >50% of all genes in each of the classes had proximal intact MMB complexes as determined by ChIP-chip analysis. For example, the genes involved in the G2/M transition and chromosome maintenance contained all five MMB members tested at sites proximal to the expressed gene, irrespective of the class into which the gene was classified by RNAi and microarray analysis (Georlette, 2007).

Levels of Myb or E2F2 were dependent on Mip120 and Mip130 but not vice versa, and hence one would not anticipate classes of regulated genes to be dependent solely on Myb or E2F2. A number of confounding issues may contribute to this paradox, including incomplete RNAi and the possibility that partial complexes, arising after loss of a factor after ablation, may remain bound to a site. Such limitations might lead to misclassification of a certain gene(s). Focusing on Myb as an example, removal of Myb may critically limit regulation of a class of genes with different kinetics than would be observed following depletion of Mip120 or Mip130. Class Q is one such class for which the depletion of only Myb (and not any other MMB member) resulted in lowered transcript levels for Class Q members. One hypothesis is that RNAi treatment to remove either Mip120 or Mip130 resulted in incomplete loss of Mip120 and Mip130 such that just enough of the MMB complex remained targeted to such sites where MMB levels were sufficient to produce normal transcript levels for that gene. It is inferred that for such sites in the Q class, Myb loss would be most limiting (Georlette, 2007).

Previous genetic and biochemical studies that linked the MMB members as one entity have been substantiated and extended by the data presented in this study. The five MMB members tested (Myb, Mip120, Mip130, E2F2, and Lin-52) had strikingly coincident genomic binding site profiles. However, among the 3538 MMB-binding sites where the five proteins colocalized, there were significant differences in the enrichment signals. The observed variations in signal strengths at different sites for MMB members by ChIP-chip provided some intriguing correlations. For Class A repressed genes, the Myb ChIP-chip signal was lower than the ChIP-chip signal for E2F2, and this relationship was reversed for the Class B repressed genes. There are many potential reasons for such differences in signal strengths including: different cofactor associations with MMB bound to a particular class of gene promoters, different subunit conformations/accessibility (resulting in altered pull-down antibody affinities), or perhaps because the formaldehyde cross-linking efficiency was highest for a protein that had a high affinity for a proximal DNA site. Moreover, MMB subunit composition in vivo may be dynamic, and off-rates for a DNA-binding factor may be lowest for those MMB factors that were tightly bound to a DNA site. Hence, for Class A genes that required E2F2 for repression, the cross-linking efficiency and potential targeting of E2F2 might dominate, whereas for Class B genes that required Myb for repression, the reverse would be found (Georlette, 2007).

Consistent with this view, a statistically significant enrichment of Myb consensus binding sites was found nearby Class B genes and E2F2 consensus binding sites nearby Class A genes. MMB targeting to one example was directly examined for both Class A and Class B genes, and for the Class A gene, E2F2 was critical for DNA binding whereas for the Class B gene, Myb was found to be a key targeting factor. Furthermore, for Class C repressed genes, where neither E2F2 nor Myb were required for repression, equivalent ChIP-chip enrichment signals were found (Georlette, 2007).

The genes repressed by MMB for both Class A and Class B were not cell cycle regulated and there was no biochemical evidence in Drosophila for the existence of two separate Myb- or E2F2-containing MMB complexes. Anti-Myb and anti-E2F2 antibodies coimmunopreciptated the entire set of MMB factors and hence each other. Furthermore, both E2F2 and Myb were stoichiometric in the defined complex - even after many different biochemical purification protocols of MMB. This point was tested in DNA binding experiments with purified MMB complex and biotinylated DNA fragments from either a Class A or B gene with a strong E2F2 site or strong Myb site, respectively. Regardless of the DNA fragment used in pulldown experiments, E2F2 and Myb stayed together. It is thus concluded that the complexity of both the targeting of MMB to DNA and the transcriptional function(s) of MMB are determined by DNA context and other proteins that may have associated with MMB. In this sense, MMB behaves like the multisubunit TFIID complex, where different TAF subunits determine DNA targeting at a specific promoter (e.g., TAFII2 at an Inr site; TAFII6 at a DPE site, and TBP at a TATA box) and the different TAF subunits interact with different coactivators (Georlette, 2007).

Many genes required for the G2/M transition were regulated by MMB. Reduced levels for many of the genes could readily account for the chromosome phenotypes that were characterized after RNAi depletion of Myb, Mip120, or Mip130 including: impaired sister chromatid cohesion, chromosome fragmentation, and condensation defects. Furthermore, transcript levels for regulatory checkpoint genes involved in spindle assembly that might indirectly lead to chromosome instability were also affected by loss of MMB. In a recent study, Goshima (2007) conducted a 'genome- wide' RNAi screen to identify factors contributing to spindle assembly in Drosophila. Among the unexpected genes revealed by this screen were Myb, Mip130, Lin-52, Mip40, and Caf1/p55. It is suggested that the MMB member genes were identified in their study, at least in part, because MMB regulates the levels of other 'expected' spindle assembly genes such as Klp61F (a kinesin) or Ial (Aurora-B kinase) or the 'unexpected' spindle assembly genes such as RacGap50C - all of which are MMB-regulated Class D genes. It will be interesting to learn if the human homologs of MMB also regulate genes required for G2/M in humans, because the oncogenic role of Myb in certain cell types may involve misregulation of spindle assembly genes that ensure normal karyotypes. The human repressor E2F4 has been shown to bind to genes involved in chromosomal stability, and one might suspect that this activity is functioning in the context of the paralogous human MMB/dREAM complex (Georlette, 2007).

Many of the genes regulated by MMB are essential, and in particular, the Class D genes are prominent in this regard. It is possible that the lethality observed for myb-null mutants is the result of misexpression of one or a set of these genes. However, even with a reduction of MMB factors >95% after sustained RNAi treatment, proliferation of Kc cells is still seen in culture. The transcript levels for genes requiring MMB for activation (Class D genes) were only modestly reduced (two- to fourfold) in the absence of MMB. Thus, the regulation of these genes by Myb may not be profound or responsible for the lethality of myb mutations in flies. Nevertheless, there are reasons to suspect that the regulation by MMB of these target genes may be relevant at least in part to the essential requirements for Myb in vivo. Recall that myb-mutant lethality is suppressed by loss of mip40, mip120, or mip130. Following along the lines of the model derived from these genetic studies one might suggest that the critical regulatory step dependent on Myb involves derepression from a quiescent state where cells need to switch on such essential genes for mitotic functions. Such a switching mechanism may be nonessential in cell culture. Hence, repression in a quiescent cell in the developing fly, perhaps mediated by Mip120 and Mip130, may require Myb for induction at a later time or in a specific tissue. In vivo, but not in cell culture, loss of Myb alone (as in myb null mutants) could result in a 'permanently repressed' essential gene whereas loss of the entire MMB complex (as in myb; mip120 mutants) may allow for suboptimal expression levels of an essential gene(s); for instance, at a two- to fourfold reduced level. Thus, a presently scored Class D gene may behave as a Class C repressed gene in another cell type, where loss of Myb would leave a repressive MMB complex that is unable to be induced (Georlette, 2007).

One important issue that needs to be explored is how MMB is targeted to such essential genes. From the genetic suppression data, it is inferred that MMB is still targeted to essential genes even in the absence of Myb, and therefore does not require Myb for targeting to these gene promoters. If the vital function of Myb is to somehow induce a MMB-repressed vital gene(s) where loss of Myb was not critical for repression, then the essential activity of Myb may not require the Myb DNA-binding domain at all. In fact, recent data showed that a transgene containing a complete deletion of the DNA-binding domain of Myb is sufficient for myb-mutant viability (Georlette, 2007).

The number of genomic binding sites for MMB far exceeded what was expected from the MMB gene regulatory network defined by the RNAi analysis. While >80% of the 3538 MMB-binding sites were proximal to promoters, only 25% of proximal genes showed any change is expression when MMB members were depleted following RNAi treatment. Similar observations have been made for other proteins; for example, the number of genes regulated by the Myc, Max, and Mad/Mnt transcriptional network is far lower than the measured number of binding sites for these factors. This type of phenomenon may be a simple consequence of biological noise. It is possible that many of the complexes that are bound to sites not regulating transcription of a nearby gene are simply 'junk' or vestigial in nature. A majority of such sites would have little selective advantage, or they may simply serve as a nonspecific binding pool to keep the levels of non-DNA-bound MMB low. Alternatively, these sites may play some role in other chromosome function(s) apart from gene expression. For instance, some of these 'silent' MMB-binding sites may be directly modulating the selection of replication initiation sites, a point suggested by the role played by the complex in follicle cell gene amplification (Georlette, 2007).

Interestingly, many of the genes that contain 'silent' promoter-proximal MMB-binding sites are expressed in Kc cells and transcript levels are unaffected following removal of MMB. Perhaps, at some of these occupied sites, MMB is simply poised to respond to signals that are absent in the culture media. Prominent among this list are the genes encoding DNA replication proteins such as Chiffon, the ORCs, and MCMs. An evolutionarily conserved multisubunit complex in human cells that contains homologs of many of the MMB/dREAM subunits, represses cell cycle-dependent genes during quiescence. It is, of course, possible that similar control in resting cells will be found for the fly complex. This prospect would then add to the established functions of MMB in repressing differentiation-specific genes, and promoting transcription of Mphase genes. Extending this general point, the Kc cells are fixed in one state through their isolation in culture and well-defined passage conditions. It is possible that some of the 'silent' MMB-binding sites may function in a developmental pathway that is dependent on the action of a new factor or signal not normally seen in Kc cells in culture (Georlette, 2007).

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 de2f1i2/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 dDPa1/dDPa2 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).

RBF binding to both canonical E2F targets and noncanonical targets depends on functional dE2F/dDP complexes

The retinoblastoma (RB) family of proteins regulate transcription. These proteins lack intrinsic DNA-binding activity but are recruited to specific genomic locations through interactions with sequence-specific DNA-binding factors. The best-known target of RB protein (pRB) is the E2F transcription factor; however, many other chromatin-associated proteins have been described that may allow RB family members to act at additional sites. To gain a perspective on the scale of E2F-dependent and E2F-independent functions, genome-wide binding profiles of RBF1 and dE2F proteins were generated in Drosophila larvae. RBF1 and dE2F2 associate with a large number of binding sites at genes with diverse biological functions. In contrast, dE2F1 was detected at a smaller set of promoters, suggesting that it overrides repression by RBF1/dE2F2 at a specific subset of targets. Approximately 15% of RBF1-bound regions lacked consensus E2F-binding motifs. To test whether RBF1 action at these sites is E2F independent, dDP mutant larvae were examined that lack any functional dE2F/dDP heterodimers. As measured by chromatin immunoprecipitation-microarray analysis (ChIP-chip), ChIP-quantitative PCR (qPCR), and cell fractionation, the stable association of RBF1 with chromatin was eliminated in dDP mutants. This requirement for dDP was seen at classic E2F-regulated promoters and at promoters that lacked canonical E2F-binding sites. These results suggest that E2F/DP complexes are essential for all genomic targeting of RBF1 (Korenjak, 2012).

The Drosophila RBF and E2F families lack the complexity of their mammalian counterparts and the distribution of RBF1- and dE2F-binding sites described in this study gives a snapshot of the Rb-E2F network in an in vivo context. The results show that RBF1 acts at > 2000 sites in the Drosophila genome and, in accordance with the idea that RB-family proteins regulate gene expression, most of these binding sites are close to promoter regions (Korenjak, 2012).

RBF1-bound regions overlap extensively with binding sites for dE2F2. Additionally, RBF1 is a component of dREAM/Myb-MuvB complexes and a large percentage (74%) of RBF1-bound regions correspond to dREAM/Myb-MuvB binding sites. In contrast, only a relatively small proportion of the regions that were bound by RBF1 were also bound by dE2F1. These findings are consistent with the idea that dE2F2-RBF1 repressor complexes are present throughout the genome, with their effects being transiently reversed by dE2F1 at a small subset of these targets in proliferating cells. One of the implications of this model is that different subsets of RBF1-repressed genes may be activated in different cellular contexts. In support of this it is noted that GO classification of RBF1-bound promoters showed enrichment for several functional categories that were absent from the lists of dE2F1-bound genes, and potentially these targets may represent groups of RBF1 repressed genes that may only be activated by dE2F1 under specific circumstances, or that may be activated by factors that are different from dE2F1. Since E2F-mediated activation can result from the transient presence of dE2F1, a caveat to this interpretation is the likelihood that RBF1 proteins that are components of a stable repressor complex will be more easily detected by ChIP than a transiently bound dE2F1-activator and it is probable that the list of dE2F1-bound promoters underestimates the overall number of dE2F1 targets. Interestingly it found that dE2F1 target genes with functions in mitosis were conspicuously underrepresented in the lists of genes bound by dE2F1 in larvae, even though they were bound by RBF1 and dE2F2, and they were bound by dE2F1 when chromatin prepared from S2 tissue culture cells was profiled. This difference is consistent with the fact that many larval cells grow via endocycles and do not need to express mitotic genes, but it also suggests that dE2F1 binds to different sets of promoters in mitotically-dividing cells and endoreduplicating tissue. Although dE2F1 is thought to be a ubiquitous driver of cell proliferation, these results suggest that different programs of dE2F1-activated transcription are used in different cell types (Korenjak, 2012).

This experimental system was used to address a key issue in E2F/RB research: how much of the RB-protein that is found on chromatin is bound via E2F, and how much is bound in an E2F-independent manner and might potentially have E2F independent functions? This has been a long-standing question, in part because pRB has been shown to associate with many different chromatin-associated proteins, and in part because mammalian cells express many different E2F complexes and it is difficult to dissect the relative contributions of these complexes on pRB chromatin association. The genome wide binding studies for pRB family members that have been carried out in mammalian cells show an enrichment of E2F consensus sequences in the regions bound by pRB, p107 and p130, but it has been unclear if these motifs are essential for the pocket proteins to associate with chromatin, and whether E2F-binding sites represent either the sole genomic location, the main location, or one of many potential locations for the set of pRB-family proteins. Previous studies have examined the distribution of pRB506 family members in cultured cells and it is unclear how accurately the distributions seen in cell lines reflect the distribution of these proteins in normal tissues (Korenjak, 2012).

In the experiments described in this study, the ability to detect chromatin-associated peaks of RBF1 by ChIP was absolutely dependent on dDP. Consistent with this, most RBF1- bound regions contained a sequence corresponding to a 'consensus' E2F-binding site, but this dependency on dDP was also seen in regions that lacked an obvious E2F-binding motif. In agreement with the ChIP results, in cell fractionation experiments no full length RBF1 was found tightly associated with chromatin in nuclear extracts from dDP mutant larvae. The simplest interpretation of these data is that the stable association of RBF1 with chromatin in Drosophila larvae is completely dependent on its recruitment by dE2F/dDP complexes (Korenjak, 2012).

