E2F
DNA POL alpha (the alpha subunit of DNA polymerase), ribonucleotide reductase large (DmRNR1) and small (DmRNR2), and Proliferating cell nuclear antigen are coordinately transcribed in transient pulses that parallel and slightly precede DNA synthesis in cells entering S phase from quiescence (Duronio, 1994). Both DmRNR2 and PCNA require DE2F function for transcription (Duronio, 1995a).
E2F recognition sequences have been identified in the promoter of Drosophila DNA polymerase alpha gene (Ohtani, 1994).
Cyclin E expression at G1-S requires E2F, activation of E2F without cyclin E is not sufficient for
S phase. Early in G1, ectopic expression of cyclin E alone can bypass E2F and induce S
phase. Cyclin E is the downstream gene that couples E2F activity to G1 control. However, not all embryonic cycles are similarly coupled to E2F activation. The rapidly proliferating
CNS cells that exhibit no obvious G1 express cyclin E constitutively and independently of E2F.
Instead, cyclin E expression activates E2F in the CNS. Thus, this tissue-specific E2F-independent
transcription of cyclin E reverses the hierarchical relationship between cyclin E and E2F. Both
hierarchies activate expression of the full complement of replication functions controlled by E2F;
however, whereas inactivation of E2F can produce a G1 when cyclin E is downstream of E2F (as in cells with a delayed S period), an E2F-independent source of cyclin E (neuroblasts) eliminates G1 (Duronio, 1995b).
Cyclin B expression is not detected in E2F-deficient clones of the eye disc, while elav is expressed normally in E2F-deficient clones (Brook, 1996)
Simultaneous overexpression of both E2F subunits, E2F and DP, stimulates the expression of multiple E2F-target genes including cyclin E, and also causes the initiation of S phase. Mutation of cyclin E prevents the initiation of S phase after overexpression of E2F/DP without affecting induction of target gene expression. Thus, E2F-directed transcription cannot bypass loss of cyclin E in Drosophila embryos. It is concluded that Cyclin E has an essential in vivo role in S phase induction other than induction of E2F activity (in mammals accomplished by phosphorylation and inactivation of Rb). Reduction of cyclin E expression blocks E2F-induced S phase in epidermis, but not in the midgut. These results suggest that different cell types have different sensitivity thresholds to cyclin E expression, and that as little as a two fold change in Cyclin E levels can have dramatic effects of cell cycle (Duronio, 1996).
Sequences similar to the transcription factor E2F recognition site are found within the
Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are
located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site.
E2F and DP cooperate and bind to the
potential E2F sites in the PCNA promoter in vitro. Thus the PCNA gene is a likely target gene of E2F. Site II plays a major role in
the PCNA promoter activity during embryogenesis and larval development, although both sites
are required for optimal promoter activity. However, for maternal expression in ovaries, either
one of the two sites is essentially sufficient to direct optimal promoter activity. These results
demonstrate an essential role for E2F sites in regulation of PCNA promoter
activity during development (Yamaguchi, 1995).
Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is
constant and ORCs are bound constitutively to replication origins (See Drosophila Orc2 for more information on the ORC). Replication is regulated by the recruitment
of additional factors such as CDC6. ORC components are widely conserved, and it generally has been
assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, it is shown
that the level of the Origin recognition complex subunit 1 (ORC1) subunit changes dramatically throughout Drosophila development. The
accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates
preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with
high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a
developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion
gene clusters. Furthermore, it has been found that overexpression of ORC1 alters the pattern of DNA synthesis in the
eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1
in Drosophila (Asano, 1999)
Late in embryonic development, most cells enter an extended quiescent period, resuming DNA synthesis upon hatching. However, replication persists in three tissues (brain, ventral nerve cord, anterior- and posterior-midgut), and the mRNAs of E2F-regulated genes (such as Ribonucleotide reductase, RNR2) accumulate in these tissues. To determine whether transcription of ORC1 is regulated by E2F, the distribution of ORC1 mRNA was examined in wild-type and E2F- embryos by in situ hybridization. The distributions of ORC1 and RNR2 mRNAs are essentially the same in wild-type embryos at stage 13. Moreover, accumulation of either RNR2 or ORC1 mRNA is largely dependent on E2F function at this stage of development. Thus, transcription of ORC1 is E2F-dependent in the embryo. It was next determined whether E2F regulates ORC1 transcription in imaginal disc cells, which have canonical four-phase cell cycles. Accumulation of ORC1 mRNA is induced following overexpression of E2F (~4-fold). By comparison, accumulation of three other E2F-regulated mRNAs - PCNA, RNR1 and RNR2 - is induced to a similar extent in these experiments (Asano, 1999).
To determine whether the regulation described above is mediated by the direct action of E2F, the ORC1 promoter was isolated. Within the 400 nt upstream of the major transcriptional start site, four candidate E2F binding sites with similarity to the canonical site in the adenovirus E2 promoter (TTTCGCGC) were identified by inspection, two at approximately -340 nt and two overlapping sites at -13 nt. Characterization of other E2F-responsive promoters has shown that binding sites close to the transcriptional start site frequently play a predominant role in regulation, and thus a focus was placed on the overlapping sites at -13. Drosophila E2F has been shown to bind to the ORC1 promoter just upstream of the start site of transcription. To test the role of these E2F sites in vivo, transcriptional reporter genes were prepared in which either the wild-type ORC1 promoter or a mutant derivative bearing substitutions within the proximal E2F binding sites drives the expression of a cDNA encoding an unstable Ftz-GFP-Myc tag fusion protein. Activity of the ORC1 promoter is dependent on the integrity of the E2F binding sites at -13 nt. In flies bearing the wild-type promoter construct, fusion protein is detectable in cells throughout most regions of the imaginal discs. However, in flies bearing a mutant promoter construct, essentially no fusion protein is detectable in any of the imaginal discs. These observations suggest that E2F acts directly by binding to the ORC1 promoter and stimulating transcription. Furthermore, the spatiotemporal pattern of ORC1 promoter activation within two specialized groups of cells in the eye and wing imaginal discs supports the idea that E2F couples transcription of ORC1 to cell cycle progression (Asano, 1999).
During the third larval instar, a developmentally regulated cell cycle transition takes place as a wave of differentiation sweeps across the eye imaginal disc. The wave front is marked by the morphogenetic furrow. During differentiation, four regions can be identified: (1) anterior to the morphogenetic furrow (including the antennal disc), undifferentiated cells cycle asynchronously; (2) as they enter the furrow, cells arrest in an extended G1 phase; (3) immediately posterior to the furrow, some cells are recruited into ommatidial pre-clusters and begin neural differentiation while others synchronously enter S phase, and (4) posterior to this synchronous wave of S phase, most cells cease cycling and terminally differentiate. The pattern of ORC1 promoter activity in the eye imaginal disc suggests that it is turned on late in G1, near the G1-S boundary. In particular, the ORC1 promoter is activated in a random pattern among the asynchronous cells in the anterior region of the disc: turned off as cells enter the morphogenetic furrow and G1, turned on in cells as they emerge from the furrow late in G1 phase, and then turned off in the quiescent cells in the posterior region of the eye. Another developmentally programmed cell cycle arrest has recently been described in the wing imaginal disc. At the dorsoventral boundary of the disc, Notch and wingless signaling establish a zone of non-proliferating cells (ZNC) in which no S phase is detectable. Cells throughout the posterior ZNC and in the center of the anterior ZNC arrest late in G1, at a point when expression of Cyclin E can drive them into S; flanking cells in the anterior ZNC arrest in G2. Among the cells of the ZNC, the ORC1 promoter is active only in G1-arrested cells and not in those arrested in G2. These observations support the idea that activation of E2F in G1 stimulates transcription of ORC1 in a variety of cell types (Asano, 1999).
In Drosophila, many genes have been shown to be transcriptionally regulated by E2F during the G1-S transition. These include Cyclin E, RNR, Polalpha , PCNA, MCM2 and MCM3 . However, only in the case of Cyclin E has it been shown that protein levels are cell cycle regulated, presumably at least in part as a consequence of E2F action. In the other cases, either the protein distribution has not been reported or the protein level is constant throughout the cell cycle. Therefore, to determine whether the level of ORC1 is modulated as a result of E2F-dependent regulation, antibodies were prepared that specifically recognize the protein, and its distribution was examined in embryos and imaginal discs. Antisera were prepared by immunizing animals with glutathione S-transferase (GST) fusion proteins bearing three different portions of ORC1. The distribution of ORC1 was examined during embryonic development. The first 13 nuclear division cycles that occur in a syncitium are parasynchronous. However, upon formation of the cellular blastoderm and the onset of gastrulation, this synchrony breaks down. Subsequent cell divisions are synchronous within cohorts of adjacent cells, but cell cycles within adjacent 'mitotic domains' are out of register. During the first 13 synchronous cell cycles, maternally synthesized ORC1 is uniformly distributed among the embryonic nuclei. However, coincident with the formation of the cellular blastoderm and the onset of gastrulation, the ORC1 distribution changes dramatically, such that different nuclei contain very different levels of protein. For example, mesodermal precursors along the ventral midline, which comprise one of the mitotic domains, accumulate relatively high levels of protein at the onset of gastrulation. During germ band extension, ORC1 levels are highest among the mitotically active neuroblasts and in domains of epidermal precursor cells. Later, in stage 13 embryos, when most cells in the embryo are cell cycle arrested, ORC1 accumulates preferentially in cells of the nervous system and midgut that continue to cycle. In summary, the level of ORC1 in the Drosophila embryo is not constant as is the case in S.cerevisiae. Instead, the protein is developmentally regulated such that high levels of protein are found in proliferating cells (Asano, 1999).
Two lines of evidence suggest that E2F-dependent transcriptional regulation is responsible (at least in part) for this differential accumulation of ORC1. (1) In stage 13 E2F- embryos, essentially no ORC1 is detectable. (Analysis of E2F-dependence in earlier embryonic stages is confounded by the maternal supply of E2F.) (2) The pattern of ORC1 accumulation is mirrored by the patterns of ORC1 promoter activity and ORC1 mRNA accumulation during embryonic development. Therefore, it is concluded that E2F-dependent transcriptional regulation, at least in part, couples ORC1 accumulation to proliferation. The distribution of ORC1 was examined in eye-antennal imaginal discs, where a developmentally regulated cell cycle transition takes place as the morphogenetic furrow sweeps across the disc. The level of ORC1 changes dramatically during this G1-S transition. The level of protein initially is low among the G1-arrested cells in the furrow. As cells emerge from the furrow late in G1, the level of ORC1 rises. Following the completion of S phase, ORC1 levels fall, returning to the basal level seen in the furrow. Two additional observations suggest that these changes in ORC1 levels are not peculiar to cells in and around the morphogenetic furrow. (1) Cells with high and low levels of ORC1 are randomly interspersed in the anterior region of the eye disc and the antennal disc where cells cycle asynchronously. (2) Within the ZNC of the wing imaginal disc, cells arrested in G1 accumulate high levels of ORC1, whereas G2-arrested cells do not. High levels of ORC1 accumulate in cells 3-4 rows to the posterior of the furrow and S phase begins in cells 5-6 rows to the posterior. Since a new row of cells emerges from the furrow every 1.5 h, this suggests that ORC1 accumulates ~1.5-3 h before the onset of S phase. ORC1 levels fall only after the completion of S phase. In summary, the level of ORC1 is cell cycle regulated, with peak accumulation during late G1 and throughout S phase. Further overexpression studies show that the abundance of ORC1 regulates DNA synthesis. In wild-type discs, cells within the morphogenetic furrow never
incorporate BrdU, and cells in the posterior region of the disc do so only rarely at this stage of development. However, in HS-ORC1 discs, some cells in both of
these regions incorporate BrdU and thus appear to have entered S phase either prematurely or inappropriately.
Ectopic ORC1 has no effect on either the onset or duration of the synchronous S phase among cells that emerge from the furrow. Nor does ectopic ORC1 have any noticeable effect on the proliferation of imaginal discs, the intensity of labeling at different BrdU concentrations or the growth rate of transgenic animals. Furthermore, the observation that endogenous ORC1 levels rise in anticipation of entry into S phase in the eye disc is consistent with the idea that high levels of ORC1 promote DNA synthesis rather than the opposite. As is the case in the imaginal discs, activity of the ORC1 promoter is E2F-dependent in the ovary (Asano, 1999).
To test if the cell cycle transcription of double parked (dup) is dependent on
E2F, embryos homozygous for a null allele of the dE2F1 subunit, dE2F91 were collected and hybridized with
dup riboprobes. The levels of dup transcript are
decreased in the endoreplicating gut and appear to be slightly
decreased in the CNS. A similar effect on dup
transcript is seen in embryos that are homozygous mutant for the
other subunit of the E2F transcription factor, of the genotype
dDPa2. Thus, dup is a downstream
target of the E2F transcription factor. Interestingly, yeast cdt1
transcription is also cell-cycle regulated. Expression of cdt1
is controlled by the G1-S transcription factor Cdc10 that, like E2F,
regulates transcription of many genes required for S phase (Hofmann, 1994). This suggests that cell cycle control of dup may
be conserved, and Dup may prove to be an important downstream target of
E2F in mammalian cells (Whittaker, 2000).
Cyclin E is required to regulate positively the transcription of S
phase genes in the nervous system and to downregulate these transcripts
in endo cycling cells.
The cyclinEl(2)305 and cyclin
EPZ5 mutations and a cyclin E deficiency,
Df(2L)TE35D1, have similar effects on double parked
transcripts. In these embryos, dup is not downregulated properly in the endoreplicating gut such that dup transcripts persist at higher levels than wild type in the anterior midgut, central midgut, and posterior midgut in later embryonic stages. In cyclin E mutant embryos dup
transcripts are reduced in the CNS, although not to as great an extent
as other S phase genes. Thus, dup expression also is regulated
by cyclin E (Whittaker, 2000).
Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. A Drosophila TRF2-containing complex has been purified by antibody affinity; this complex contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding leads to potential target genes containing TRF2-responsive promoters. A combination of in vitro and in vivo assays has been used to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. Additional TRF2-responsive target genes involved in DNA replication and cell proliferation have also been identified. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila (Hochheimer, 2002).
Having identified DREF as a tightly associated component of the TRF2 complex, it was next asked whether TRF2 can function as a true core promoter recognition factor and selectively initiate transcription at a promoter that is documented to be stimulated by the DRE/DREF system. The DREF-responsive PCNA promoter, which contains at least three promoter-proximal regulatory elements including an upstream regulatory element URE, DRE and two E2F recognition sites located within 200 bp upstream of the start site, was chosen (Hochheimer, 2002).