This finding is surprising given the number of reports that have proposed that RB family proteins have E2F independent functions. There are several potential explanations for this paradox. First, the experiments described here provide a picture of the general properties of RBF1 in larval chromatin, but do not exclude the possibility that RBF1 has E2F-independent targets in specific subsets of cells, or at other stages of animal development. It is noted that loss of RBF1 causes defects in chromosome compaction in neuroblasts that were not seen in de2f1, de2f2 or dDP mutants. This role might not require a stable association of RBF1 with chromatin, or alternatively there may be types of RBF1-DNA complexes that were not detected using the ChIP conditions and were disrupted by the fractionation procedure (Korenjak, 2012).

A second possibility is that E2F proteins may play a larger role in the differentiation-specific functions of pRB than has been appreciated. For example, the fact that pRB and Runx2 co-localize at osteoblast-specific promoters during osteogenic differentiation, and the fact that Runx2 is required for pRB to be recruited to these promoters in Saos-2 cells does not exclude the possibility that E2F proteins might also be involved in this process. A recent study demonstrated that the osteogenesis defects resulting from the conditional deletion of murine Rb can be suppressed by the combined inactivation of E2F1. An alternative possibility is that some of the differentiation defects seen when pRB-family members are inactivated are due primarily to problems in establishing permanent cell cycle exit rather than to a direct role for pRB in differentiation (Korenjak, 2012).

A third scenario is suggested by the observation that pRB may act in some contexts by blocking the function of inhibitors of differentiation, such as EID-1, RBP2 and Id2. Id2 is targeted for degradation by the anaphase promoting complex (APC/C) upon cell cycle exit. Interestingly, during cell cycle exit pRB promotes a physical interaction between the APC/C and Skp2 that results in the targeted degradation of Skp2. Although such activities are not E2F-dependent, it is possible that they normally occur in the vicinity of E2F-recruited complexes. Alternatively, they might not occur in the context of chromatin at all (Korenjak, 2012).

Fourth, almost all of the E2F-independent functions of Rb-family proteins have been discovered using mammalian cells and it is possible that there is a basic difference in the roles of these proteins between flies and mammals. Perhaps, during evolution, mammalian pRB has acquired an ability to interact with additional proteins, while the Drosophila orthologs have remained specific partners of dE2Fs (Korenjak, 2012).

These results provide a cautionary note to the use of 'consensus' E2F-binding motifs. 'Consensus' E2F-binding sequences are widely used to predict sites of E2F regulation. However, human E2F proteins bind to a large number of promoter regions and many of these regions do not harbor classical E2F consensus sequences. Approximately 15% of RBF1-binding sites lacked a clear E2F-binding motif. Further analysis of examples of these promoters showed not only that dE2F/dDP proteins could be detected at these regions, but also the analysis of dDP mutant larvae showed that that these complexes were essential for the recruitment of RBF1 (Korenjak, 2012).

These observations raise the question whether additional sequence motifs (and binding proteins) mediate or assist the binding of E2F complexes to DNA. As a component of dREAM/Myb-MuvB complexes, RBF1 is associated with proteins such as dMyb and Mip120 that have known DNA binding activites, and recent work suggests that several dREAM/Myb-MuvB subunits may assist the binding of dE2F2 and RBF1 to E2F target genes. In contrast to a previous study, which identified a consensus DNA binding site for Myb in a small subset of dREAM/Myb-MuvB binding sites, this study failed to detect an enrichment of this motif in dE2F/RBF bound regions. However a sequence motif with similarity to the CHR element was significantly enriched at dE2F/RBF binding sites. CDE/CHR elements have recently been linked to the sequence-specific DNA binding of the human and C. elegans Lin54 proteins, which are the orthologs of Mip120. Moreover, human DREAM binds the cyclin B2 promoter preferentially through the CHR, but not the CDE element. Given that the majority of E2F binding in Drosophila appears to be in the context of dREAM/Myb- MuvB, CHR elements may play an important role in dE2F DNA binding and be useful in predicting functional E2F binding sites (Korenjak, 2012).

Interestingly, dE2F/RBF bound promoters that lack an E2F consensus motif show a strong enrichment for a sequence resembling the DNA replication element (DRE). DRE's are often found in core promoter sequences in Drosophila and are bound by the DNA replication element binding factor (DREF). dE2F and DREF regulate the expression of an overlapping set of genes involved in DNA replication and both proteins are required for cell proliferation in vivo. DREF forms a complex with TATA-box-binding protein-related factor 2 (TRF2) that regulates the expression of several E2F target genes, including PCNA. In the PCNA promoter the DRE was found to be required, in addition to an E2F-binding site, for dE2F1/dDP-dependent transcriptional activation. It seems likely therefore that, at some promoters, DRE/DREF facilitates the binding and action of dE2F/RBF1 complexes (Korenjak, 2012).

Despite the presence of these additional motifs and their potential role in dE2F/dDP DNA binding, the complete lack of RBF1 binding to chromatin in dDP mutants shows that no additional proteins can compensate for the absence of dE2F/dDP in Drosophila larvae and allow the recruitment of RBF1 to chromatin. This suggests that RBF1 and dE2F/dDP function are intimately linked in Drosophila. In support of this, larval lethality due to the homozygous inactivation of rbf1 can be rescued by a mutation in dE2F1 that deletes sequences in the RBF1-binding and transactivation domains. The binding of Mip130, another dREAM/Myb-MuvB subunit, to polytene chromosomes was only marginally affected in dDP animals, suggesting that submodules of dREAM/Myb-MuvB complexes are recruited to chromatin independent of one another. The results are consistent with the idea that protein/protein interactions with dREAM/Myb-MuvB subunits and DREF may help to recruit dE2F/dDP/RBF complexes to promoters, and this may be especially relevant at promoters that lack a consensus E2F motif, but even in this situation the presence of RBF1 at these promoters is completely dependent on dE2F/dDP complexes. When it becomes possible to completely inactivate all of the different classes of E2F complexes in mammalian cells, it will be interesting to learn how this finding compares with the recruitment of pRb, p107 and p130 to chromatin in humans (Korenjak, 2012).

The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex (see Rbf) has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue (Clavier, 2014).

Protein Interactions

E2f2 encodes a protein of 370 amino acids containing an N-terminal DNA-binding domain, followed by a leucine zipper dimerization domain and a C-terminal pRB-interacting domain, each of which are similar to the corresponding region of other members of the E2F family. A yeast two-hybrid assay was used to determine which regions of E2F2 are required for interaction with DP. Both full-length E2F2 and E2F21-218, which contain only the DNA-binding and leucine zipper dimerization domains, were able to interact with DP as well as or better than E2F. A mutant capable of expressing the first 188 residues of E2F2 could also bind DP. This mutation was tested because E2f21-188 was used as an allele in genetic studies. As expected, a mutant capable of expressing only the first 62 amino acids of E2F2 (i.e. truncated before the DNA-binding domain) was not able to interact with DP in this assay. Thus, the basic architecture of E2F2 is similar to other members of the E2F protein family (Cayirlioglu, 2001).

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. RBF2, a second family member, represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. E2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to E2F2, a form of E2F that functions as a transcriptional repressor. RBF2-mediated repression requires E2F2. Moreover, RBF2 and E2F2 act synergistically to antagonize E2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and E2F1 or E2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in Drosophila (Stevaux, 2002).

The Drosophila EST database contains two entries (LD02737 and LD15806) that showed similarities to the N-terminal portions of RBF and human p107. Sequencing of these cDNAs and the corresponding genomic region confirmed the presence of an RBF-related gene (DDBJ/EMBL/GenBank accession No. AF197059 and AF195899). The original retinoblastoma family member is referred to as rbf1, and the later isolate as rbf2. rbf2 is an intronless gene coding for a 782 amino acid protein with extensive homology to RBF1 and mammalian pocket proteins. RBF2 contains sequences corresponding to the A and the B halves of the pocket-domain, and has potential CDK phosphorylation sites on both sides of the pocket region. RBF2 also contains an N-terminal sequence (the N-box) that is highly conserved in human p107, human p130 and both Drosophila pocket proteins, but is absent in pRB. In addition, and unlike RBF1, RBF2 contains a short spacer sequence between the two halves of the pocket domain that is homologous to sequences in the p107 and p130 spacer regions. However, RBF2 lacks sequences homologous to the high affinity cdk-binding sites found in the N-terminal and the spacer regions of p107 and p130. Thus, the RBF2 protein shares more sequence identity with RBF1 than with any other pocket protein, and like RBF1, appears more closely related to p107 and p130 than to pRB (Stevaux, 2002).

An N-terminal portion of RBF2 was expressed in bacteria and used to generate monoclonal antibodies that specifically recognize RBF2 and do not cross-react with RBF1. On SDS-PAGE, RBF2 migrates slightly faster than RBF1 and separates as a single band of ~85 kDa. The expression of RBF1 and RBF2 was examined by Western blot analysis using extracts prepared at various stages of Drosophila development. The levels of RBF1 are relatively constant; the highest level of RBF1 is seen in 0- to 4-h-old embryos, and this drops slightly during the later stages of embryogenesis. In contrast, RBF2 levels vary considerably during development. RBF2 levels increase 4-fold in the first 8 h of development, and drop by one to two orders of magnitude at later stages of embryogenesis. Comparatively low levels of RBF2 were detected in whole larval extracts, and on long exposures of the Western blots, in adult females, but not in males. Higher levels of RBF2 were found in dissected larval imaginal discs and in tissue culture cells. These patterns suggest that RBF2 may be most highly expressed in rapidly cycling cells, a fact that has previously been for p107. The RBF2 present in female extracts is provided exclusively by the ovary, raising the possibility that RBF2 may function during oogenesis (Stevaux, 2002).

Repression of E2F transcription is a hallmark of RB family proteins. To determine whether RBF2 regulates E2F-dependent transcription, its ability to repress E2F-regulated reporter constructs in SL2 cells was assayed. RBF1 and RBF2 expression constructs were generated and transfected into SL2 cells together with a PCNA reporter that had been previously used to measure the activity of dE2F1 and dE2F2. In order to monitor the expression levels from both constructs, RBF1 and RBF2 were HA-tagged on their N-terminal ends. Titration experiments showed that RBF2 represses transcription from the wild-type PCNA promoter but has no effect on the mutant PCNA reporter construct lacking E2F-binding sites. The repression properties of RBF1 and RBF2 were examined. RBF2 represses transcription from the PCNA promoter, as well as the MCM3 and DNA Polalpha promoters, two other E2F-regulated genes. In these reporter assays, RBF1 proved a more effective repressor than RBF2, when expressed at the same level. The reason why RBF1 and RBF2 expression plasmids give different levels of protein expression is not known. This difference may reflect a property of the endogenous proteins, since quantitative blots show that SL2 cells contain ~30 times more RBF1 than RBF2. It is concluded that RBF2 can repress E2F-dependent transcription, but in a less efficient manner than RBF1 (Stevaux, 2002).

To determine whether RBF1 and RBF2 are present at these promoters in vivo under physiological conditions, a chromatin immunoprecipitation (ChIP) assay was used with specific RBF1 or RBF2 antibodies. DNA sequences from the PCNA and the DNA Polalpha promoters are selectively enriched in the RBF1 and RBF2 immunoprecipitations. It is concluded that RBF1 and RBF2 are both able to repress transcription from E2F-regulated promoters, and that the endogenous RBF1 and RBF2 proteins are normally found at these promoters in vivo. The presence of both Drosophila pocket proteins at E2F promoters suggests that RBF1 and RBF2 may have overlapping functions in the regulation of E2F targets genes (Stevaux, 2002).

Since the overexpression of RBF2 is able to repress E2F-dependent transcription, it seemed likely that RBF2 would repress dE2F1-mediated activation in a manner similar to that previously shown for RBF1. To test this possibility, RBF2 and dE2F1 expression constructs were co-transfected together with a PCNA reporter plasmid in SL2 cells. To ensure that dE2F1 was not saturating, small quantities of the dE2F1 expression plasmid were used that resulted in a 4.5-fold activation of the PCNA reporter. Co-transfection of HA-RBF1 completely blocked this dE2F1-induced transcriptional response, and a significant degree of repression was observed when a low amount of HA-RBF1 was transfected (Stevaux, 2002).

Surprisingly, however, HA-RBF2 had no effect on dE2F1-activated transcription. RBF2 also failed to repress the dE2F1 activation of the DNA Pola reporter. In keeping with these transfection results, it was noted that in transgenic animals, contrary to RBF1, RBF2 overexpression fails to suppress phenotypes caused by elevated levels of dE2F1 in various tissues. Since overexpression of RBF2 does not inhibit dE2F1-driven transcription and does not suppress dE2F1-induced phenotypes in vivo, it appears that RBF1 and RBF2 regulate E2F-dependent transcription in a distinct manner (Stevaux, 2002).

To understand the relationship between RBF1, RBF2, and the E2F proteins, the pattern of dE2F-RBF protein interactions that exist in Drosophila SL2 cells was examined. Specific antibodies for dDP, dE2F2, dE2F1 were used to immunoprecipitate protein complexes from SL2 extracts. These immune complexes were analyzed by Western blotting with monoclonal antibodies specific for RBF1 or RBF2. A single 85 kDa band was detected by an anti-RBF2 monoclonal antibody in dDP and dE2F2 immunoprecipitates, but not in the dE2F1 or the control immunoprecipitates. The blot was stripped and re-probed with an anti-RBF1 monoclonal antibody and, as expected, RBF1 was detected in each of the test lanes. In the reciprocal experiment, dE2F1 was detected in RBF1 immune complexes, but not in RBF2 immune complexes, whereas dE2F2 was found in both RBF1 and RBF2 immune complexes. These results indicate that, under physiological conditions, RBF1 forms complexes with dE2F1 or dE2F2. RBF2, however, does not bind dE2F1, the activator Drosophila E2F, but associates exclusively with the repressor Drosophila E2F, dE2F2 (Stevaux, 2002).

This pattern of interactions could explain, in a very simple way, why RBF2 is unable to block dE2F1-mediated activation. This arrangement also predicts that the effects of RBF2 on E2F-regulated transcription are likely to be mediated via dE2F2. To test this hypothesis, the ability of RBF2 to repress transcription in cells depleted for dE2F2 was assessed by RNA-mediated interference (RNAi). Cells were treated with control or dE2F2-specific double-stranded (ds) RNA and subsequently transfected with an E2F reporter construct and RBF1 or RBF2 expression plasmids. Western blots of the dsRNA-treated cells showed that the level of dE2F2 was substantially reduced by the RNAi treatment after 4 days. The ability of RBF2 to repress transcription from the MCM3 promoter is completely inhibited in cells treated with dE2F2 dsRNA. In contrast with RBF2, the ability of RBF1 to repress transcription is unaffected by the depletion of dE2F2, presumably because of the presence of dE2F1, the other E2F partner of RBF1. This experiment indicates that dE2F2 is necessary for the proper repression of E2F target genes by RBF2. To test whether dE2F2 is required for the detection of RBF2 at E2F-regulated promoters, ChIP assays was performed on cells treated with dE2F2 dsRNA. The depletion of dE2F2 from SL2 cells eliminates the binding of dE2F2 and RBF2 to the DNA Pola promoter, while leaving dE2F1 binding unaffected (Stevaux, 2002).

These experiments show that RBF2 can no longer be localized to E2F-regulated promoters or repress transcription in the absence of the DNA-binding activity provided by dE2F2. While the levels of HA-RBF2 expressed in transient transefction assays were unaffected by depletion of dE2F2, it was noticed that levels of endogenous RBF2 protein were slightly reduced when dE2F2 was depleted by RNAi. Interestingly, Western blots of larval extracts prepared from wild-type and de2f2 mutant larvae show that a long-term consequence of removing dE2F2 is that RBF2 becomes barely detectable. The reduction of RBF2 protein is due to post-transcriptional effects, since rbf2 transcripts are readily detectable in total RNA preparations from de2f2 mutant larvae and, indeed, are present at elevated levels in the de2f2 mutants. The most likely explanation is that RBF2 becomes less stable in the absence of its binding partner. At present, the formal possibility that dE2F2 influences the synthesis of RBF2 cannot be excluded. Nevertheless, these observations all support the notion that the function of RBF2 depends on the presence of dE2F2 (Stevaux, 2002).

Previous studies have revealed that dE2F1 and dE2F2 have distinct biochemical and functional properties. Both proteins can be found at endogenous E2F-regulated promoters, but dE2F1 functions primarily as an activator of transcription, whereas dE2F2 is a transcriptional repressor. Studies of de2f1, de2f2 and de2f2;de2f1 double mutants demonstrate that the normal expression patterns of E2F-target genes depends on the integrated activities of both dE2F1 and dE2F2. Thus far, it has been observed that (1) RBF2 interacts specifically with dE2F2, (2) dE2F2 is able to antagonize dE2F1 in a manner dependent upon its interaction with RBF proteins, and (3) RBF2-mediated repression requires dE2F2. These results suggest that transcriptional repression by RBF2 is not mediated via dE2F1. Rather, RBF2 appears to form an RBF2-dE2F2 complex that antagonizes dE2F1 indirectly, by altering the balance between the transcriptional activities of dE2F1 and dE2F2 (Stevaux, 2002).

This idea was tested directly in SL2 cells, where constant levels of transfected dE2F1 were challenged with increasing amounts of transfected dE2F2. The overall level of transcription generated by both dE2Fs at the PCNA promoter were compared with and without co-transfected RBF2. The combined expression of dE2F2 and RBF2 is able to reduce dE2F1-mediated activation of the PCNA reporter far more effectively than dE2F2 alone. Thus, in SL2 cells, RBF2 and dE2F2 cooperate to antagonize the transcriptional activity of dE2F1 (Stevaux, 2002).

The combined expression of dE2F2 and RBF2 generates a small rough eye phenotype. This small eye phenotype is similar to, but weaker than, what is observed with an ey-Gal4/UAS-rbf1 transgenic line. Wing discs overexpressing RBF2 and dE2F2 were examined to determine the physiological basis of the observed phenotype. The co-expression of RBF2 and dE2F2 causes a significant decrease in DNA synthesis. Overexpression of RBF2 and dE2F2 gives a phenotypic range that varies from discs that are shrunken and abnormal to those discs that have relatively normal morphology but display reduced BrdU incorporation. The defects do not appear until sufficient amounts of dE2F2 and RBF2 have accumulated in sensitive tissues (Stevaux, 2002).

To further assess the effects of RBF2-E2F2 expression on the cell cycle, clones of dE2F2-RBF2-overexpressing cells were generated in the wing discs and marked with GFP. The cell cycle profile of these cells was determined by FACS analysis and compared with wild-type cells from the same discs. The overexpression of dE2F2 or RBF2 alone has no effect on cell cycle distribution. However, the co-expression of dE2F2 and RBF2 causes a significant increase in the population of cells with a G1 DNA content, and a decrease of S phase and G2 cells. Taken together, these experiments demonstrate that, when overexpressed, dE2F2 and RBF2 act synergistically to antagonize dE2F1-mediated transcriptional activation and to block S phase entry in vivo (Stevaux, 2002).

It is concluded that although RBF1 and RBF2 are both able to repress E2F-dependent transcription, they appear to act in markedly different ways. RBF1 was originally identified by virtue of its ability to physically interact with the transcriptional activation domain of dE2F1. RBF1 is a potent inhibitor of dE2F1-mediated activation and it readily suppresses dE2F1-induced phenotypes in vivo. Unlike RBF1, RBF2 does not associate with dE2F1 in vivo, and it is unable to suppress the effects of overexpressed dE2F1. In vivo, RBF2 associates specifically with dE2F2. The recruitment of RBF2 to E2F-regulated promoters, and its ability to repress transcription, requires dE2F2. In support of the idea that RBF2 acts in a stable complex with dE2F2, these proteins act synergistically when overexpressed in SL2 cells or in transgenic animals, and that RBF2 levels are strongly reduced in de2f2 mutant larvae (Stevaux, 2002).

Of the four proteins, only dE2F1 activates transcription. It is suggested that RBF proteins modulate dE2F1-mediated activation in two distinct ways. (1) dE2F1 can be directly regulated through a physical interaction with RBF1. (2) dE2F1-mediated activation can be antagonized by the presence of RBF2-dE2F2 and RBF1-dE2F2 repressor complexes at the promoter of E2F-regulated genes. Because E2F-regulated promoters often contain multiple E2F-binding sites, it is unclear whether these complexes compete for the same binding element or whether they act antagonistically through adjacent sites. The results described here suggest that there is a hierarchy of effects, with RBF1 being a stronger antagonist of dE2F1 than either of the dE2F2-containing complexes. Nevertheless, the level of dE2F1-dependent transcription is influenced by dE2F2 and RBF2, and changing the levels of RBF2 alters the balance between dE2F1-mediated activation and dE2F2-mediated repression. One of the implications of this arrangement is that a pocket protein does not need to bind directly to an E2F subunit in order to influence its activity (Stevaux, 2002).

Strikingly, a similar arrangement of E2F/pocket proteins exists in mammalian cells. Although mammalian cells contain multiple E2F and pRB family members, recent studies have suggested that the different forms of E2F can be subdivided into two groups depending on whether they appear to be primarily involved in activation or repression. Intriguingly, pRB, like RBF1, interacts with both sets of E2Fs, whereas p107 and p130, like RBF2, interact specifically with the co-repressor E2Fs. Consistent with this, the loss of RBF1 function induces phenotypes that are remarkably similar to the effects of mutating pRB (deregulation of E2F, ectopic S phases, increased apoptosis), and the mutation of dE2F1 gives phenotypes that are very similar to those recently described for the combined mutation of the murine E2F1, E2F2 and E2F3 genes (G1 arrest and loss of E2F-dependent transcription). Furthermore, the genetic interactions observed between RB and E2f1 or RB and E2f3 alleles in mice are reminiscent of the genetic interactions between rbf and de2f1 in flies. The logical extension of this homology is that the roles of RBF2-E2F2 in Drosophila may be similar to the repressor complexes formed by p107/p130 and E2F4/E2F5 in mammalian cells. Despite the attractions of this analogy, it is noted that the phylogenetic tree of Retinoblastoma-related proteins shows that RBF1 and RBF2 are more closely related to one another than they are to any mammalian protein. This suggests that RBF1 and RBF2 were generated by a gene duplication event, most likely from an ancestral protein that resembled p107 or p130. Consequently, similarities between the arrangement of Drosophila and mammalian RB-E2F complexes are more likely to result from a convergent evolutionary process, rather than the conservation of functional differences between pocket proteins (Stevaux, 2002).

What is the advantage of such an arrangement of complexes, and why might it be selected? The distinction between activator and repressor E2Fs is most important if one considers the effects when the complexes are disrupted. Since dE2F2 appears to be unable to activate transcription, the release of RBF1 or RBF2 from a dE2F2 complex is predicted to de-repress E2F target genes. In contrast, the release of RBF1 from a dE2F1-containing complex would liberate a strong activator of transcription. These types of E2F complexes therefore allow three different types of E2F regulatory transitions: (1) from repression to de-repression (in which RBF2-dE2F2 or RBF1-dE2F2 complexes are disrupted and removed from the promoters); (2) from repression to activation (in which RBF1-dE2F1 complexes are disrupted by phosphorylation liberating dE2F1, a strong activator of transcription); and (3) from repression to de-repression to activation (in which RBF2-dE2F2 or RBF1-dE2F2 repressors are disrupted and replaced by dE2F1) (Stevaux, 2002).

The evolution of multiple E2F-RB complexes may offer several additional advantages. Individual complexes may preferentially regulate different subsets of targets. This specificity might be achieved by different DNA-binding subunits (e.g. E2F1-RBF1 versus E2F2-RBF1) or by different protein-protein interactions with adjacent factors at the promoter. Indeed, several studies have suggested that mammalian E2F/pocket proteins may target specific promoters. A second possibility is suggested by the fact that many chromatin-remodeling activities have been linked to pocket proteins, potentially allowing a wide variety of activities to be recruited to E2F-regulated promoters. Perhaps RBF1 and RBF2 provide a bridge to different types of complexes. A third possibility is that the pocket protein-E2F complexes may be differentially regulated. pRB appears to be uniquely required for DNA damage-induced cell cycle arrest. pRB is specifically required in TGFß and p16-induced cell cycle arrest. In a similar way, RBF2 may be required for cell cycle arrest at a specific stage of development or in particular tissues. The high levels of RBF2 protein in early embryos and in dissected ovaries may reflect specific roles in embryonic cell cycle and oogenesis. Alternatively, RBF1- and RBF2-containing complexes might occupy E2F sites during different phases of the cell cycle. E2F has recently been shown to control the expression of genes encoding mitotic functions whose transcription is induced later than the G1 to S transition. RBF2, like p107, is expressed at higher levels in actively cycling cells and is a likely E2F target gene. Potentially, this newly synthesized pocket protein may provide a repressor activity during S phase on mitosis-specific promoters or during G2 at S phase-specific promoters (Stevaux, 2002).

The answer to many of these questions will stem from a careful comparison between rbf1 mutants, rbf2 mutants and rbf1;rbf2 double mutants. This analysis will be needed to separate the specific functions of RBF1 and RBF2 in vivo and to uncover the functions that are redundant between the two Drosophila pocket proteins (Stevaux, 2002).

Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes

In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. The second Drosophila Retinoblastoma family gene (Rbf2) has been mutated, and the in vivo molecular functions of RBF2 have bee contrasted with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. This study finds that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. The profiles were compared of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated; RBF2 and dE2F2 were found to control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues (Stevaux, 2005).

In order to study the unique functions of Rbf2 advantage was taken of a P-element insertion, P2000, located 5 kb downstream of the Rbf2 ORF, and it was used to generate Rbf2 deletion alleles. Western blot analysis showed that stocks homozygous for two alleles, rbf2M2 or rbf2M3, lack the full-length RBF2 protein and express a 30kD amino-terminal fragment of RBF2 (DRBF2). This fragment lacks the entirety of the RBF2 pocket domain and is thus unable to perform any of the functions associated with this signature domain. Hence, animals carrying the rbf2M2 or rbf2M3 deletion alleles are deficient for E2F binding and transcriptional repression functions of RBF2 (Stevaux, 2005).

Stocks homozygous for rbf2M2 and rbf2M3 are viable and do not display visible developmental abnormalities. Because de2f2 mutant flies exhibit fertility defects, tests were performed to see whether rbf2 mutants also have reduced fertility. It was found that rbf2 mutant females have no fertility defect: they lay an increased number of eggs as compared to wild-type flies and none of the eggs exhibit morphological abnormalities. Furthermore the hatching rate of eggs laid by rbf2 mutant females is comparable to the hatching rate of eggs laid by wild-type flies (Stevaux, 2005).

To test for functional overlap between RBF1 and RBF2 in vivo, the rbf2M2 mutant was crossed with two rbf1 mutant strains. First a weak loss-of-function allele of rbf1 (rbf1120a), which harbors a P-element insertion in its promoter region, was used. The rbf1120a viable allele exhibits normal eye architecture but interacts genetically with mutations in cell cycle regulators such as p21Cip/Kip and de2f1. Flies carrying rbf1120a and homozygous for rbf2M2 displayed a rough eye phenotype that was absent in the single rbf2M2 and rbf1120a mutant strains indicating that mutation in both genes has a cooperative effect. This rough eye phenotype was rescued by an Rbf2 genomic rescue construct. Next, the genetic interaction between rbf1 and rbf2 was examined using the null allele rbf1D14. rbf1D14 larvae are sub-viable, never reach the late pupal stages of development, and display a developmental delay of up to two days at the third instar larval stage. rbf1D14; rbf2M2 double mutant larvae exhibit significantly poorer viability than rbf1D14 larvae at late larval and early pupal stages. Moreover, the double mutants had a greater developmental delay, taking up to nine days to reach the early pupal stages (Stevaux, 2005).

These results show that loss of rbf2 enhances the defects observed in rbf1 mutant animals. Identical genetic interactions were observed with the rbf2M3 mutant. These results are consistent with previous experiments showing that RBF2 has many of the same properties as RBF1 and with the fact that RBF1 and 2 redundantly repress a number of E2F-regulated genes (Stevaux, 2005).

RBF2 and dE2F2 control minimally overlapping transcriptional programs in vivo. Since rbf2 mutant flies are viable, these animals provided an opportunity to examine the role of RBF2 in transcriptional regulation in vivo. Such experiments have not been possible using null alleles of de2f1 or rbf1 because these mutations cause dramatic developmental defects. Previous studies in tissue culture cells showed that RBF2 acts redundantly with RBF1 at E2F-repressed genes. The fact that RBF2 levels vary greatly during development suggests that its function in vivo may be tissue or stage specific. In adult flies RBF2 protein levels are barely detectable in males but are markedly elevated in ovaries (Stevaux, 2002). To assess the potential role of RBF2 in transcriptional regulation in this tissue, the transcriptional profiles of wild type and rbf2 mutant ovaries were compared (Stevaux, 2005).