To test the responsiveness of the PCNA promoter in vitro and to map the transcription start site(s), a -580 PCNA (-580 to +56) promoter fragment, which contains all known regulatory elements, and a -64 PCNA (-64 to +56) promoter fragment, which lacks all regulatory elements except for the E2F-binding sites were used as DNA templates for in vitro transcription. Increasing amounts of a partially purified Drosophila embryo nuclear extract (H.4) that contains all the necessary basal factors were added, as well as both the TRF2 complex and limiting amounts of TFIID to the transcription reaction. Using the -580 PCNA template two distinct transcription start sites separated by 63 nucleotides were detected. Promoter 1 (with start site at position +1) was stimulated with increasing amounts of H.4 supplemented with TFIID whereas promoter 2 (with start site at position -63) was detected only with the lowest amounts of H.4 added. Using the truncated -64 PCNA, template, transcription from promoter 2 was essentially abolished, whereas a weak activity could be detected from promoter 1 by adding the maximum amount of H.4 + TFIID. In vitro and in vivo results suggest that promoter 2 might be TRF2- and DRE-dependent, whereas promoter 1 appears to be mediated by TFIID (Hochheimer, 2002).
It was next asked whether TRF2 can contribute to the enhancer-dependent activated transcription of the PCNA promoters by E2F and DP, which cooperate in DNA-binding and transcriptional activation. The co-expression of just the trancriptional activators E2F and DP in the absence of exogenous TRF2/DREF results in a substantial transcriptional activation of the PCNA reporter. It is likely that this activation by E2F/DP is mediated by endogenous TRF2. This activation is abolished with the -64 PCNA reporter, which lacks the DRE-binding sites but still contains the E2F-binding sites. As expected, inducing the co-expression of all three promoter recognition factors -- TRF2, DREF and E2F -- results in a strong synergistic activation of the PCNA promoters (80-fold) in a DRE-dependent fashion. These results suggest that in SL2 cells TRF2 and DREF can work together to stimulate the PCNA reporter in a DRE-dependent fashion. This is consistent with the finding in vitro that the TRF2 complex can selectively initiate transcription from promoter 2 of the PCNA gene in a DRE-dependent manner (Hochheimer, 2002).
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).
Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. This study found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. This study used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase (Heriche, 2003; full text of article).
In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).
Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG–SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG–SD dimer activity, thereby providing a feedback control (Legent, 2006).
Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG–SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG–SD dimers. Therefore, sd induction may efficiently restrain VG–SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG–SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).
The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG–SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG–SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG–SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG–SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG–SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).
The results also demonstrate that dE2F1-DP regulates VG–SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).
Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG–SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).
In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).
Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG–SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG–SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG–SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).
Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).
Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG–SD, but also via additive impairment of VG–SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG–SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG–SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).
At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG–SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG–SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).
Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG–SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).
Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG–SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).
Retinoblastoma (Rb) family proteins control E2F-dependent transcription and restrict cell proliferation. In the early G1 phase of the cell cycle, Rb family proteins bind to E2F family members, inhibiting their ability to activate transcription and recruiting repressor complexes to DNA. In late G1 to S phase, cyclin-dependent kinases (CDK) phosphorylate Rb family proteins, liberating E2F and activating E2F-dependent transcription. One of the least-well-understood aspects of in vivo studies of Rb function is the fact that the inactivation of Rb often sensitizes cells to apoptosis.
The extent of apoptosis caused by the inactivation of Rb is highly cell type and tissue specific, but the underlying reasons for this variation are poorly understood. This study characterizes specific time and place during Drosophila development where rbf1 mutant cells are exquisitely sensitive to apoptosis. During the third larval instar, many rbf1 mutant cells undergo E2F-dependent cell death in the morphogenetic furrow. Surprisingly, this pattern of apoptosis is not caused by inappropriate cell cycle progression but instead involves the action of Argos, a secreted protein that negatively regulates Drosophila epidermal growth factor receptor (EGFR [DER]) activity. Apoptosis of rbf1 mutant cells is suppressed by the activation of DER, ras, or raf or by the inactivation of argos, sprouty, or gap1, and inhibition of DER strongly enhances apoptosis in rbf1 mutant discs. RBF1 and a DER/ras/raf signaling pathway cooperate in vivo to suppress E2F-dependent apoptosis and the loss of RBF1 alters a normal program of cell death that is controlled by Argos and DER. These results demonstrate that a gradient of DER/ras/raf signaling that occurs naturally during development provides the contextual signals that determine when and where the inactivation of rbf1 results in dE2F1-dependent apoptosis (Moon, 2006).
This study takes advantage of the observation that the inactivation of rbf1 in the Drosophila eye results in a distinctive pattern of apoptosis that is tightly linked to eye development, and this model system was used to define a cellular context in which RBF1 is needed to protect cells against dE2F1-dependent cell death. The results show that the cellular response to the inactivation of rbf1 involves a combination of signals. Deregulated dE2F1 provides one function that is required for apoptosis. However, in most situations, deregulation of the endogenous dE2F1 is not sufficient to induce apoptosis. In addition, a second condition, the down-regulation of an EGFR/Ras/Raf signaling pathway, is also necessary. In the eye imaginal disc, the EGFR/Ras/Raf signaling pathway is down-regulated at the region immediately anterior to the 'intermediate group' (IG) of cells, from which the R8 founder cell will be selected. When rbf1 mutant cells pass through this gradient, they become highly sensitive to dE2F1-dependent apoptosis. Elevation of the level of DER/Ras/Raf signaling by a variety of means suppresses apoptosis in rbf1 mutant cells. Conversely, expression of a dominant-negative mutant of DER strongly synergized with mutation of rbf1 to induce apoptosis (Moon, 2006).
Before starting this work, several different ways were considered in which the inactivation of RBF1 might result in apoptosis. If differentiated/differentiating cells try to reenter the cell cycle following the inactivation of RBF1, then an abnormal or inappropriate S-phase entry might cause apoptosis. Alternatively, one could argue that rapidly proliferating cells contain the highest levels of E2F transcriptional activity, and hence these cells ought to be most sensitive to E2F-induced apoptosis when RBF is removed. Although both models were plausible, in fact, neither explanation fits the data. rbf1 mutant eye discs display little apoptosis in either the population of differentiated cells or in actively cycling cells. Instead, rbf1 mutant cells are sensitive to apoptosis in the MF, at a time when some cells exit the cell cycle and initiate a differentiation program. This apoptosis was not accompanied by inappropriate cell cycle progression. Indeed, when rbf1 mutant cells were rescued from apoptosis, they showed no indication of S-phase entry. Hence rbf1 mutant cells were not dying because they were inappropriately progressing through the cell cycle. Instead, apoptosis was dependent on a specific developmental context. This need for the correct context may be particularly significant when designing cell culture-based screens for treatments that are synthetic lethal with the inactivation of Rb (Moon, 2006).
Several studies have shown that E2F complexes regulate the expression of proapoptotic genes, but why would the effects of losing RBF1 be sensitive to EGFR signaling? While it seems likely that deregulated dE2F1 activates transcription of several proapoptotic targets, the results indicate that an important part of the explanation lies in the regulation of the proapoptotic gene hid. hid transcripts are up-regulated in rbf1 mutant eye discs and halving the gene dosage of hid dramatically reduced apoptosis. Previous studies have shown that HID-induced apoptosis is highly sensitive to EGFR/Ras/Raf signaling. Signaling through this pathway suppresses transcription of hid and is thought to induce an inhibitory phosphorylation on the HID protein. This provides a simple model, in which the loss of RBF1 results in the elevated expression of a proapoptotic protein, which is then held in check by EGFR/Ras/Raf-mediated signaling. Apoptosis would then occur when EGFR signals are reduced. Consistent with this model, it was found that the region of the eye disc that is most sensitive to loss of RBF1 is also highly sensitive to low levels of ectopic hid expression (Moon, 2006).