Transcripts displaying a statistically significant two-fold increase in rbf2 mutants, as compared to wild type extracts, were compiled and analyzed. The list of genes with increased expression shows that, in ovaries, RBF2 is required for the repression of genes with a wide variety of functions, some of which, remarkably, are male-specific testis differentiation markers. These results reveal a unique role for RBF2. Unlike RBF1, RBF2 does not appear to regulate cell cycle genes. Rather, RBF2 functions to repress the expression of differentiation markers (Stevaux, 2005).

It was asked if the transcripts that are repressed by RBF2 in vivo are also repressed by dE2F2, the only known partner of RBF2. de2f2 mutant flies have reduced viability and fertility. However, the extent and nature of these defects differ for the two reported de2f2 null alleles, suggesting that animals deficient for dE2F2 are highly sensitive to mutations at other loci in the genome. To exclude genetic background-specific and/or allele-specific effects, de2f2 mutant females were used that carry trans-heterozygous combinations of the two alleles. Although these animals are homozygous null for dE2F2, they lay normal eggs and do not exhibit the morphological defects previously described for the homozygous alleles of de2f2. They also display much less pronounced fertility defects -- slightly reduced hatching rate and lower number of eggs laid. Genes that have elevated expression in de2f2 mutant ovaries were also identified. No deregulation of genes with functions in DNA replication were detected, as was observed by Cayirlioglu (2001) who performed an analysis of E2F regulated transcripts in ovarian follicle cells. Two reasons may have precluded detecting these changes: (1) the fold change reported by Cayirlioglu was small, between 1.5 and 2.0, while this study considered only genes that exhibited a fold change of 2.0 or more; (2) the other ovarian cell types may have masked the deregulation of these genes, which occurs in a subset of ovarian cells (Stevaux, 2005).

By comparing the lists of RBF2 and dE2F2 target genes, an unpredicted feature was noted. Although RBF2 physically associates with dE2F2, and the ability of overexpressed RBF2 to repress E2F transcription requires dE2F2 the overlap between the RBF2-dependent genes and the dE2F2-dependent genes was unexpectedly small. Comparisons of the transcripts elevated in de2f2 and rbf2 mutant ovaries show that only 16 transcripts (~8%) were elevated in both mutants. The expression patterns of these differentially regulated genes underline the complexity of dE2F/RBF transcriptional control in vivo with individual target genes requiring either dE2F2 (CG5250), RBF2 (CG17470) or both gene products (CG8316, CG4250, CG10654) to maintain their normal patterns of expression (Stevaux, 2005).

Given the observed functional redundancy between RBF1 and RBF2 at dE2F2-regulated promoters in S2 cells, it is not surprising to find some transcripts upregulated in de2f2 mutant ovaries that remain unchanged in rbf2 mutant ovaries.12 However, the presence of transcripts elevated in rbf2 mutants but unchanged in de2f2 mutants raises a number of possibilities. First, RBF2 could be recruited to particular promoters by factors other than dE2F2. Second, a dE2F1/RBF1 complex might repress these targets in the absence of dE2F2, but be precluded to bind when RBF2 is missing due to the retained presence of dE2F2 at these promoters. Third, these changes in gene expression could be indirect effects of mutating either dE2F2 or Rbf2. To distinguish between these possibilities, chromatin immunoprecipitation (ChIP) assays were performed with ovary chromatin extracts on a number of RBF2- and dE2F2-regulated promoters (Stevaux, 2005).

ChIP analysis revealed that dE2F and RBF proteins bind at the promoters of genes from every category, verifying that these genes are directly regulated by dE2F/RBF. As evidenced by the lack of dE2F1 binding to their promoters, these genes appear to be specifically regulated by dE2F2-mediated repression similar to the genes repressed by dE2F2/RBF1 and dE2F2/RBF2 in S2 cells. Interestingly, dE2F2 and RBF2 were found at these promoters, regardless of whether they are upregulated in de2f2 mutant ovaries, rbf2 mutant ovaries, or both. The fact that dE2F2 and RBF2 are part of a large multi-subunit complex, the dREAM complex, raises the possibility that RBF2 can be retained within this complex via another subunit and maintain repression in the absence of dE2F2. Studies with de2f2 mutant ovaries rule out this possibility since RBF2 can no longer be detected at any promoter in de2f2 mutant ovaries indicating that RBF2 is recruited to promoters via dE2F2. It is thought unlikely that dE2F1/RBF1 can redundantly repress transcription of these genes, since no binding to their promoters is detected in de2f2 mutant ovaries, even though dE2F1 can bind to some dE2F2-regulated genes in the absence of dE2F2. It is likely that a repression mechanism independent of dE2F1 can function in the absence of dE2F2 to inhibit the expression of these genes (Stevaux, 2005).

These results suggests that the different regulation of these target genes is not determined by the differential binding of dE2F/RBF proteins to their promoters. Rather, the importance of the individual components most likely results from the differential requirement of additional cofactors. RBF2 and dE2F2 control cell-type specific transcriptional programs. Since its discovery, E2F has been viewed as a regulator of cell proliferation. Because the genes regulated by E2F encode core components of the cell cycle machinery, and E2F is a ubiquitous factor, it has been generally assumed that E2F regulates the transcription of these genes in most, if not all cell types. E2F has been studied in a wide variety of tissue culture cells. These tissue culture studies have, for a large part, placed very little emphasis on the types of cells used for experimentation. Although the activity of E2F is understood to vary between cell lines, and at different stages of the cell cycle, many reviews of the E2F literature contain an implicit assumption that E2F and RB like proteins target the same sets of genes in all cell types. This assumption was tested by comparing the ovary RBF2-dependent targets genes with target genes previously identified in S2 cells following RBF2 specific RNA interference. Remarkably, minimal overlap was found between the transcripts that are elevated in RBF2-deficient ovaries and in S2 cells. Only 1 gene (<1%) is commonly elevated in rbf2 mutant ovaries in vivo and in RBF2 depleted S2 cells. This analysis was extended to dE2F2, and using the same methodology the transcripts that have elevated expression in de2f2 mutant ovaries were compared with the genes upregulated in dE2F2-depleted S2 cells. In keeping with the results obtained for RBF2, a small overlap of only 15 genes was found between these two lists. The limited overlap between the genes regulated by dE2F2 and RBF2 in S2 cells and in ovaries indicates that dE2F2 and RBF2 are needed to regulate distinct sets of genes in different cell types (Stevaux, 2005).

In order to understand the molecular basis for the differential effects observed following removal of dE2F2 and RBF2 in ovaries and S2 cells, focus was placed on genes that were upregulated in de2f2 mutant ovaries, but that were unchanged in the equivalent microarrays of S2 cells following the depletion of dE2F or RBF proteins, and the occupancy of the promoters of such targets was compared in ovaries and in S2 cells. Arp53D served as a positive control for this experiment since it was de-repressed in the absence of dE2F2 in both S2 cells and ovaries. As one would expect, the Arp53D promoter is occupied by dE2F2 and RBF2 in both settings (Stevaux, 2005).

Clear-cut results were obtained for the four promoters examined. The promoters of two targets, CG10654 and CG14610, are occupied by RBF2 and dE2F2 in ovaries but not in S2 cells. These changes in promoter occupancy provide a very simple explanation for why their expression levels change in ovaries but not in S2 cells following dE2F2/RBF2 removal: these promoters are not occupied by dE2F2/RBF2 repressor complexes in S2 cells, hence removing these proteins in these cells has no effect; but they are occupied by dE2F2/RBF2 complexes in ovaries, hence in this organ, removing these repressor proteins results in increase gene expression. These changes illustrate that dE2F/RBF repressor proteins are physically redistributed to different promoters in different cell types (Stevaux, 2005).

Promoter occupancy analysis of the CG5245 and CG15267 promoters provided an equally clear result, but a different answer. The ChIP experiments show that these promoters are occupied by dE2F2 and RBF2 in ovaries as well as in S2 cells. However, in ovaries the binding by dE2F2 and RBF2 is functionally important since the transcription of these genes is elevated when these proteins are removed. In contrast, removing dE2F2 and RBF2 in S2 cells had no effect on the expression of these genes. Thus, the functional importance of RBF2 and dE2F2 at these promoters varies in different cell types. It has been shown that in S2 cells, dE2F2 and RBF2 can be found at promoters of genes whose expression did not change in the absence of either dE2F2 or RBF2. The results from the current study demonstrate that the functional importance of individual dE2F/RBF proteins varies not only between different promoters in a given cell type, but also at the same promoter in different cell types (Stevaux, 2005).

Thus, in vivo promoter-binding studies point to two fundamentally different mechanisms that contribute to the existence of cell-type specific dE2F/RBF transcriptional programs. First dE2F/RBF repressor complexes are physically relocalized to different promoters in different cell types, and second the functional importance of individual RBF and dE2F family members in regulating particular target genes changes in different cell types. Rbf2 and dE2F2 control changing sets of target genes during development. As a further test of the idea that RBF2 controls the expression of different sets of genes in different cellular and developmental contexts, the RBF2 transcriptional program was examined at a different developmental stage. Given that RBF2 is part of the recently described dream embryonic transcriptional repression complex and that RBF2 levels are markedly increased in embryos 4 to 8 hours after egg deposition (Stevaux, 2002), the transcriptional profiles of wild type and rbf2 mutant embryos were compared using microarrays at this early embryonic developmental stage (Stevaux, 2005).

Numerous transcripts were found to be elevated in early rbf2 mutant embryos, including a number of ovary- and testis-specific differentiation markers. In keeping with earlier observations, the overlap was minimal between transcripts elevated in rbf2 mutant embryos, rbf2 mutant ovaries and S2 cells depleted for RBF2. Interestingly, S2 cells, which are of embryonic origin, do not display upregulation of the genes elevated in de2f2 and rbf2 mutant embryos. The lack of deregulation in S2 cells may stem from the fact that RBF2 and dE2F2 control different sets of genes at different developmental stages: S2 cells are derived from 20-24 hour-old embryos, whereas in vivo target gene were observed to be deregulation in 4-8 hour-old embryos. Alternatively, these differences could be due to the fact that the cell lineage in which the dE2F2/RBF2 embryo-specific transcripts are elevated in vivo is different than the lineage from which S2 cells are derived (Stevaux, 2005).

Transcripts identified in the rbf2 embryo array were further analyzed by Northern blot. This figure demonstrates that transcripts that are repressed in the embryo by RBF2 and dE2F2 (Kek1, CG8607 and CG3509) do not require RBF2 and dE2F2 in ovaries and S2 cells for their proper transcriptional regulation. Conversely, a target gene that requires RBF2/dE2F2 in ovaries (CG4250) no longer requires them in S2 cells and embryos for its repression. The results lead to the conclusion that RBF2 and dE2F2 do not control a single static transcriptional program but, rather, they regulate different sets of genes, not only in different cell types, but also at different stages of development (Stevaux, 2005).

Taken together these results demonstrate that RBF2 is required in vivo for the direct transcriptional repression of dE2F2 targets encoding a wide array of biological functions, including genes encoding sex-specific differentiation markers. Remarkably, the dE2F2/RBF2-dependent transcription program appears to be variable, rather than constant (Stevaux, 2005).

Previous work has reported that individual dE2F family members have common as well as specific transcriptional target genes. Among the common targets are genes with functions in cell cycle progression and DNA synthesis. Recent work suggests that the type of regulation of these targets depends on the cellular context. While in S2 cells and in the developing eye disk dE2F1 and dE2F2 play antagonistic roles, activating and repressing their expression respectively, in ovarian follicle cells dE2F1 and dE2F2 cooperate to repress transcription of DNA synthesis genes. This study confirms this idea by showing that dE2F2/RBF2 complexes control the expression of changing sets of genes in different cell types and/or different developmental contexts. Collectively this data indicates that dE2F/RBF proteins provide a diversity of transcriptional regulatory inputs at different target gene promoters in different contexts. Thus, a variety of transcriptional outputs is achieved by a relatively simple set of proteins. This regulation is likely conserved throughout evolution and it is tempting to speculate that the more complex mammalian E2F/RB network will generate even a greater variety of transcriptional responses. Clearly the next challenge will be to identify the cofactors that act in concert with E2F/RB proteins in each context to confer functional importance. Likely candidates include E-Box transcription factors that have recently been shown to associate with E2F3 and allow it to specifically target subsets of promoters (Stevaux, 2005).

The ability of E2F and RB family members to regulate distinct sets of genes in different cell types means that E2F/RB functions should be viewed only in the context of a particular cellular environment and imply that E2F microarray studies based on a single cell type most likely underestimate the full range of E2F target genes. This regulatory plasticity could also explain why the biological responses to perturbations the E2F/RB pathway can be seen to vary so much between different tissues, be that in the context of normal development or in tumorigenesis (Stevaux, 2005).

Slimb regulates E2f2 in Drosophila egg chamber development

Substrate-specific degradation of proteins by the ubiquitin-proteasome pathway is a precise mechanism that controls the abundance of key cell regulators. SCF complexes are a family of E3 ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome. These complexes are composed of three constant polypeptides -- Skp1, Cullin1/3 and Roc1/Rbx1 -- and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. Slmb participation in egg chamber development was analyzed and slmb was found to be required in both the follicle cells and the germline at different stages of oogenesis. In slmb somatic clones, morphogenesis of the germarium and encapsulation of the cyst are altered, giving rise to egg chambers with extra germline cells and two oocytes. Furthermore, in slmb somatic clones, ectopic Fasciclin 3 expression was observed, suggesting a delay in follicle cell differentiation, which correlates with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. Later in oogenesis, Slmb is required in somatic cells to specify the position, size and morphology of dorsal appendages. Mild overactivation of the Dpp pathway causes similar phenotypes that are antagonized by simultaneous overexpression of Slmb, suggesting that Slmb normally downregulates the Dpp pathway in follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer trap reveals that the Dpp pathway is upregulated in slmb somatic clones and, consistent with this, ectopic accumulation of the co-Smad protein, Medea, occurs. By analyzing slmb germline clones, it was found that loss of Slmb provokes a reduction in E2f2 and Dp levels, which correlate with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte (Muzzopappa, 2005).

Thus Slmb is required for oogenesis in both the germline and FC. In the germline, Slmb plays a role in the control of mitotic cycles during cyst formation, in regulation of nurse cell endoreplication and in nurse cell dumping. Recent reports have demonstrated that Slmb can control cell cycle progression in different experimental settings. Following DNA replication, Slmb is required in larval wing discs for proteolysis of the cell cycle modulator E2f1. Remarkably, the E2f complex is implicated in cell cycle control of ovarian germ cells, in nurse cell transition from polyteny to polyploidy and in dumping of the nurse cell content into the oocyte. This study shows that two subunits of the E2f complex, Dp and E2f2, are downregulated in ovaries bearing slmb germline clones, while E2f1 does not change. Differences in Cyclin E levels, another cell cycle regulator involved in cyst formation, could not be detected in these clones. A good correlation exists between the phenotypes observed in slmb germline clones and in Dp germline clones; in both cases an additional round of cystocyte mitotic divisions occurs. In order to understand the molecular mechanism causing Dp and E2f2 reduction in slmb germline clones, a detailed analysis of the alterations of the network regulating the cell cycle is required (Muzzopappa, 2005).

tMAC, a Drosophila testis-specific meiotic arrest complex paralogous to Myb-Muv B, contains Myb, E2F2, Caf1/p55 and Aly, and can activate or repress gene transcription

The Drosophila Myb-Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. A mutation has been identified in mip40, and a mutation has been generated in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. Mip40-containing complexes were purified from testis nuclear extracts and tMAC, a new testis-specific meiotic arrest complex was identified that contains Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types (Beall, 2007).