Why does this pattern of apoptosis occur? It is likely that several different factors are needed to establish the gradient of DER activity. Important regulators of DER activity in the eye include Gap1, Sprouty, and Argos. In this particular context, the ability of Argos to diffuse and act at a distance from the p-Erk-positive cells appears to be important. It is suggested that the pattern of Argos expression in the developing eye disc generates a zone in which cells that have failed to exit the cell cycle and inappropriately inactivate RBF1 become prone to undergo apoptosis. In essence, this could be viewed as a developmental failsafe mechanism against inappropriate proliferation. In support of this, it is noted that E2F1 levels are transiently elevated in G1 phase cells in the MF, even though E2F regulation is not needed for S phase entry in the second mitotic wave. Consistent with the idea that this region of the disc may be more sensitive to apoptosis, it was found that a transient pulse of cyclin E expression, which drives ectopic S phases throughout much of the disc, generates a similar stripe of apoptosis in the MF. It is curious that this sensitivity occurs at the time when the role of EGFR is apparently changing from being needed for cell proliferation in the anterior part of the disc to being required for differentiation in the posterior part of the disc. It will be interesting to discover whether similarly sensitive regions exist in other discs (Moon, 2006).
As seen with rbf1, the effects of mutating Rb in the mouse are most evident at points in development when cells attempt to exit the cell cycle and differentiate. Rb-null mouse retinas show increased cell death during the transition from proliferation to differentiation. Whether this is due to an analogous interaction between Rb/E2F and EGFR/Ras signaling has not been tested but is an interesting possibility. It is also tempting to speculate that some of the different cellular responses to the inactivation of Rb in the mouse retina may be caused by differences in EGFR/Ras-mediated differentiation signals (Moon, 2006).
There are several indications that the general phenomenon described in this study is likely to be conserved in mammalian cells. For example, recent studies have shown that apoptosis in cultured fibroblasts lacking Rb family proteins (TKO) can be suppressed by activation of Ras/Raf. Interestingly, a functional homologue of Hid, Smac/Diablo, was recently shown to be a direct target of E2F1 in mammalian cells, raising the possibility that mammalian cells may contain a regulatory loop that directly parallels the regulation of Hid. However, a connection between the proapoptotic function of Smac/Diablo and EGFR pathway has yet to be described (Moon, 2006).
The molecular events underlying the convergence of EGFR signaling and Rb/E2F may be different between flies and humans. It is noted that Akt activation suppresses E2F1-induced apoptosis in mammalian tissue culture cells, while neither the overexpression of dAKT1 nor the mutation of dPTEN is sufficient to prevent cell death in rbf1 mutant eye discs. This may reflect a difference between an in vivo analysis and tissue culture conditions, or it may reflect species-specific differences in the regulation of apoptosis. It is known, for example, that caspase activation is regulated differently between species. In vertebrates, cytochrome c release from mitochondria is a key step in the promotion of caspase activation, while in Drosophila, this step is largely dispensable. It is possible that EGFR activity converges on E2F-dependent cell death through a previously identified E2F target whose activity is regulated by Raf/Erk- and/or AKT-mediated signals, such as Bim. In order to define this circuitry, it is first necessary to identify the appropriate in vivo context in mice or humans in which Rb/E2F and EGFR activity cooperate to regulate cell survival. Once the appropriate context is found, then it may be possible to identify the molecular mechanism linking E2F-dependent cell death to survival signals (Moon, 2006).
Both EGFR family and Rb pathways are often altered in cancer. Given that developmentally controlled fluctuations in EGFR signaling have dramatic effects on the sensitivity of rbf1 mutant cells to apoptosis, it is speculated that therapeutic cancer drugs that target EGFR family proteins may induce cell death most efficiently in tumor cells that have the highest levels of E2F1 activity. One of the curious features of human retinoblastoma is that, unlike many other cancers, these tumors rarely contain mutations in p53, suggesting that either these cells do not need to mutate p53 or that they find a more effective way to suppress apoptosis. Identification of the critical components that protect premaligant Rb mutant cells from apoptosis may lead to new ways to target these cells for treatment (Moon, 2006).
Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).
Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).
How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).
More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).
Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).
In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).
How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).
There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).
The initiation and maintenance of G1 cell cycle arrest is a key feature of
animal development. In the Drosophila ectoderm, G1 arrest first
appears during the seventeenth embryonic cell cycle. The initiation of
G117 arrest requires the developmentally-induced expression of
Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G117
arrest requires Rbf1-dependent repression of E2f1-regulated replication factor
genes, which are expressed continuously during cycles 1-16 when S phase
immediately follows mitosis. The mechanisms that trigger Rbf1 repressor
function and mediate G117 maintenance are unknown. This study shows
that the initial downregulation of expression of the E2f1-target gene
Ribonucleoside diphosphate reductase small subunit (RnrS), which occurs during cycles 15 and 16 prior to entry into
G117, does not require Rbf1 or p27Dap. This suggests a
mechanism for Rbf1-independent control of E2f1 during early development. E2f1 protein is destroyed in a cell cycle-dependent manner during S
phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and
requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal
cells arrested in G117, and in these cells the induction of
p27Dap activates Rbf1 to repress E2f1-target genes to maintain a
stable G1 arrest (Shibutani, 2007).
The finding that p27Dap expression is not necessary for the
downregulation of E2f1 targets was unexpected, based on the known regulatory
circuitry of the pRb-E2F pathway. This result led to the hypothesis that mechanisms in addition to Rbf1 binding are used to control E2f1 activity in the early embryo. It was found that E2f1 is destroyed during S phase of the post-blastoderm divisions
in the embryonic epidermis, has been reported for cells in wing and
eye imaginal discs. E2f1 destruction first occurs during S15, at the
same time that E2f1-regulated transcripts such as RnrS begin to
decline. Because E2f1 functions as a transcriptional activator, and because Rbf1 is not required for the initial decline in RnrS transcripts, it is proposed that the loss of E2f1 protein contributes to the
initial termination of replication factor gene expression. Rbf1 is first
required during development for the maintenance of G117 arrest and
the continued repression of E2f1-target genes. The data suggest that Rbf1 is
converted to a repressor after the developmentally-induced expression of Dap,
most likely because the consequent inhibition of CycE-Cdk2 results in the
accumulation of hypophosphorylated Rbf1. Dap expression accompanies the
downregulation of Cyclin E transcription, and each of these
mechanisms of inhibition of CycE-Cdk2 contributes to G1 arrest (Shibutani, 2007).
The high level of E2f1 protein in G117 epidermal cells may
permit the formation of E2f1-Rbf1 complexes necessary to actively and stably
repress replication factor genes during G1 arrest, and
also provides a simple explanation for why the loss of Rbf1 function results
in the ectopic expression of E2f1 targets. After
hatching, and in response to the first instar larvae beginning to feed, the
epidermal cells start to endoreduplicate. Thus, the accumulation of Rbf1-E2f1
complexes during G1 arrest may prepare cells for rapid production of
replication factors and efficient re-entry into the cell cycle upon activation
of G1 Cyclin-Cdk complexes after growth stimulation (Shibutani, 2007).
RnrS expression is lost in E2f1 zygotic mutant embryos,
but not until cell cycle 17. One interpretation of this result is that maternal
stores of E2f1 are sufficient for the early induction of replication gene
expression in the post-blastoderm divisions. Consistent with this, maternal
E2f1 protein persists into cycle 14, coincident with the commencement of
zygotic transcription of E2f1 targets such as RnrS. In addition,
mutation of the E2f1-binding sites in the regulatory region of the
Pcna gene (mus209 - Flybase) is sufficient to abolish
zygotic Pcna expression. However, the data do not demonstrate a requirement
for E2f1 for early zygotic RnrS expression, and E2f1 may be only one
of several factors necessary for early zygotic expression of genes encoding
replication factors. For instance, the transcription of Cyclin E
requires E2f1 in embryonic endocycles, but also occurs independently of E2f1
via tissue-specific enhancer elements such as those operating in the CNS. Thus, any
control of replication factor gene expression by E2f1 abundance may be
modulated by other transcription factors, or bypassed entirely in certain cell
types by E2f1-independent modes of expression (Shibutani, 2007).
The data suggest that E2f1 destruction is coupled to DNA synthesis.
CycE-Cdk2 has been suggested as a possible cell cycle input for E2f1
destruction in imaginal cells because it is activated at the G1-S transition
when E2f1 is destroyed. However, CycE-Cdk2 is continuously active during the
embryonic post-blastoderm cell cycles, whereas E2f1 is destroyed only during S
phase. Thus, CycE-Cdk2 is unlikely to be the only signal, and actively replicating
DNA may provide a necessary input into E2f1 destruction. This model is
consistent with the observation that E2f1 destruction occurs after DNA
synthesis begins, resulting in cells that are positive for both E2f1 and BrdU
incorporation in early interphase (Shibutani, 2007).
Previous studies have suggested that mammalian E2f1 is degraded by the
ubiquitin-proteasome pathway. In this pathway, E3 ubiquitin ligases bind to and mediate the ubiquitylation of specific proteins. The SCF class of cullin-dependent E3 ligases has been implicated in E2F1 destruction. In
Drosophila, evidence from genetic and cell biology studies suggest
that SCFSLMB mediates E2f1 destruction at the G1-S transition in
wing imaginal disc cells (Heriche, 2003). Although there is no evidence implicating a specific E3 ligase in the destruction of embryonic E2f1, there are interesting parallels with recent experiments describing the destruction of Cdt1/Dup. Like E2f1, Cdt1/Double parked is degraded at the G1-S transition and cannot be detected during S phase. In vertebrates, Cdt1 destruction is mediated by two independent and apparently redundant mechanisms: direct Cdk2 phosphorylation that targets Cdt1 to
SCFSKP2, and binding of PCNA to the Cdt1/Dup amino-terminus that
targets Cdt1 to Cul4DDB1. This latter result is consistent with a recent study
indicating that Drosophila Dup hyperaccumulates in cells where DNA
synthesis is attenuated. Thus, more than one E3 ubiquitin ligase may participate in
E2f1 destruction. Determining the molecular mechanism of E2f1 destruction
should permit a direct test of whether prevention of E2f1 destruction would
affect replication factor gene expression in the embryo (Shibutani, 2007).
E2F is necessary for the development of worms, flies and mice. Remarkably,
however, pRb is not needed for the entirety of mouse embryonic development. This could in part be due to redundancy with other pRb family members, such as p107 and
p130. Alternatively, a pRb-independent mechanism of regulating E2F activity may
control S phase gene expression and cell cycle progression during early
mammalian development. This idea is supported by experiments modeling the cell
cycles of early vertebrate development in cell culture using murine embryonic
stem cells. These pluripotent cells have a cell cycle composed mostly
of S phase that is characterized by ubiquitous Cdk activity and the absence of
CKIs. As in the
Drosophila embryo, E2F-regulated transcripts are also ubiquitous even
though pRb family members are expressed.
Differentiation requires the lengthening of G1 and the negative regulation of
Cdk2 activity, which is accomplished both by increases in the level of CKIs
and by the downregulation of Cyclin E1 expression via inhibition of E2F. Thus,
evolutionarily conserved regulatory mechanisms operating in early development
may mediate the conversion from rapid cell cycles driven by intrinsic cues to
slower, more highly regulated cycles that are influenced by extrinsic
developmental and environmental cues (Shibutani, 2007).
E2F is really a heterodimer, consisting of E2F itself and DP protein. A Drosophila
DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding
component of Drosophila E2F.
Some aspects of the mechanisms which integrate early cell
cycle progression with the transcription apparatus are thus conserved between Drosophila and
mammalian cells. The distinct expression patterns of Drosophila DP and E2F suggest that the formation
of DP/E2F heterodimers, and hence E2F, is subject to complex regulatory cues (Hao, 1995).
Drosophila DP and human DP-1 proteins share 61% identity over a region between residues 91 and 315 that includes the putative DNA-binding domain. Drosophila DP binds to Drosophila E2F fusion proteins containing amino acids 225-805 or 1-432, but fails to bind to a fusion protein that includes only aa 47-343. Thus a region of Drosophila E2F between aa225 and 432 may be required for interaction with Drosophila DP. This region of the protein is highly conserved. Drosophila DP has a weak nonspecific DNA-binding activity, while Drosophila E2F alone is unable to bind DNA. When the two proteins are mixed, E2F acquires DNA-binding activity, and the affinity of DP for DNA is dramatically enhanced. The enhanced binding is site-specific (Dynlacht, 1994).
Whereas the ectopic expression of Cyclin E activates dE2F-dependent
transcription, it has been proposed that Cyclin E does not act directly on E2F but targets a negative
regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins
(pRB, p107, and p130) that associate with E2F in humans. Drosophila Retinoblastoma-family protein) combines several of the
structural features of pRB, p107, and p130, suggesting that it may have evolved from a common
ancestor to the three human genes. RBF associates with Drosophila E2F and DP in vivo and is a
stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed
E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of
dE2F and dDP in the developing Drosophila eye. RBF is phosphorylated by a cyclin
E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF
overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively
regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor
that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF
plays a critical role in the regulation of cell proliferation in Drosophila (Du, 1996a).
The E2F family of transcription factors contributes to cell
cycle control by regulating the transcription of DNA
replication factors. Functional 'E2F' is a DNA-binding
heterodimer composed of E2F and DP proteins. Drosophila
contains two E2F genes (E2F and E2F2) and one DP
gene. Mutation of either E2F or DP eliminates
G1-S transcription of known replication factors during
embryogenesis and compromises DNA replication.
However, the analysis of these mutant phenotypes is
complicated by the perdurance of maternally supplied gene
function. To address this and to further analyze the role of
E2F transcription factors in development mitotic clones of DP, mutant
cells in the female germline have been
phenotypically characterized. DP has been shown to be required for several essential processes during
oogenesis. In a fraction of the mutant egg chambers the
germ cells execute one extra round of mitosis, suggesting
that in this tissue DP is uniquely utilized for cell cycle
arrest rather than cell cycle progression. Mutation of DP
in the germline also prevents nurse cell cytoplasm transfer
to the oocyte, resulting in a 'dumpless' phenotype that
blocks oocyte development. This phenotype likely results
from both disruption of the actin cytoskeleton and a failure
of nurse cell apoptosis, each of which is required for
normal cytoplasmic transfer. DP is
required for the establishment of the dorsal-ventral axis, since
loss of DP function prevents the localized expression of the
Egfr ligand Gurken in the oocyte: Gurken initiates dorsal-ventral
polarity in the egg chamber. Thus new functions for E2F transcription factors
during development have been uncovered, including an unexpected role in
pattern formation (Myster, 2000).