Coordinating developmentally regulated transcription and replication patterns in metazoans is critical for differentiation of tissue-specific cell types. Central to these processes is the modification and/or remodeling of chromatin by multisubunit complexes through association of site-specific DNA-binding proteins. A multisubunit complex in Drosophila, the Myb-Muv B (MMB) or dREAM complex contains the previously identified five-subunit Myb complex (containing Myb, Caf1/p55, Mip40, Mip120, and Mip130), in addition to E2f2, Rbf1 or Rbf2, DP, and Lin-52. Curiously, the complex contains both activator (Myb) and repressor (Mip130, Rbf1, Rbf2, and E2f2/DP) proteins. That MMB is widely expressed in different tissues has led to the idea that MMB may function both as an activator or repressor at specific chromosomal locations. Depending on the developmental pathways in particular tissues or cell types, different signals might regulate switching of function at a particular site. It has been suggested that at sites known to be repressed by MMB, Myb is a silent member not participating in the transcriptional repression, even though Myb itself is present at the cis-acting site, and that activation of MMB at a subsequent time would depend on Myb function (Beall, 2007).

The finding that Lethal (3) Malignant Brain Tumor [L(3)MBT], NURF, and the histone deacetylase Rpd3 associate with MMB suggests that histone binding and/or modification are possible mechanisms by which MMB acts to repress transcription and/or replication. Thus, MMB and individual subunits are poised to change their measured role by switching off repression (or activation) in a given cell lineage by post-transcriptional modifications or association of coactivators (or repressors) (Beall, 2007).

The genes regulated by MMB in Drosophila tissue culture cells are primarily differentiation and development-specific genes, and most often, MMB is a transcriptional repressor. Recent genomic profiling in Kc cells of five MMB members (Mip130, Mip120, Myb, E2F2, and Lin-52) showed that these proteins were bound together at thousands of chromosome sites, and RNA interference (RNAi) experiments revealed that MMB participated in either transcriptional repression or activation for many genes. In cell culture and in vivo, the accumulation of Myb and E2F2 proteins, but not mRNAs, depends on the integrity of MMB: Loss of Mip130 dramatically affects the levels of both proteins. These data, together with the biochemical finding that essentially all of Myb is found in complex with MMB, led to the proposal that most if not all of the phenotypes previously identified as Myb-specific (or E2F2-specific) must be evaluated in terms of loss of MMB function in either myb or e2f2 mutants (Beall, 2007).

myb is an essential gene in Drosophila. However, mutations in the largest subunit of MMB, mip130, are viable and suppress myb lethality. Furthermore, homozygous mip130 mutant females have drastically reduced fecundity. Cytological and developmental studies of egg chambers from several MMB subunit mutants were critical in building a heuristic model for MMB function. Normally in the ovary, a developmentally controlled replication program occurs in the somatic follicle cell nuclei surrounding the developing oocyte. In these nuclei, overall genomic replication ceases at stage 9 during egg chamber development and is followed at stage 10 by specific DNA replication at four loci that results in amplification of the genes critical for egg shell formation. Myb binds to the well-defined enhancer for one such amplicon in vivo. When myb is removed by genetic manipulation, replication no longer occurs at the four foci, demonstrating a direct and positive role for Myb in replication at these sites. In contrast, mip130 mutant ovaries display global genomic replication in amplification-stage follicle cell nuclei, indicating a negative role for mip130 in replication at sites other than at the chorion origins. Based on these observations, it has been suggested that MMB functions as either an activator or repressor of chromosomal functions depending on the chromosomal and developmental context. Critically in this model, the essential function of Myb is to activate a repressive complex to which it belongs. In its absence, this unchecked repressive activity by the partial MMB complex is lethal. The presumption was that animals lacking MMB (as in mip130 mutants) maintained expression (or repression) of normal target genes through less robust or redundant mechanisms, resulting in viability of these mutants. Furthermore, a critical, but previously untested prediction of this model is that an MMB complex devoid of Myb could still be targeted to chromosomes (Beall, 2007).

In order to gain further insight into the role(s) for MMB in vivo, a mutation was generated in the second largest subunit, mip120. In addition, a P-element-induced allele was identified in another subunit, mip40. As with mip130, it was found that mip40 and mip120 mutants were viable and displayed either sterility (mip120) or reduced fecundity (mip40) of mutant females. Moreover, mip40 and mip120 suppressed myb lethality, again suggesting that the essential function of Myb in vivo is to counter a repressive activity of MMB. Immunostaining of polytene chromosomes revealed that the association of Myb with specific chromosomal sites was dependent on Mip120, reinforcing the idea that Myb needs MMB for chromatin binding. Conversely, Mip120 and Mip130 did not require Myb for polytene chromosome binding (Beall, 2007).

Unanticipated and in contrast to mip130 mutants, it was found that mip40 and mip120 mutant males were sterile, thus defining a new role for these proteins in male fertility. Given that Mip130 has a paralog in the testis called Always early (Aly), the possibility was investigated that Mip40 and/or Mip120 might either function in a testis-specific Aly complex or that one or both might have testis-specific paralogs (Beall, 2007).

A combination of affinity, ion-exchange, and gel filtration chromatography was used to isolate Mip40-containing complexes from testis nuclear extracts. In addition to MMB, tMAC, a new testis-specific meiotic arrest complex in which the only MMB subunits found were Mip40 and Caf1/p55, was identified, in addition to the testis-specific proteins Aly, Cookie monster (Comr), Matotopetli (Topi), and Tomb. It is suggested is that MMB functions as a cell-type- and developmental-stage-specific regulator of transcription and replication with various subunits contributing to a 'Swiss Army Knife' type of versatility: the ability to interact specifically with numerous cis-elements, and to interact with numerous coactivators or corepressors as determined by context. This versatility extends beyond MMB itself, as some subunits are part of other tissue-specific complexes involved in gene expression (Beall, 2007).

These genetic findings that clearly separate developmental functions for Mip40 and Mip120 do not provide mechanistic insights into how the pleiotropic effects are manifested. For example, partial MMB complexes, resulting from loss-of-function alleles, may assume neomorphic activity. It has been argued that myb lethality in Drosophila is a consequence of such effects. The model that rationalizes 'silent subunits' present at a given location to promote switching from repression to activation (or vice versa) adds genetic complexity to the timing of critical execution functions for different MMB factors. This model anticipates that loss-of-function alleles of genes for different MMB subunits would manifest different arrest points. The possibility was considered that MMB subunits do not always function as a unit. The finding that mip40 mutant males displayed a staining pattern for Mip40 protein in testes quite distinct from Mip130 or Mip120 was curious. Epitope masking of one or another protein in MMB, due to changing coactivator or corepressor association, might explain such staining patterns. However, further work led to the search for a putative testis-specific complex that contained Mip40, where it reasonably would act in a distinct way from its functions in MMB (Beall, 2007).

A complex was purified from testis nuclear extracts that contains MMB members Mip40 and Caf1/p55, in addition to the testis-specific meiotic arrest proteins Aly, Comr, Topi, and Tomb. This complex was named tMAC, because aly, comr, topi, and mip40 mutants all display the same testis phenotype: an arrest at the primary spermatocyte stage of development, consistent with the notion that they are all acting together in a complex to promote differentiation and meiotic cell cycle progression. It seems likely that other proteins might interact with tMAC to aid in the regulation of testis-specific transcripts. This idea stems from what is already known about MMB, where proteins such as Rpd3 and L(3)MBT physically associate with MMB only during early steps in the biochemical purification process and are critical for function at different DNA sites. To date, no other alternative or subcomplexes containing members of MMB have been isolated from either embryo or tissue culture nuclear extracts. Furthermore, genomic profiling in KC cells of key MMB members (Myb, E2F2, Lin-52, Mip120, and Mip130) substantiates the hypothesis that these proteins work together as a group rather than as isolated factors on DNA. Had testis-enriched starting material had not been examined, it would have been impossible to identify tMAC. Thus, despite the present data showing that the MMB core factors always work as an ensemble, it is possible that some of the pleiotropic phenotypes observed for different MMB subunit mutants could reflect the activity of a subunit functioning outside of MMB (Beall, 2007).

It is striking that both tMAC and MMB contain proteins, other than Mip40 and Caf/p55, that are similar to each other in domain architecture: Aly (tMAC) or Mip130 (MMB) and Tomb (tMAC) or Mip120 (MMB). Given that MMB and tMAC contain multiple site-specific DNA-binding proteins (Myb, E2F2/DP, Mip120, Mip130 in MMB; and Tomb and Topi in tMAC), a potentially large number of genes may be regulated by tMAC as is now know is true for MMB (Beall, 2007).

Antisera against the MMB subunit, Lin-52, failed to coimmunoprecipitate Comr or Aly; therefore, it is not likely a tMAC subunit. However, it is interesting to note that there is another Lin-52 family member in Drosophila (CG12442). The adult Drosophila gene expression database indicates that this gene is, indeed, highly expressed in testis relative to other tissues. Given that this protein is extremely small (predicted molecular weight of 16 kDa), it is possible that it was not present in sufficient quantities to be detected in the mass spectrometry analysis and may in fact be part of tMAC. If so, that would be the third MMB subunit to have an alternative 'testis-specific' form present in tMAC (Beall, 2007).

Gonad-specific forms of proteins that are ubiquitously expressed and generally found in complexes that regulate transcription may, indeed, be a common theme. For example, gonad-specific components of the basal RNA polymerase II transcription machinery are crucial for developmentally regulated gene expression programs in these tissues. Five testis-specific TATA-binding protein-associated factors (TAFs) have been identified in Drosophila (encoded by the can, sa, mia, nht, and rye genes). All are required in spermatocytes for the normal transcription of target genes involved in post-meiotic spermatid differentiation (the so-called can class of genes). It is thought that these testis-specific TAFs may associate with some of the general TAF subunits to create a testis-specific TFIID (tTFIID) that carries out the developmentally regulated transcriptional program in spermatocytes. The mip40-null allele is also in the can class, suggesting that tMAC may interact with tTFIID at can class gene promoters. It will be interesting to explore the possibility that tMAC is a testis-specific coactivator with tTFIID. Other tMAC subunits fall into the aly class and might be what is expected for a large complex paralogous to MMB, where one or another subunit may be silent and subsequently required at a later stage or developmental pathway (Beall, 2007).

Mip130 family members, such as Aly, share a domain that is called a 'DIRP' (domain in Rb-related pathway) domain that is thought to be responsible for interaction with Rb. The DIRP domain of human-Lin-9 (Mip130) is necessary for association with Rb; however, the interaction between hLin-9 and Rb may be indirect as hLin-9 may exist in a complex with other proteins that directly touch Rb. Neither of the two Drosophila Rb proteins was in tMAC-containing fractions. When alignments were made with Mip130 family members, a region was noticed within the DIRP domain that was conserved between all family members except Aly. It is possible that this divergent region within the DIRP domain is critical for Rb interaction in other family members and has been lost in Aly. Although no direct understanding is available of how Aly works for transcriptional activation, it is possible that tMAC contains both activating and repressing components similar to MMB and that repression at particular loci does not require E2F/Rb (Beall, 2007).

When examined for replication profiles in mutant ovaries, an absence of amplification-stage egg chambers was found in mip120 mutants, and widespread BrdU incorporation and Orc2 staining in mip40 mutant amplification-stage follicle cell nuclei. The mip40 egg chamber phenotype is similar to that of mip130 and is consistent with a negative regulatory role for these proteins in genome-wide replication at these stages. It is suggested that both Mip40 and Mip120 are functioning in complex with MMB in ovaries and that both are required for normal patterns of replication in amplification-stage follicle cell nuclei. It is speculated, based in part on unpublished studies of the intricate regulatory network of MMB in Kc cells, that the different mutant phenotypes may simply reflect differences in gene expression profiles that result when individual MMB complex members are missing. A key role for Mip120 in the stability of chromatin-bound MMB might, therefore, explain the more severe phenotype of mip120 mutants. More specifically, MMB may regulate the expression of genes critical for amplification-stage egg chamber development either directly or indirectly, and Mip120 is required for targeting MMB to these gene promoters at a particular developmental stage prior to amplification stages. In contrast to Mip120, Myb, and E2F2/DP, Mip40 has no direct DNA-binding ability. Mip40 may be required for repression or activation only after MMB is targeted to chromosomal sites (Beall, 2007).

As with myb; mip130 mutants, it was found that myb; mip40 and myb; mip120 double mutants were viable, further demonstrating that function(s) of MMB without Myb are responsible for myb lethality. Myb protein was no longer associated with chromatin in mip120 and mip130 mutant polytene spreads. However, staining of polytene chromosomes demonstrated that Mip120 and Mip130 proteins were still bound to chromatin in myb mutants in such a way that may prove lethal in the absence of myb. Together, these data support a model in which Myb is critically dependent on members of the MMB complex for both stability and association with chromatin (Beall, 2007).

It is suggested that the presence of MMB at the replication enhancer ACE3 in stage 7-9 egg chambers may actively repress DNA replication here and at other sites in the genome. MMB at ACE3 at these early stages seems poised to await signals for initiation of amplification. The conversion of a repressive MMB complex into an activating complex may require cyclin E/Cdk2 activity, which is required for amplification. In this context it is likely that Rbf association with MMB will persist during amplification as Rbf association with MMB remains unchanged even after saturating hyperphosphorylation by Cdk:cyclin E in vitro. In the future, determining the cell-type-specific signals that target MMB at well-defined cis-regulatory elements at both the follicle cell amplicons and in other tissues will help unravel how MMB functions in vivo (Beall, 2007).

A dual role for the dREAM/MMB complex in the regulation of differentiation-specific E2F/RB target genes>

E2F and RB proteins regulate the expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. Recent studies indicate that they function as part of an evolutionarily conserved multiprotein complex termed dREAM/DREAM/LINC. This study characterizes the role of the Drosophila complex, dREAM [(Drosophila RBF, dE2F2, and dMyb-interacting proteins), three Myb-interacting proteins (Mip40, Mip120, and Mip130), and p55CAF1 (RbAp46/48)], in the regulation of differentiation-specific E2F target genes in actively proliferating cells. These genes are regulated differently from cell cycle-related E2F targets, they do not depend on E2F activation, and E2F/RB repression is maintained throughout the cell cycle. In proliferating cells, their repression is dependent on dREAM. dREAM was found to play a dual role in their regulation. First, it is required for the stability of the repressive dE2F2/RBF complexes at their promoters during S phase. dREAM was found to be indispensable for both transcriptional repression mechanisms employed at these genes (Lee, 2012).

The identification of native pocket-protein associated complexes in flies, worms and humans called dREAM/MMB/DRM/LINC indicates that such complexes play important roles in RB functions. The mechanisms of action of the complex are not very well understood. The complex has been shown to regulate the expression of G1/S, G2/M, as well as differentiation-specific E2F target genes, and to potentiate RBs tumor suppressive functions. It has been reported to repress as well as activate transcription, regulate site-specific DNA replication, and has been located at a large number of genomic sites. These observations indicate that dREAM/MMB/DRM/LINC plays a vital role in RB functions but also raise the question of how it can support all these diverse E2F/RB activities (Lee, 2012).

This study has explored the means by which the Drosophila complex, dREAM, represses differentiation-specific E2F/RB targets in actively proliferating cells. Differentiation-specific target genes (Group D/E) differ in their regulation from classic, cell cycle regulated targets, and it was found that the function of dREAM is required for some of the unique features of this gene regulation. Specifically, dREAM is not responsible for the target specificity of E2Fs. Instead, it is required for the stability of dE2F2/RBF complexes at Group E gene promoters during S-phase. Our results indicate that it may function, at least in part, by protecting RBF1 from phosphorylation. In addition, the complex is also essential to maintain both mechanisms of repression at these gene (Lee, 2012).

One of the distinctive features of Group E gene regulation is the lack of dependence on E2F activation - the activator dE2F1 does not bind to their promoters or regulate their expression. This poses the question of how dE2F2-specificity is achieved at these promoters. While a comparison of E2F binding sites did not reveal any major differences between sites found at Group E and cell cycle gene promoters, dE2F1 is unable to bind to Group E gene promoters even in the absence of dE2F2. This finding suggests that either dE2F1 is inherently incapable of binding to these promoters or that some factor(s) other the dE2F2 prevents it from binding. It is also possible that dE2F2 is not capable of binding without the assistance of another factor. One candidate for such a factor is the dREAM complex. It could either assist dE2F2 or prevent dE2F1 from binding. This study found that disruption of dREAM did not lead to dE2F1 binding, but disrupted dE2F2 binding, suggesting that neither E2F is capable of binding to these promoters without assistance. However, upon close examination, it was discovered that dREAM is dispensable for dE2F2 binding outside of S-phase, demonstrating that dE2F2 can bind to Group E gene promoters without the assistance of dREAM. These results indicate that dE2F1 is not capable of binding at these promoters. It is possible that there are some subtle differences in the E2F binding sites; alternatively the two E2F proteins may have different affinities for a particular chromatin landscape (Lee, 2012).

In S-phase, dE2F/RBF complexes are replaced by dE2F1 at cell cycle regulated promoters, yet remain bound and functional at Group E gene promoters. Several observations in mammals also indicate that E2F/pocket-protein complexes exist irrespective of cell cycle stage and can function in a CDK-independent manner. It is well established that E2F/RB complexes are disrupted at cell cycle genes at the G1/S transition. Are E2F/RB complexes then protected from CDK phosphorylation in some cases or is the regulation of E2F/pocket-protein interactions even more complex? Structural studies of pRB have suggested that the C-terminus of pRB functions as a molecular sensor that recognizes CDK mediated phosphorylation. When pRB is hypo-phosphorylated, its C-terminal region stabilizes the interaction with E2F1, phosphorylation by CDKs, causes a conformational change, and this intra-molecular interaction is thought to inhibit the interaction between pRB and E2F1. However, it is not clear whether that this is true for other RB family members or that all E2Fs interact in the same manner with pocket proteins. Furthermore, it has been shown that pRB has two distinct E2F binding sites, one being specific for E2F1 and linked to its ability to regulate E2F1-dependent apoptosis. This indicates that pRB interacts with individual E2F proteins in different ways and that regulation of distinct E2F functions are physically separable. Studies in Drosophila also indicate that G1 CDKs are not sufficient to disrupt repressive E2F/RB complexes at cell cycle genes. In the case of differentiation-specific target genes in flies, a simple explanation was found of the stability of E2F/RB complexes in S-phase, namely dREAM. In the absence of a functional dREAM complex, dE2F2 and RBFs exhibit reduced binding in S-phase. The results show that expression of a phospho-mimic RBF mutant results in impaired binding at both cell cycle regulated and differentiation-specific (Group E) gene promoters. Conversely a mutant RBF1 that cannot be phosphorylated is sufficient to bypass the need for a functional dREAM at Group E gene promoters; E2F/RB complexes are stable in S-phase even in the absence of dREAM. These findings indicate that CDK phosphorylation can disrupt dE2F2/RBF repressive complexes at Group E gene promoters and that necessitates their assembly into the dREAM complex. But how does assembly into dREAM ensure stability of E2F/RB complexes? The simplest model is that the phosphorylation sites are blocked, inaccessible to CDKs. Alternatively, dREAM could stabilize either fully or partially phosphorylated RBF protein. Previous studies have shown that Cyc E/cdk2 can phosphorylate RBF1 as part of the dREAM complex in vitro and that phosphorylation does not disrupt the association. However, RBF1 is regulated by both CycD/cdk4 and Cyc2/cdk2 and it is likely that when assembled into dREAM in vivo not all phosphorylation sites are modified. In agreement with this idea, it was found that in cells lacking functional dREAM hyper-phosphorylated RBF1 is increased. Thus, the findings suggest that the complex functions, at least in part, by protecting RBF1 at these promoters from being phosphorylated by CDKs (Lee, 2012).

dREAM, possibly in association with additional factors, may also modify RBF1. For instance, pRB is known to be acetylated during differentiation of monoblastoid cells and keratocytes. Additional post-translational modifications of the protein could either prevent phosphorylation and/or induce a conformational change to promote stability of phosphorylated E2F/RB complexes. It will be interesting to investigate if RBF proteins bound in the dREAM complex have modifications other than phosphorylation (Lee, 2012).

What is the role of the dREAM complex in the regulation of differentiation-specific 424 E2F/RB target genes? The initial identification and characterization of the dREAM/MMB complexes has led to the speculation that dREAM is a repression complex that functions by affecting chromatin structure. The complex is not stably associated with any enzymatic activity, yet this study has demonstrated that Group D/E genes are repressed via two distinct mechanisms in a dREAM-dependent manner. Is the observed dependence on dREAM the result of its ability to affect chromatin structure or is it an indirect consequence of its role in dE2F2/RBF binding? The idea is favored that dREAM plays a direct role in the repression of Group E genes for two reasons. Binding of dE2F2/RBF1 complexes does not require dREAM outside of S-phase. Only about 10% of asynchronously growing SL2 cells are in S-phase, yet disruption of dREAM leads to the same level de-repression as the removal of dE2F2 or RBFs. Moreover, while the expression of phospho-mutant RBF1 restores the binding of dE2F2/RBF1 in S-phase, it does not restore the repression in cells lacking dREAM. A dual role for dREAM in the regulation of these genes is also more consistent with its reported involvement in other RB functions. It would suggest that the complex is capable of potentiating diverse RB activities (Lee, 2012).

dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes

dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts, the distribution and localization of Drosophila E2F and dREAM proteins were examined. This study reports a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains (Korenjak, 2014).

The large number of E2F proteins has precluded all attempts to generate a comprehensive set of E2F binding sites in the human genome. This study has taken advantage of the simplicity of the Drosophila E2F family to examine the genome-wide distribution of E2F proteins. An unexpected finding from this analysis is that dE2F proteins strongly co-localize with insulator-binding proteins. The extent of overlap between dE2F2/dREAM and CP190 binding sites is comparable to the co-localization previously described for dCTCF and CP190, which are both required for insulator function at common binding sites. Furthermore, the co-localization between dREAM and Beaf-32 is even greater than that observed for Beaf-32 and CP190. This striking overlap of dREAM binding sites with proteins involved in nuclear architecture has exciting new implications for the function of dREAM complexes (Korenjak, 2014).

An interesting feature of dREAM bound genes is their strong enrichment in divergently paired genes (DPGs), genes that are transcribed in opposite direction, with their TSS separated by less than 1000 bp. Moreover, the set of genes de-regulated upon loss of dE2F2/dREAM include mostly DPGs that are differentially expressed, with one gene of the pair being stably repressed whereas its partner is actively transcribed. Inactivation of dREAM complex subunits or CP190 results in the loss of transcriptional integrity at these differentially expressed DPGs (Korenjak, 2014).

Several different models could account for the observed transcriptional up-regulation of the stably repressed and down-regulation of the actively expressed gene. First, dREAM/CP190/Beaf-32 sites might act as boundary elements at DPGs, separating an active from a repressed chromatin domain. Genome-wide binding maps for insulator-binding proteins revealed enriched binding to DPGs. In addition, these studies have shown that CP190 and Beaf-32 binding are significantly enriched at differentially expressed DPGs. The exact role of insulator-binding proteins at differentially expressed DPGs is still unclear. A recent study has shown that, upon inactivation of the SOX14 transcription regulator, DPGs that lack Beaf-32 binding show a significantly higher likelihood of concerted de-regulation of the two genes within a pair (up or down) than when Beaf-32 is present at the DPG. This suggests a role for Beaf-32 in the maintenance of independent regulation of gene expression at DPGs, consistent with a function as boundary factor. Moreover, CP190 binding sites are commonly found at the borders of large H3K27me3 domains, which are a hallmark of Polycomb-mediated silencing. These studies further show that inactivation of CP190 can, at a subset of regions, result in local spreading of H3K27me3 beyond the CP190 binding site. Although the repressive mechanisms might vary at different dREAM-regulated DPGs, several of the stably repressed genes display H3K27me3 over the length of the gene body. The possibility of dREAM and CP190 being important for the physical separation of distinct chromatin domains was tested by assessing the distribution of H3K27me3 over selected gene pairs. In agreement with the observed de-repression of the inactive gene, mutant animals displayed loss of H3K27me3 in the gene body. However, spreading of the mark into the active gene was not observed, suggesting that the down-regulation is achieved by a different mechanism or the level of reduction in gene expression is below the detection limit of our H3K27me3 ChIP (Korenjak, 2014).

Second, dREAM, together with CP190 and possibly Beaf-32, may be involved in the silencing of stably repressed genes. The repressive mechanism might include specific activities provided by CP190/Beaf-32. Both CP190 and Beaf-32 have been shown to be critical for the establishment of long-range chromatin interactions through looping mechanisms, which could be utilized to physically separate a stably repressed gene from the surrounding transcriptionally active chromatin environment. It is intriguing to speculate that dREAM, either by facilitating chromatin association of CP190 or even more directly, could be involved in the formation of these chromatin loops. Interestingly, the CP190 and Beaf-32 binding sites involved in long-range chromatin interactions are also prominent dREAM binding sites. Alternatively, Beaf-32 is known to compete for DNA binding with the transcriptional activator DNA replication-related element factor (DREF) (70). It has further been shown that, upon inactivation of Beaf-32, bound genes are specifically de-repressed when they also contain a DREF consensus site. In this scenario, loss of the repressive mechanism by inactivation of dREAM or CP190/Beaf-32 might either generate a vacant binding site for a transcription activator or result in the re-distribution of the general transcription machinery or a specific transcription activator from the actively expressed to the repressed gene (Korenjak, 2014).

Third, the possibility cannot formally be ruled out that dREAM acts on both components of differentially expressed DPGs, serving as a repressor for one and as an activator for the other gene. dREAM has been implicated in transcriptional repression as well as activation. However, dREAM complexes containing dE2F2 do not appear to be involved in gene activation and, based on genome-wide binding studies for dREAM subunits, there is no evidence for the presence of two independent dREAM peaks at differentially expressed DPGs (Korenjak, 2014).

Genome-wide association studies have identified a large number of binding sites for insulator-binding proteins, but only a few studies have addressed the potential enhancer-blocking activity of these DNA fragments. In these experiments, CP190 and dCTCF co-bound regions display strong enhancer-blocking activity compared to Su(Hw) bound sites. Moreover, it has been shown that sites associated with CP190 or CP190 and Beaf-32 exhibit strong enhancer-blocking activity, whereas sites occupied by any other combination of insulator-binding proteins show only weak or no enhancer blocking. In agreement with the importance of CP190 for the definition of elements with enhancer-blocking activity, dREAM-CP190 co-bound regions display robust enhancer-blocking in a cell-based assay system. The notion that dREAM functions as an enhancer-blocker may explain why a complex that is best known as a transcriptional repressor is almost exclusively found in euchromatic regions (Korenjak, 2014).

The dREAM subunits Mip130 and Mip120 are important for the observed enhancer-blocking activity, but the underlying mechanism is unclear. Work in Drosophila suggests that the propagation of a nucleosome-free region in insulator elements is required for enhancer blocking. It is possible that dREAM is needed for the establishment of nucleosome-free regions through recruitment of chromatin modifying activities or their maintenance through binding and stabilization of these regions, which in turn might be important for the recruitment of CP190 (Korenjak, 2014).

A detailed analysis of the enhancer-blocking function of the 1A2 insulator, which harbors Su(Hw) binding sites, shows that the region adjacent to the Su(Hw) sites is important for full enhancer-blocking activity of 1A2, even though this element by itself lacks any activity. It is conceivable that dREAM binding sites fulfill a similar 'facilitator' function for CP190 and/or Beaf-32 (Korenjak, 2014).

Although cell-based assays have been effectively used to measure the enhancer-blocking activity of characterized insulator elements, transfected plasmids are only partially chromatinized. In order to address the enhancer-blocking function of dREAM/CP190-bound regions in more detail and dissect the underlying mechanism it will, therefore, be interesting to test the identified elements in an in vivo enhancer-blocking assay (Korenjak, 2014).