Drosophila oogenesis relies on communication between the
nurse cells and the oocyte; this includes transfer of nurse cell
cytoplasm to the oocyte. There are two successive phases of
cytoplasmic transport during oogenesis. Early transport is slow
and highly selective. Later in oogenesis a rapid phase of
cytoplasmic transfer occurs as nurse cells contract to squeeze
their contents into the oocyte. DP mutant egg chambers are
defective in this rapid transport. They are also defective in
nurse cell apoptosis. How might dDP affect both nurse cell
cytoplasmic transport and apoptosis? Recent evidence reveals
that entry into apoptosis may be coupled to rapid cytoplasm
transport. In wild-type egg chambers several events indicate an
apparent initiation of a nurse cell apoptotic pathway just prior
to rapid cytoplasm transfer. The nurse cells undergo actin
cytoskeleton rearrangements and their nuclear membranes become permeabilized. Delays in apoptosis seen in dumpless mutants that disrupt the actin cytoskeleton, such as chickadee
(profilin) and kelch (a ring canal component), suggest that an
apoptotic pathway and a rapid cytoplasmic transport pathway
are interconnected. Germline mutant
clones of Drosophila dcp-1, a CED-3-related caspase, display defects in actin bundle assembly, nuclear
envelope permeabilization and cytoplasmic transport. Thus, it has been suggested
that activation of an apoptotic pathway in nurse cells may lead
to the formation of the actin bundles and subsequent nurse cell
to oocyte cytoplasmic transfer. This model places a signal to
enter apoptosis at the start of the pathway, followed by dcp-1
function, followed by chickadee, quail (villin) and singed
(fascin) functions in bundling actin, and ending with cytoplasm
transfer. In the context of this model, DP could act either by
directly regulating genes involved in actin cytoskeleton
function (e.g., chickadee) or an apoptotic pathway (e.g., dcp-1). The well-documented ability of both mammalian and
Drosophila E2F to induce apoptosis suggests
the latter hypothesis, i.e. that dDP is required for the initiation
of nurse cell apoptosis, which triggers subsequent cytoplasmic
dumping (Myster, 2000).
Surprisingly, loss of dDP function significantly inhibits the
establishment of dorsal-ventral polarity during oogenesis.
Germline cells mutant for either of two alleles of dDP
prevent the normal accumulation of Grk protein. In DP oocytes, Grk protein fails to accumulate to wild-type levels.
Grk protein is detected at higher levels in DP mutants
than DP mutants, but fails to properly localize to the
oocyte plasma membrane. These data suggest that both Grk
accumulation and intracellular localization are disrupted by
mutation of DP. Because Grk is required for an oocyte-follicle
cell signaling system that initiates dorsal follicle cell
fate, the DP mutants displayed a ventralized eggshell.
Defects in Gurken accumulation may
result from disruption of the cell cycle caused by loss of DP.
There is already evidence that Gurken expression is linked to
cell cycle control. Mutation of the 'spindle' class genes spnB
and okra prevent normal Grk accumulation and establishment
of the dorsal-ventral axis. spnB and okra encode proteins with
sequence similarity to yeast genes required for repair of double
strand breaks (DSBs). The failure to accumulate Grk in spnB and okra mutants is a downstream developmental consequence of a meiotic checkpoint activated
in response to the persistence of DSB generated during meiotic
recombination. Perhaps loss of DP function alters replication sufficiently to activate a checkpoint pathway. Alternatively, the dorsal-ventral defect in
DP mutants may reflect a DP requirement for expression of
spnB and okra during meiosis (Myster, 2000).
The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila E2f1 and DP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Mutants of Rbf (an retinoblastoma protein homolog) fail to limit DNA replication. DP, E2f1 and Rbf proteins are located in a complex with ORC, and E2f1 and ORC are bound to the chorion origin of
replication in vivo. These results indicate that E2f and Rbf function together at replication origins to limit DNA replication through interactions with ORC (Bosco, 2001).
To explore the possibility that E2f-Rbf is directly involved in controlling ORC activity, a test was performed to see whether a female-sterile mutant of Rbf (Rbf120a) has DNA replication and gene amplification defects in follicle cells of the Drosophila ovary. TheRbf120a mutation is due to a P-element insertion that causes reduced levels of wild-type Rbf protein, and Rbf14 is a null mutant. Ovaries from mutant Rbf120a/Rbf14 and heterozygous Rbf14/+ females were double labelled with 5-bromodeoxyuridine (BrdU) and anti-ORC2. Wild-type Drosophila follicle cells undergo endoreduplication cycles (endo cycles), reaching 16n ploidy by stage 9 or 10A of egg-chamber development. In stage 10B follicle cells, endo cycles have ceased, ORC has been cleared from the nucleus, and ORC is localized to discrete genomic regions undergoing amplification. Amplification is detected by BrdU incorporation at ORC localized foci. By contrast, the Rbf120a/Rbf14 mutant egg-chambers have a mosaic of follicle cells exhibiting striking replication defects: (1) some mutant follicle cells have inappropriate total nuclear ORC2 staining and continued endo cycles instead of amplification; (2) some follicle cells with specific ORC2 localization to replication origins have undergone gene amplification; and (3) some cells perform both amplification and genomic replication. Staining ovaries with anti-Rbf antibodies reveals a uniform absence of Rbf protein, and thus the mosaic phenotype cannot be explained by stochastic differences in Rbf protein levels (Bosco, 2001).
The Rbf120a/Rbf14 follicle cells undergoing gene amplification have large BrdU foci relative to sibling controls. Quantitation has confirmed that Amplification control element ACE3 DNA is amplified ~26-fold in mutant stage 13 egg-chambers, compared with ~16-fold in heterozygous egg-chambers. This phenotype in the Rbf120a/Rbf14 mutant is similar to the overamplification observed in the E2fi2 truncation mutant in which ACE3 is amplified 32-fold. Thus both Rbf and E2f are negative regulators of gene amplification (Bosco, 2001).
There is also a cell-cycle defect in the Rbf120a/Rbf14 mutant follicle cells. Inappropriate genomic replication seen in the mutant follicle cells results from the continuation of S/G endo cycles beyond the developmental stage at which they would normally cease. This predicts the presence of mutant follicle cells with greater DNA content than the wild-type 16n DNA. Fluorescence-activated cell sorting (FACS) analysis was carried out on purified ovarian nuclei, and heterozygous Rbf14/+ ovaries gave follicle cells with 2n, 4n, 8n and 16n DNA content. Rbf120a/Rbf14 ovaries, however, had cells with 32n DNA content, indicating that they had undergone at least one extra S-phase. It is concluded that stage 10B Rbf120a/Rbf14 mutant follicle cells undergo an ectopic S phase, and genomic replication in stage 10B cells is not due to a developmental delay. This result parallels that obtained with mutations in dDP (Bosco, 2001).
DNA replication also persists in later stages of mutant follicle cells. Wild-type stage 13 egg-chambers have no detectable ORC localization and little or no BrdU incorporation. In contrast, Rbf120a/Rbf14 stage 13 egg-chambers continue to undergo amplification and genomic replication that is consistent with persistent nuclear ORC staining. Some stage 13 cells exhibit characteristics of G1 cells, with nuclear ORC but no BrdU staining. This observation also supports the conclusion that Rbf120a/Rbf14 follicle cells continue bona fide G/S endo cycles (Bosco, 2001).
Tests were performed to see whether misregulation of important E2f target genes might account for the replication defects observed in the Rbf mutant follicle cells. Four important E2f target genes, Cyclin E, PCNA, RNR2 and ORC1, as well as ORC2 transcripts, are not normally induced in wild-type stage 10 follicle cells, and their transcripts are not elevated in the truncation E2fi2 mutant follicle cells. However, because overexpression of ORC1 is sufficient for initiating an ectopic endo cycle in stage 10 follicle cells, ORC1 and ORC2 transcripts were analyzed by in situ hybridization in Rbf mutant follicle cells. No significant differences were found in the amount of messenger RNA levels for either gene in Rbf120a/Rbf14 stage 9, 10 or 13 egg-chambers, as compared with Rbf14/+ sibling controls (Bosco, 2001).