The underlying mechanism(s) by which dREAM cooperates with CP190 is likely to be connected with the ability of dREAM to help establish or maintain CP190 chromatin association. Interestingly, CP190, but not Beaf-32 chromatin binding was reduced in dE2f2 mutant animals. Beaf-32 can bind to DNA in a sequence-specific manner through recognition of the CGATA motif. In contrast, CP190 does not possess known DNA-binding activity and is thought to get recruited indirectly by DNA-binding factors. This view is based on physical and functional interactions with sequence-specific insulator-binding proteins as well as the high degree of co-localization of these factors in genomic binding studies. Despite the high degree of overlap in their genomic binding profiles, the significance of Beaf-32 for CP190 chromatin association is unclear. Recent studies found contrary results regarding the significance of Beaf-32 for CP190 recruitment. CP190 chromatin association is reduced, however, in ctcf mutant animals and upon dCTCF knockdown in tissue culture cells. A recent study in Drosophila cells has shown that CP190 associates with the majority of its binding sites in distinct combinatorial patterns with other insulator-binding proteins. At a subset of binding sites, however, it does not co-localize with any known DNA-binding protein, suggesting that CP190 either has intrinsic DNA-binding activity or depends on a not yet identified factor for recruitment. Given the physical interaction between dREAM complex subunits and CP190, it is speculated that dREAM complexes may be directly involved in the recruitment of CP190 to these sites (Korenjak, 2014).

Interestingly, the strong enrichment of DPGs among dREAM-bound genes is conserved in human cells, but, to date, CTCF is the only known mammalian ortholog of fly insulator-binding proteins. Based on the extensive co-localization among insulator-binding proteins in the fly genome and the presence of CP190 as a common insulator co-factor, which suggests a shared mechanism, it has been proposed that the functions of the Drosophila proteins were integrated in CTCF. CP190 is a chromatin architectural protein, and it is conceivable that another protein with similar properties has adopted its role. Interestingly, the function of mammalian CTCF, including its role in chromatin looping, has been intimately linked to the Cohesin complex. Remarkably, inactivation of pRB in mammalian cells results in reduced chromatin association of Cohesin and the functionally related Condensin II-subunit Cap-D3. Furthermore, pRB physically interacts with Condensin II-subunits in fly and human cells and RBF1 co-localizes extensively with Cap-D3 on polytene chromosomes, raising the fascinating possibility that these specialized architectural protein complexes have taken over a CP190-related role in higher organisms (Korenjak, 2014).

Over the past years, several studies have shown that a variety of different proteins co-localize with insulator-binding proteins and/or contribute to insulator function. These factors range from different combinations of insulator proteins to factors like L(3)MBT, Topoisomerase II, the ubiquitin ligase dTopors, Ago2, the Rm62 helicase and exosome components. Further studies are clearly needed to determine how dREAM function is integrated with the array of factors acting in concert with insulator-binding proteins and to delineate a potential role of dREAM complexes in the organization of chromosome architecture (Korenjak, 2014).



E2f2 is expressed throughout embryonic development, with the highest levels detected in cycling cells. This includes mitotically active cells during germband extended stages, as well as proliferating cells of the CNS and endoreduplicating cells of tissues such as the midgut at later embryonic stages (Sawado, 1998; Cayirlioglu, 2001).

Effects of Mutation or Deletion

Mutation of either E2f or Dp inhibits the embryonic expression of several well established target genes (e.g. RNR2, Cyclin E, or Mcm3) that are used as indicators of 'E2F' function in vivo. Genetic analysis of E2f2 was begun by examining embryos homozygous for a deficiency (Df(2L)DS8) that should remove the E2f2 locus, located at cytological position 39B2-3 on chromosome 2. In situ hybridization experiments were used to confirmed that Df(2L)DS8 deletes E2f2 (Cayirlioglu, 2001).

In spite of the disruption of E2f2 gene expression in homozygous deficiency embryos, there was no alteration to the pattern of expression of RNR2, Cyclin E or Mcm3. This indicates either that E2F2 does not regulate these genes in the embryo, or that a maternal pool of E2F2 is sufficient to provide function during embryogenesis. Indeed, maternal E2f2 mRNA can be detected either by Northern analysis (Sawado, 1998) or by in situ hybridization. This maternal mRNA is destroyed by the cellular blastoderm stage, indicating that any E2F2 activity in homozygous Df(2L)DS8 deletion embryos would have to be provided by perdurance of maternal E2F2 protein (Cayirlioglu, 2001).

In order to generate E2f2 mutations, a P-element insertion, l(2)16402a, was identified located 856 and ~650 nucleotides upstream of the E2f2 translation and transcription start sites, respectively. Transposase-mediated excision of l(2)16402a was used to recover a small deletion [Df(2L)E2f2329] containing one breakpoint at the P-element insertion point and the other 11 bp downstream of the E2f2 stop codon near the end of the transcription unit, thereby removing all E2f2 coding sequence. Both l(2)16402a and Df(2L)E2f2329 are 100% lethal in trans to each other, or in trans to other deletions (Cayirlioglu, 2001).

Flies that completely lack E2f2 or are hemizygous for another allele, E2f21-188 eclose at near wild-type Mendelian frequency with no overt morphological defects, indicating that zygotic expression of E2f2 is not essential for development. While adult E2f2 mutant males are fully fertile, mutant females have significantly reduced fertility, suggesting that oogenesis is perturbed by loss of E2f2 function. The ovaries of E2f2 mutant females do not mature as rapidly as wild type after adult eclosion: late stage egg chambers are not visible in dissected mutant ovaries until ~5 days after eclosion, compared with ~1.5 days for wild type. After 6 days, the mutant females lay eggs at a rate similar to wild type. However, only 10% and 8% of eggs laid by Df(2L)E2f2329/Df(2L)E2f2329 and Df(2L)E2f2329/e2fsnd1-188 females hatch, respectively. The latter phenotype is substantially rescued by an E2f2 transgene. The eggs that do hatch are able to develop and produce viable adult flies. More than 90% of the eggs laid by E2f21-188/Df(2L)E2f2329 and Df(2L)E2f2329/Df(2L)E2f2329 females have a defective eggshell, or chorion, and the null phenotype is rescued by a E2f2 transgene. The mutant chorions appear far more translucent than the normally opaque wild-type chorion, which is indicative of a thin eggshell. The eggs are more fragile than wild type, and many appear collapsed (Cayirlioglu, 2001).

Oogenesis was examined in E2f2 mutant females, focussing in particular on follicle cells, because of the chorion defects. The Drosophila eggshell is a crosslinked protein matrix composed of several chorion proteins produced by a single epithelial follicle cell layer that surrounds the developing oocyte. As described above, the chorion protein biosynthetic capacity of the follicle cells relies on a developmentally controlled cell cycle program that includes both polyploidization and amplification of two clusters of chorion genes, one on the X chromosome and the other on chromosome 3. The 60- to 80-fold amplification of chorion genes assures that sufficient amounts of chorion proteins are rapidly synthesized during eggshell formation. Southern hybridization experiments were carried out to investigate whether E2f2 mutants had a reduced level of chorion gene amplification. DNA isolated from stage 13 egg chambers dissected from wild-type and Df(2L)E2f2329/Df(2L)E2f2329 mutant ovaries was probed with a 3.8 kb chromosome 3 chorion gene cluster probe. Four independent experiments revealed that amplification was reduced two to three times by mutation of E2f2 when compared with wild type (Cayirlioglu, 2001).

The reduced chorion gene amplification in E2f2 mutant females suggests that E2F2 plays a role in the developmentally regulated cell cycle program in follicle cells. Moreover, female sterile alleles of Dp, E2f and Rbf, each cause follicle cell replication defects. DNA synthesis occurring during follicle cell endocycles and chorion gene amplification can be visualized in situ by BrdU pulse labeling of dissected ovaries. By stage 10B of oogenesis in wild type, all follicle cells have exited endoreduplication cycles and have begun chorion gene amplification. While follicle cell endocycles within each egg chamber are asynchronous, gene amplification occurs simultaneously throughout the epithelium. Consequently, at stage 10B, BrdU incorporation is detected within every follicle cell in at least four subnuclear foci, the two largest of which correspond to the chorion gene clusters on chromosomes X and 3 (the others remain unidentified). Gene amplification continues through stage 13 (7 hours older than stage 10B), at which point BrdU incorporation at the chromosome 3 chorion cluster predominates. E2f2 mutant follicle cells have a very different profile of BrdU incorporation. In stage 10B and later Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f21-188 egg chambers, BrdU incorporation is observed throughout the entire nucleus, rather than in the characteristic subnuclear foci. There is cell to cell variability in the intensity of ectopic genomic BrdU labeling at stage 10B, with some nuclei having quite little or no ectopic replication. This variability is slightly more pronounced in the E2f21-188 hemizygote nuclei compared with the Df(2L)E2f2329 deletion, suggesting that E2f21-188 is not null. By stage 13 every follicle cell nucleus in the null situation displays intense genomic replication. Similarly, the severity of the Df(2L)E2f2329/E2f21-188 phenotype increases by stage 13, but again there is more cell to cell variability in the intensity of labeling compared to E2f2 null cells. Inclusion of a wild-type E2f2 transgene restores the aberrant BrdU incorporation pattern in Df(2L)E2f2329/Df(2L)E2f2329 egg chambers to normal. These data suggest that at the time DNA synthesis is normally restricted to chorion gene amplification, replication is instead occurring throughout the entire genome in the E2f2 mutant follicle cells (Cayirlioglu, 2001).

The penetrance of the ectopic genomic replication phenotype is virtually 100% in either Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f21-188 mutants, in that all egg chambers contain many cells with inappropriate genomic replication. By contrast, egg chambers of the genotype E2f216402a/e2fsnd1-188 have a strong but less penetrant phenotype: 88% of the egg chambers scored had some follicle cells with inappropriate genomic replication at or after stage 10B. In addition, of those E2f216402a/e2fsnd1-188 egg chambers that scored as mutant, the number of follicle cells that were undergoing genomic replication was clearly less than 100%. That is, some of the cells displayed genomic replication while other cells displayed normal amplification foci. In addition, two other classes of aberrant BrdU incorporation patterns were observed in E2f216402a/e2fsnd1-188 follicle cells. In some nuclei, both gene amplification foci and genomic BrdU incorporation were apparent in the same nucleus, while in others no BrdU incorporation was detected, suggesting that neither gene amplification or genomic replication was occurring. This variable penetrance and expressivity suggests that the l(2)16402a mutation is hypomorphic for E2f2 function, perhaps because the P-element insertion reduces E2f2 gene expression. Consistent with this interpretation, eggs laid by E2f216402a/e2fsnd1-188 mothers have a less severe phenotype compared to those from null allele combinations when scoring hatching frequency and the proportion of eggs with a defective chorion (Cayirlioglu, 2001).

One possible cause of the ectopic genomic replication in stage 10B-13 E2f2 mutant egg chambers is that the cells inappropriately enter an additional endocycle in the later developmental stages. In this case, follicle cell ploidy should increase from 16C to 32C. This was observed in an Rbf female sterile mutant, which cytologically causes a similar BrdU incorporation phenotype to E2f2 mutants (Bosco, 2001). FACS profiles of DAPI- or PI-stained nuclei isolated from whole ovaries can be used to determine follicle cell ploidy. Using this method, no significant difference could be distinguised in the FACS profile between Df(2L)E2f2329/Df(2L)E2f2329 or Df(2L)E2f2329/E2f21-188 mutant nuclei and wild-type controls. Each profile contained four prominent peaks representing 2C-16C follicle cell nuclei. While a small 32C peak was occasionally observed in the mutants, it was not reproducible. Moreover, other cell types can lead to the appearance of a small 32C peak in some wild-type preparations. To circumvent these problems, pure populations of follicle cells were analyzed at stage 9 and beyond. This was achieved by FACS analysis of trypsin dissociated ovaries using a follicle cell-specific GAL4 driver (c323) to induce a UAS-GFP transgene. The driver is activated initially at stage 9 in wild-type egg chambers, and persists until the end of oogenesis. Importantly, the expression of c323-induced GFP expression in E2f2 mutant egg chambers occurs during the same developmental stages as wild type. This indicates that mutation of E2f2 does not affect developmental control of the c323 driver, allowing a direct comparision of ploidy values between the same population of wild-type and mutant follicle cells. Using this technique, wild-type 8C and 16C GFP-positive follicle cells were readily distinguished as a subset of the entire FACS profile obtained by staining the cells with the DNA binding dye Hoechst 33342. Interestingly, in the Df(2L)E2f2329/E2f21-188 mutant profile there was no indication of a 32C cell population. By contrast, follicle cells isolated from Rbf120/Rbf14 mutant egg chambers clearly contain a 32C population that is not detected in wild type preparations. Taken together, these data indicate that the ectopic genomic BrdU incorporation seen in E2f2 mutant follicle cells does not result from an additional, complete endocycle S phase (Cayirlioglu, 2001).

The conversion from asynchronous follicle cell endocycles to synchronous gene amplification is under developmental control. This can be seen at successive stages of wild-type egg chamber development, where the proportion of BrdU-positive cells within the follicle cell epithelium decreases until very few cells are replicating at stage 10A, immediately before the onset of gene amplification. To determine whether E2f2 mutant follicle cells are responding to the developmental cues that terminate endocycle DNA synthesis, the number of BrdU positive nuclei in pre-stage 10B wild-type and E2f2 mutant egg chambers was determined. The number of BrdU positive follicle cell nuclei in pre-stage 10B Df(2L)E2f2329/Df(2L)E2f2329 mutant egg chambers was similar to wild type. Importantly, stage 10A mutant egg chambers contained very few BrdU-positive cells, just as in wild type. This observation suggests that E2f2 mutant follicle cells terminate the endocycles at the normal developmental time. From these data and the FACS analysis, the inappropriate BrdU incorporation in E2f2 mutant follicle cells is interpreted as a failure to adequately restrict DNA synthesis to sites of gene amplification, rather than as a continuation of endocycles. Thus, E2F2 appears to facilitate the conversion to the gene amplification phase of follicle cell development by preventing the cells from initiating genomic DNA replication in response to signals that trigger the onset of synchronous gene amplification (Cayirlioglu, 2001).

Proteins of the ORC complex assemble at origins of DNA replication and recruit factors (e.g. CDC45L) required to initiate bi-directional DNA synthesis. During oogenesis, localization of different ORC proteins within the follicle cell nuclei is dynamically regulated, coincident with changing patterns of DNA replication. ORC1, ORC2, ORC5, and CDC45L are distributed throughout the entire follicle cell nucleus when these cells are performing genomic replication during endocycle S phase, and after stage 10, these proteins are detected in foci that correlate with sites of chorion gene amplification. Because E2f2 mutant follicle cells fail to restrict DNA replication to gene amplification foci, the localization of replication factors was determined in follicle cells. Anti-ORC2, -ORC5 and CDC45L antibodies each label sites of chorion gene amplification in wild-type stage 10B egg chambers, presumably because these are the major sites of active DNA synthesis. This is consistent with the known role of these proteins in replication origin firing, and indeed female sterile mutants of Orc2 have reduced chorion gene amplification. In E2f2 mutant egg chambers, the distinct localization pattern of these replication proteins is lost, resulting in the detection of all three proteins throughout the entire nucleus. This phenotype is rescued by an E2f2 transgene. These data are consistent with the firing of origins in addition to those at the chorion loci causing inappropriate genomic DNA synthesis. Whether the mis-localization of ORC components and CDC45 in E2f2 mutants is a direct cause or a consequence of the ongoing ectopic replication is not known (Cayirlioglu, 2001).