Transcription of the reaper gene is highly induced in the follicle cells of wild-type stage 9 and 10 egg-chambers, and thus reaper levels were used as a measure of general transcriptional activity in an experiment in which attempts were made to block transcription of all genes. Egg-chambers were cultured in vitro for up to 6 h with or without alpha-amanitin. Rbf120a/Rbf14 egg-chambers cultured in the presence of alpha-amanitin abolish visible transcript levels of reaper in stage 10B follicle cells, whereas the Rbf120a/Rbf14 controls induce reaper normally. However, alpha-amanitin does not change the pattern of BrdU labelling in Rbf120a/Rbf14 stage 10 or 13 egg-chambers. Thus, the Rbf mutant replication defects persist even when general transcription is inhibited in follicle cells. It is possible that the in situ analysis of transcript levels or the inhibition of transcription by alpha-amanitin fail to uncover an effect of the Rbf mutant. Taken together, however, these data suggest that the gene amplification phenotype seen in Rbf120a/Rbf14 or E2fi2 follicle cells is not due to a misregulation of E2f target gene transcription in stage 10 egg-chambers (Bosco, 2001).
Whether E2f-Rbf complexes execute an S-phase function through a direct interaction with ORC was tested. Immunoprecipitations were carried out on ovary extracts; immunoblots of the pellets show that E2f and Rbf co-immunoprecipitate with Drosophila ORC when either anti-ORC2 or anti-ORC1 antibodies were used. The E2f-Rbf-ORC interaction could also be detected when immunoprecipitation reactions were performed with anti-E2f polyclonal or anti-DP monoclonal antibodies. This complex could be specifically immunoprecipitated from ovary extracts with five different antibodies. It is possible that in extracts the dDP-E2f-Rbf and ORC interaction might be due to dDP-E2f and ORC binding next to each other on DNA fragments. Therefore, immunoprecipitation reactions were carried out in the presence of ethidium bromide or micrococcal nuclease to disrupt protein-DNA interactions or cleave DNA fragments. Treatment of immunoprecipitation reactions with either reagent failed to disrupt the E2f-Rbf-ORC interaction. Furthermore, a mutation in DP predicted to reduce the DNA-binding activity of E2f did not abolish the E2f-Rbf-ORC interaction. It is therefore concluded that E2f and Rbf can co-immunoprecipitate with ORC through interactions that are independent of their respective DNA-binding activities (Bosco, 2001).
What is the functional relevance of this E2f-Rbf-ORC complex? One possible mechanism is that E2f-Rbf helps localize ORC to E2f-binding sites near the chorion replication origin. Another possibility is that ORC localization to the chorion replication origin is independent of its interaction with E2f-Rbf, and instead E2f-Rbf when bound next to an origin regulates replication initiation through its interaction with ORC. ORC binds the critical amplification control element ACE3 in vivoat a specific time in follicle cell development (stages 10A and 10B). Using anti-ORC2 antiserum, ACE3 has been specifically enriched relative to a control locus that does not bind ORC and is not amplified by using chromatin immunoprecipitation (CHIP). Using CHIP it was asked whether E2f also could be shown to localize specifically to ACE3 in vivo. Stabilization of protein-DNA interactions in live tissue is achieved by formaldehyde crosslinking. Subsequent CHIP enriches for specific trans-factors that are bound to genomic loci. The relative amounts of these loci are quantified by polymerase chain reaction (PCR). Sequence analysis reveals that there are several potential E2f-binding sites within 2.5 kilobases (kb) of ACE3. Using anti-E2f antibodies, it has been shown that ACE3 DNA is enriched ~15-fold relative to the rosy locus in stage 10 egg-chambers. Similarly, anti-ORC2 antibodies also enriched ACE3 DNA ~20-fold relative to the rosy locus. Thus, both E2f and ORC localize to ACE3 when amplification is occurring, and E2f binding is limited to sequences immediately adjacent to ACE3. This observation is consistent with E2f-Rbf functioning at replication origins and possibly controlling ORC activity (Bosco, 2001).
Since transactivation and RB-binding activities are known to be located in the C-terminal domain of mammalian E2F, a truncated form of Drosophila E2f predicted to lack the C-terminal transactivation and Rbf-binding domains was characterized. The E2fi2 mutation produces a stable, truncated E2fi2 protein that can still interact with DP. Truncated E2fi2 does not bind Rbf, as it does not co-immunoprecipitate, even when more than 10% of the total Rbf protein is immunoprecipitated. This failure to pellet the truncated E2fi2 protein is not due to low Rbf levels in mutant extracts because comparable amounts of Rbf in wild-type extracts can immunoprecipitate full-length E2f. Failure to detect this interaction is not due to low levels of truncated E2fi2 protein, because the amount of truncated E2fi2 in the supernatant represents 10% of total E2fi2 in the immunoprecipitation reaction and is comparable to full-length E2f that does interact with Rbf (Bosco, 2001).
Previous work has shown that mutant follicle cells producing this truncated E2fi2 protein specifically localize ORC to the amplification regions as in wild type, but that such cells have elevated levels of ACE3 amplification. This elevated level of amplification is probably due to extra rounds of origin initiation events, suggesting that both E2f and Rbf have a negative regulatory function in origin firing during amplification. The DNA-binding domain of the truncated E2fi2 protein might be sufficient to localize ORC, if it could still interact with ORC. Therefore, whether or not the truncated E2fi2 protein complexes with ORC was tested. Immunoprecipitation experiments show that truncated E2fi2 does not interact with ORC. This means that the C-terminal domain of E2f is necessary for its interaction with ORC, and possibly requires Rbf to mediate this interaction. In contrast to the stated hypothesis, however, localization of ORC to the amplification region does not require a physical complex with E2f (Bosco, 2001).
Thus, the Drosophila E2f-Rbf complex functions during S phase, specifically to regulate DNA replication initiation at origins. It is thought that DP-E2f-Rbf are bound near ORC at the amplification origin and regulate initiation by forming a complex with ORC. Although E2f does not direct ORC binding, it restricts its activity through Rbf. Five lines of evidence form the basis for this model: (1) reduced levels of Rbf result in increased gene amplification levels and genomic replication without measurable effects on transcription of E2f target genes; (2) a complex of dDP-E2f-Rbf-ORC is present in ovary extracts; (3) this complex is independent of DNA binding; (4) truncation of the C terminus of E2f eliminates this complex, and (5) in this truncation mutant, ORC is localized but increased amplification occurs. The mechanism by which the dDP-E2f-Rbf complex limits replication initiation at the chorion locus remains to be determined. It is possible that the dDP-E2f-Rbf proteins inhibit the activity of the ORC subunits through a physical interaction. Alternatively, E2f-Rbf might inhibit loading of other replication factors at origins, such as MCM proteins. Finally, Rbf might alter the local chromatin configuration, for example by histone deacetylation, and thereby affect origin firing. Although ORC does not need to be in the E2f-Rbf complex to bind specifically to the chorion replication origin, a mutation in the DNA-binding domain of E2f does result in loss of ORC localization in the follicle cells. This observation needs to be evaluated in the context of the result that ORC is localized in the E2fi2 mutant, in which the truncated E2f protein is able to bind DNA but does not complex with ORC. Thus, DNA binding by E2f seems to be a prerequisite for ORC localization, but ORC localization does not require complex formation with E2f. This may be because when E2f is not bound to the chorion region, E2f2 can bind to sites at ACE3 normally occupied by E2f, and E2f2-Rbf may repel ORC and preclude localization or antagonize ORC binding activity (Bosco, 2001).