Chorion gene amplification is characterized by the repeated firing of an origin of DNA replication within the chorion locus. The mechanisms by which chorion replication origins are repeatedly used while other origins are not, is unclear. Two general possibilities exist: either the assembly of the pre-RC required for initiating DNA synthesis is confined to sites of gene amplification, or pre-RC assembly occurs throughout the genome and initiation is confined to amplification sites. Two distinct phases of follicle cell gene amplification have been detected. The first phase occurs during endocycles, where at least the third chromosome chorion locus amplifies to low levels during each endo S phase. The second phase occurs synchronously during stage 10B at several loci, including both of the two chorion gene clusters, after the termination of endocycles. E2f2 mutant egg chambers display defects in this second phase, failing to restrict DNA synthesis to amplification foci. This cellular phenotype is associated with an approximate halving in chorion gene copy number and production of eggs with a thin chorion. However, this apparent amplification defect is much less severe than other mutations (e.g. Orc2 and chiffon/dbf4), which virtually eliminate chorion gene amplification and cause a thin eggshell phenotype. Consequently, while a small reduction in chorion gene copy number caused by mutation of E2f2 could contribute somewhat to defective chorion biosynthesis, this may not be the sole cause of the observed chorion defects. Similarly, the reduced fertility of E2f2 mutant females may not result entirely from desiccation caused by a thin eggshell, owing to follicle cell defects. Although it is highly likely that the follicle cell replication defects are an indication that E2f2 activity is required in this cell type, E2f2 may play additional roles in the germline. Indeed, Dp mutants have germline defects that disrupt oogenesis. Thus, determining the cellular basis for the reduced fertility of E2f2 mutant females will require an analysis of genetically mosaic ovaries (Cayirlioglu, 2001).

Possible interpretations of the DNA synthesis phenotype depend on when during follicle cell development E2f2 primarily functions. If, for example, E2f2 acts during the endocycles, which in wild type generate cells with a 16C DNA content, then the ectopic replication in E2f2 mutants could represent the inappropriate continuation of endocycle S phase into late stages of oogenesis. The data do not support this model, since a mutant follicle cell population with a 32C or greater DNA content was not observed. In addition, the number and pattern of BrdU-positive nuclei in pre-stage 10B egg chambers is the same in mutant and wild type, including a cessation of endocycles just before the onset of gene amplification. This suggests that the early program of endocycles terminates on schedule in the E2f2 mutants. An alternative explanation for the E2f2 mutant phenotype is that endocycles are actually delayed, relative to egg chamber stage. In this scenario, genomic DNA synthesis occurring at stages 10B and later would represent an endo S phase of cells with less than a 16C DNA content. Consequently, if genomic replication was delayed relative to morphological development, then when compared with wild type, a larger fraction of the GFP-positive cell population in E2f2 mutants should contain cells with less than a 16C DNA content, possibly including 4C cells. This is true because only those cells within stage 9 and older egg chambers in both mutant and wild type are GFP positive. No evidence could be found by FACS analysis of a GFP-positive 4C follicle cell population in E2f2 mutants, nor a reproducible increase in the number of cells with less than 16C DNA content compared with wild type. While a minor delay in late endocycles cannot be ruled out, the interpretation is favored that the consequences of E2F2 function are manifested specifically during the synchronous phase of gene amplification either to prevent pre-RC assembly at ectopic locations or to confine firing to sites of amplification (Cayirlioglu, 2001).

How might loss of E2f2 cause this? One possibility is that E2F2 is part of a transcription repressor complex, and that loss of E2f2 function leads to an increase in the expression of genes encoding limiting replication components. An increase in the level of Orc2 and Orc5 mRNA was detected by RT-PCR, suggesting that inappropriate DNA synthesis could be triggered by increased accumulation of pre-RC components. Consistent with this hypothesis, a two- to three-fold increase in the expression of ORC1 using a heat shock promoter stimulates an ectopic genomic replication phenotype in follicle cells similar to that caused by mutation of E2f2. How an increased abundance of ORC proteins could trigger ectopic replication is not clear, but the observation fits a model in which limited assembly of pre-RC helps confine DNA synthesis to sites of gene amplification. Alternatively, E2f2 could positively regulate the expression of a 'specificity factor' that acts to confine replication to sites of gene amplification. These questions could be addressed through comprehensive analyses of mRNA abundance between mutant and wild-type follicle cells (Cayirlioglu, 2001).

There are alternatives to gene expression based models for modulation of DNA synthesis in follicle cells. E2F/DP/RBF complexes control the extent of chorion gene amplification (Bosco, 2001), and may do so via a direct interaction with the pre-RC. E2F, DP and RBF co-immunoprecipitate with ORC2 (Bosco, 2001), and both E2F and ORC2 associate with chorion DNA in chromatin IP assays (Bosco, 2001). In addition, direct regulation of replication origins by chromatin bound pRB/E2F complexes may be conserved: in cultured primary mammalian cells, pRB, p107 and p130 are localized to sites of DNA synthesis early in S phase and may help control the nuclear organization of replication. Perhaps DNA-bound E2F2 controls the localization and/or assembly of replication factors at origins throughout the genome. Indeed, E2f2 is required for the proper localization of ORC2, ORC5 and CDC45L to amplification foci. However, it is difficult to distinguish whether mislocalization of replication factors is the cause, or simply a consequence, of the ectopic genomic replication seen in E2f2 mutant follicle cells. More definitive answers to these questions await the characterization of the location of the E2F2 protein within follicle cells, and whether it directly associates with replication factors (Cayirlioglu, 2001 and references therein).

Two pieces of evidence suggest that disruption of E2F2/DP/RBF complexes contributes to the phenotypes reported in this study. (1) E2f21-188 appears to be a loss of function allele, since it causes a phenotype very similar to complete deletion of E2f2. E2f21-188 produces a truncated protein capable of binding DP but lacking the RBF interaction domain, suggesting the possibility that much of the functions of E2F2 require association with an pRb family member. (2) Dp and Rbf female sterile alleles each cause a cytological phenotype in BrdU-labeled follicle cells similar to that seen by mutation of E2f2 (Bosco, 2001). In each case, genomic replication was observed at a stage when only gene amplification should be occurring. There are, however, notable differences between the E2f2, Rbf and Dp mutant phenotypes. The E2f2 null phenotype is 100% penetrant, whereas the Rbf and Dp follicle cell replication phenotypes are not fully penetrant (Bosco, 200). The difference in penetrance could be explained by partial loss of Dp and RBF function, since the Dp and Rbf alleles used in these experiments are hypomorphic (null alleles are zygotically lethal). But more significantly, the E2f2 and Rbf mutant FACS profiles are different. Bosco (2001) detected a 32C population in nuclear preparations from Rbf mutant ovaries, which this study has confirmed. A 32C population of similar size relative to wild type could not be detected in either nuclear or intact follicle cell preparations of E2f2 mutant ovaries. These data suggest that the BrdU labeling observed cytologically in E2f2 mutants does not represent an additional endocycle S phase, which is expected to generate a 32C peak. It is possible that the ectopic replication in the E2f2 mutants is actually much less than in the Rbf mutants, such that by the completion of oogenesis and egg laying a 32C ploidy value is never reached. Since RBF can bind either E2F2 or E2F (Bosco, 2001), mutation of Rbf and E2f2 could have overlapping but distinct phenotypes, perhaps because each mutation would affect the distribution of cellular pRB/E2F complexes differently (Cayirlioglu, 2001 and references therein).

E2F, DP and RBF also play a role in establishing the extent of chorion gene amplification. Viable, hypomorphic mutations of E2f and Dp that encode proteins predicted to bind DNA poorly result in a decreased level of chorion gene amplification. By contrast, both a viable, hypomorphic allele of Rbf and the E2fi2 nonsense allele, which produces a truncated protein lacking the RBF-binding domain, cause an increase in chorion gene amplification (Bosco, 2001). These data suggest that E2F/DP stimulates gene amplification, whereas the E2F/DP/RBF complex attenuates it. Both E2f2 null and E2f21-188 (lacking the RBF binding domain) alleles cause similar phenotypes, including reduced chorion gene amplification. This could be because E2F2 function is mediated mostly, or even exclusively, via complexes with a pRb protein, in contrast to E2F. Moreover, the opposite amplification phenotypes caused by E2fi2 and E2f21-188 suggest a different role in amplification for the two wild-type proteins. One possibility is that loss of E2f2 indirectly affects gene amplification, perhaps because a limiting replication factor is used at multiple, ectopic replication origins, thereby reducing its availability for use at the chorion loci and interfering with efficient gene amplification. Alternatively, the lack of E2F2 could make more RBF available to bind E2F, thus driving the formation of excess E2F/DP/RBF complexes, which would limit the extent of gene amplification (Cayirlioglu, 2001 and references therein).

Irrespective of the mechanism by which E2F2 regulates DNA replication in follicle cells, E2f2 is not absolutely required for much of Drosophila development. This is despite prominent E2f2 gene expression beginning at embryonic stages in cycling cells (both dividing and endocycling) and continuing throughout development (Sawado, 1998). E2f2 and E2f are the only two E2F-like genes in Drosophila, and the lack of an obvious phenotype in E2f2 single mutants is not due to redundancy with E2f. E2f is an essential gene, with homozygous embryos hatching into slow growing larvae that die before pupation. The E2f lethal phase is due at least in part to E2F2 activity, since E2f; E2f2 double mutant progeny grow at a normal rate and survive until mid- to late-pupal development (Frolov, 2001). This indicates that E2F and E2F2 perform opposing roles during development, and suggests that E2F2 acts to inhibit growth and cell cycle progression, but only when E2F is limiting or absent. Nevertheless, the ability of E2F2 to antagonize E2F is not absolutely essential. While E2F2 does in fact inhibit replication and cell cycle progression in other contexts, this function would be nonessential if it is redundant with other mechanisms that inhibit cell cycle progression during Drosophila development, such as the activation of CDK inhibitors, transcriptional downregulation or an increased rate of protein degradation. It follows from this model that the follicle cells specifically rely more heavily on E2F2 than other mechanisms to inhibit genomic DNA replication (Cayirlioglu, 2001).

Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development

E2F transcription factors can activate or actively repress transcription of their target genes. The role of active repression during normal development has not been analyzed in detail. dE2F1su89 is a novel allele of Drosophila E2f that disrupts E2f's association with RBF Drosophila retinoblastoma protein (Rb) homolog but retains its transcription activation function. Interestingly, the dE2F1su89 mutant, which has E2f activation by dE2F1su89 and active repression by E2f2, is viable and fertile with no gross developmental defects. In contrast, complete removal of active repression in de2f2;dE2F1su89 mutants results in severe developmental defects in macrochaetae and salivary glands, tissues with extensive endocycles, but not in tissues derived from mitotic cycles. The endoreplication defect results from a failure to downregulate the level of cyclin E during the gap phase of the endocycling cells. Importantly, reducing the gene dosage of cyclin E partially suppresses all the phenotypes associated with the endoreplication defect. These observations point to an important role for E2f-Rb complexes in the downregulation of cyclin E during the gap phase of endocycling cells in Drosophila development (Weng, 2003).

A novel allele of E2f1, dE2F1su89, was identified from a genetic screen for suppressors of the Rbf overexpression phenotype. Sequence analysis revealed that dE2F1su89 contains a single base pair mutation in the conserved Rb-binding domain that converts the conserved amino acid leucine at position 786 to glutamine. To test whether this mutation disrupts the interaction between Rbf and E2f, a yeast two-hybrid interaction assay was performed. E2F1su89 is unable to bind to Rbf. To demonstrate further the effect of this mutation with endogenous proteins, a co-immunoprecipitation experiment was carried out. While both E2f and Dp co-immunoprecipitate with Rb from wild-type embryo extracts, no E2f co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, even though similar levels of E2f protein are present in the two extracts. These results indicate that the endogenous dE2F1su89 and Rb proteins do not form a complex. Interestingly, Dp still co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, indicating that Dp can still form a complex with Rb in the dE2F1su89 mutant background, probably through the other Drosophila E2F protein, E2f2 (Weng, 2003).

The decreased number of endocycles could be due to a lengthening of the S phase or a lengthening of the gap phase. Lengthening of the S phase would lead to an increased number of cells that are in the S phase, while lengthening of the gap phase would decrease the number of cells that are in S phase at any given time. A decreased number of S phase nuclei was observed in e2f2;dE2F1su89 salivary glands compared with that in wild-type salivary glands. Thus the gap phase of the endocycles in the e2f2;dE2F1su89 mutants is significantly lengthened. e2f2;dE2F1su89 but not wild-type salivary gland cells accumulate high levels of cyclin E in some gap phase cells (cells that are not incorporating BrdU). Since downregulation of cyclin E levels is required for continuous endoreplication, the failure to downregulate cyclin E levels properly in these gap phase cells probably inhibits endoreplication and leads to severe defects in tissues that require extensive endoreplication during development. The observation that decreasing the gene dosage of cyclin E partially suppresses the e2f2;dE2F1su89 phenotypes such as salivary gland endoreplication defects, macrochaetae defects and lethality provides strong support for the idea that the failure to downregulate cyclin E levels in these gap phase cells is a cause for the observed defects in e2f2;dE2F1su89 endocycle tissues (Weng, 2003).

Although previous results established that cyclin E oscillation is critical for continuous endoreplication, it is not clear how cyclin E oscillation in endocycle cells is achieved. No cyclin E oscillation defect is observed in salivary gland cells in the dE2F1su89 mutants, suggesting that active repression by the E2f2-Rb complexes is sufficient to downregulate the level of cyclin E during the gap phase, even in the presence of the unregulated dE2F1su89. In contrast, removal of the dE2F2-Rb complexes in the dE2F1su89 background results in extensive defects in endocycle tissues and defective cyclin E downregulation in the gap phase of endocycling cells. These results argue strongly that the E2f-Rb complexes are required for the normal downregulation of cyclin E in the gap phase of endocycling cells. These results, in conjunction with the observation that E2F activity is required for cyclin E expression and S phase progression of endocycle cells, suggests a model in which E2f activation is required for S phase of the endocycles and active repression by E2f-Rb complexes is required during gap phase. It is interesting to note that even in the complete absence of Rb-E2f active repression, there are still significant levels of endoreplication, suggesting that the oscillation of cyclin E activity, although defective, can still occur to some extent in e2f2;dE2F1su89 mutants. It is possible that additional mechanisms such as protein degradation or binding to inhibitor proteins such as Dacapo can also contribute to the downregulation of cyclin E activity (Weng, 2003).


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E2F transcription factor 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 November 2014

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