The Rbf mutant provides insights into the controls leading to the cessation of the endo cell-cycle during follicle cell development. Both the female-sterile Rbf mutant and the dDP female-sterile mutant show inappropriate continuation of the endo cell cycle beyond stage 10 of egg-chamber development. In contrast, an ectopic S phase does not occur in either of the female-sterile E2f mutants. Like the dDPa1 mutant, the Rbf120a/Rbf14 mutant is expected to have effects on both E2f-Rbf and E2f2-Rbf complexes. Thus, it seems that DP-E2f2-Rbf is needed to exit endo cycles, whereas DP-E2f-Rbf is involved more directly in regulating ORC and gene amplification. Identification of mutations in E2f2 will permit direct analysis of the roles of E2f2 in the endo cell cycle and amplification. Although it has not been shown whether any other specific replication origins may be regulated in this manner, the E2f-Rbf-ORC complex has been found in embryonic extracts, indicating that E2f-Rbf may be a general repressor of replication origins in embryonic tissues. Notably, a region between the DmPolalpha and E2f genes, containing several known E2f-binding sites, has been identified as a replication initiation region. Human RB (and associated HDACs) co-immunolocalize to BrdU foci in early S phase of primary cells, suggesting that RB may have a role in replication initiation. This observation is consistent with the model that suggests that Drosophila E2F1-Rbf localizes to replication origins and regulates ORC activity through a direct protein-protein interaction. It will be of great value to determine whether mammalian E2F-RB complexes can interact with ORC. Such an interaction would allow for a better understanding of how E2F and RB function to regulate DNA replication and cell proliferation during tumor progression (Bosco, 2001).
During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to
the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. This study examines the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. RBF-280 retains the wild-type RBF ability to inhibit transactivation by E2F1. Expression of RBF-280 in the developing eye reveals that RBF-280 does not inhibit G1/S transition in the second mitotic wave; rather, it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB and E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth shows that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the non-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs (Xin, 2002).
e2f1 null mutant eye discs undergo a second mitotic wave when the level of RBF is reduced, indicating that transcription activation by E2F1 is not required for S-phase entry in the second mitotic wave. There are two possible explanations for this observation: one possibility is that derepressed basal E2F target gene expression in the second mitotic wave is sufficient to drive S-phase entry. Alternatively, it is possible that cells in the second mitotic wave are driven into S phase through an E2F-independent mechanism. The results presented in this report suggest that S-phase entry in the second mitotic wave is probably driven by an E2F-independent mechanism, since overexpression of a non-regulated RBF inhibits E2F target gene expression but does not inhibit S phase there. These results are consistent with the observation that Hh signaling is required for S phase entry in the second mitotic wave through direct induction of Cyclin E. Since Hh signal is known for its role in neuronal differentiation and in pattern formation of the developing eye, it appears that developmentally regulated G1/S transition in the second mitotic wave is controlled by the same signal that also controls differentiation and pattern formation to coordinate the cell proliferation with differentiation. Although RB/E2F does not control the S-phase entry in this case, RB and E2F appear to be important for the rapid progression through S phase in the second mitotic wave. The observation that RBF-280 delays S-phase completion in the second mitotic wave and severely disrupts normal eye development indicates the importance of coordinating the rate of cell proliferation and differentiation in the developing eye (Xin, 2002).
Besides the observation that RB and E2F play important roles regulating S-phase progression in the developing eye, RB/E2F has also been shown to affect S-phase progression in developing embryos and wing discs. Thus, RB and E2F appear to regulate S-phase progression in multiple developmental settings. Similarly, RB and E2F have also been shown to regulate G1/S transition in a number of other developmental settings. The question is when do RB and E2F regulate G1/S transition and when do they regulate S phase progression? It appears that RB and E2F often play important roles in the G1 arrested cells (such as the G1 arrested cells in the embryos and in the eye discs) to prevent ectopic S phase entry. In these cases, Rb and E2F probably function through inhibiting the expression of Cyclin E. By contrast, developmentally regulated cell proliferation (G1/S transition) appears to be tightly linked to the developmentally regulated transcription of cyclin E, which is controlled by a large cis-regulatory region containing tissue- and stage-specific components. Temporal and tissue specific Cyclin E expression will drive cells into S phase and lead to the inactivation of RB and the coordinated E2F target gene expression, which might be required for the timely progression through S phase. The observation that RBF-280 expression inhibits E2F target gene expression and delays the completion of S phase supports a role for RB/E2F in S phase progression in the second mitotic wave (Xin, 2002).
There are at least two possible mechanisms that may contribute to the function of RBF in regulating S-phase progression. One mechanism is through the inhibition of E2F target gene expression besides cyclin E. Because several E2F target genes such as PCNA, RNR2, Orc1 and DNA polalpha are components of the DNA replication machinery, inhibition of E2F target gene expression may result in an insufficient amount of DNA replication machinery, which may delay the completion of S phase. A second possibility is that RBF may regulate DNA replication directly. Recently, it has been shown that the E2F1/RBF complex is localized to the DNA replication origin, and interacts with ORC proteins directly (Bosco, 2001). In addition, mammalian RB can interact with MCM7 and regulate DNA replication directly. Thus, it is possible that RBF can regulate S-phase progression directly by controlling firing at replication origins (Xin, 2002).
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).
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).
Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway, which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger, can reversibly suppress DNA synthesis and cell division. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however.
Transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors were used to address this issue. Manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. These data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway (Kuzin, 2000).
To regulate ectopic production of NO during development, transgenic lines of Drosophila were generated in which the expression of dNOS1 cDNA was controlled either by the heat-shock-inducible hsp70 promoter (hs-dNOS1 flies) or by the eye-specific GMR promoter, which functions in all cells of the eye imaginal disc in, and posterior to, the morphogenetic furrow [19] (GMR-dNOS1 flies). Examination of scanning electron micrographs of the eyes of, and thin sections of the retinas of, different transgenic lines did not reveal obvious differences among eyes of wild-type flies, transgenic hs-dNOS1 flies with or without heat-shock and GMR-dNOS1 flies. This indicates that a moderate increase in NO production on its own does not noticeably affect eye development (Kuzin, 2000).
To investigate the relationship between NOS activity and cell cycle progression, NOS activity was manipulated in transgenic flies ectopically expressing genes of the Rb pathway in the developing eye. Drosophila RBF is structurally related to the mammalian proteins of the Rb family and, like the Rb proteins, RBF is a negative regulator of cell cycle progression. The RBF transgene was placed under control of the GMR promoter and flies with either two (GMR-RBF2) or four (GMR-RBF4) copies of the transgene were used in these experiments. The eyes of adult flies with two copies of the RBF transgenes (GMR-RBF2) appear normal, indicating that at this dosage the RBF transgene does not noticeably disturb cell division in the eye disc. When GMR-RBF2 flies are crossed to hs-dNOS1 flies and the progeny larvae are treated with heat shock before pupariation, however, the resulting adults have multiple defects in the eyes, including missing bristles and pigment cells. Pigment cells, which comprise the boundaries of each ommatidia, appear as a characteristic honeycomb pattern in thin sections of normal eyes. A lack of the regular number of pigment cells in GMR-RBF2 + hs-dNOS1 flies results in the appearance of many fused ommatidia and a rough eye phenotype. Thus, hs-dNOS1 and GMR-RBF2 flies, both of which do not affect the development of eye structure when overexpressed on their own, nevertheless yield eye defects when overexpressed together. This transgenic interaction suggests that NOS and RBF genes interact synergistically during the development of ommatidia. A similar effect was observed employing a different genetic strategy. This time, the overexpression of dNOS1 was restricted to the developing eye by crossing GMR-RBF2 flies with GMR-dNOS1 flies. These double-transgenic flies also display eye defects similar to those of heat-shocked GMR-RBF2 + hs-dNOS1 flies ó missing pigment and bristle cells and fused ommatidia. Thus, regardless of the promoter that drives the expression of the dNOS1 transgene, elevated levels of NO and RBF synergize to limit cell number in the developing eye, supporting the notion of interaction between dNOS1 and RBF genes (Kuzin, 2000).
Both RBF and NOS act to suppress cell division. If indeed NOS acts in concert with RBF during eye development, then inhibition of NOS might suppress RBF function and restore the normal number and shape of ommatidia to GMR-RBF4 flie