In syncytial embryos E2F is uniformly distributed and loosely clumped around migrating nuclei. Around the time of cellularization and activation of zygotic gene expression, E2F mRNA shows a concentration of distribution in two distinct regions: a broad band at the posterior end of the embryo and a narrower (and sharper) band at the anterior end. The narrow anterior band is approximately coincident with the pro-cephalic neuroregion. By early gastrulation (stage 6), the anterior band become more restricted to two narrower regions and the broader posterior band forms a number of individual stripes of expression. By stage 12, RNA is scattered in cells in the ventral cord, the greatest levels occurring in the posterior brain region. By stage 13 expression is restricted to the brain. This pattern contrasts with that of DP, which at this stage in development, is expressed throughout the CNS (Hao, 1995).


dE2F is detected throughout the eye disc. A low level of dE2F is seen in the anterior portion of the disc. Protein staining increases anterior to the furrow and this eleveated level is maintained to the posterior of the disc. Strongest staining is seen in two stripes on opposite sides of the furrow. Individual nuclei that stain strongly posterior to the furrow may represent S phase cells (Brook, 1996).

Endogenous E2F falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated G1-S-transition in the eye disc. Ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Thus an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase, and the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis (Asano, 1996).

dE2F expression persists in post-mitotic cells of the eye disc of third-instar larvae. The loss of dE2F function in these cells causes a novel phenotype characterized by loss of photoreceptors and abnormal rhabdomere cell morphology (Brook, 1996).

Three discrete populations of neuroblasts in the larval brain show stereotypic temporal and spatial patterns of cell cycle arrest and activation. All three populations are quiescent upon larval hatching and begin cell division as specific times post hatching (ph): the central brain neuroblasts (CBNBs) begin division at about 8-10 h ph; the optic lobe neuroblasts (OLNBs) at about 10-12 h ph, and the thoracic neuroblasts (TNBs) at about 28 h ph. In contrast, the mushroom body neuroblasts (MBNBs) and the ventral lateral NBs divide continously from larval hatching through pupariation, and provide an internal control to differentiate between developmental regulation of proliferation and the process of cell division. The trol locus of Drosophila regulates the timing of neuroblast proliferation. In trol mutants, quiescent neuroblasts fail to begin division. The trol gene product is required specifically for initiation of OLBN and CBNB proliferation. Activation of OLNB and CBNB cell divsion by mutation of the proliferation repressor ana bypasses the requirement for trol, consistent with the hypothesis that trol is not required for the maintenance of the proliferative state. This trol mutation induced cell cycle arrest was investigated in order to examine trol function. Induced expression of cyclin E or DP/E2F in trol mutants results in normal levels of dividing neuroblasts, while cyclin B expression has no effect. cyclin E expression is lower in the trol mutant larval CNS as assayed by quantitative RT-PCR, suggesting that trol neuroblasts are arrested in G1 due to lack of Cyclin E. Neither cyclin E nor E2F expression can phenocopy ana mutations, indicating that arrest caused by lack of Trol is different from Ana-mediated arrest. Neither the induction of cyclin E nor DP/E2F is capable of bypassing Ana mediated repression. It is concluded that trol neuroblasts are arrested in mid-G1 stage (Caldwell, 1998).

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).

These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).

The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).

Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).

In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).

The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions and increases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).

Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).

The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).

Control of cell proliferation in the Drosophila eye by Notch signaling

Cell proliferation in animals must be precisely controlled, but the signaling mechanisms that regulate the cell cycle are not well characterized. A regulated terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila eye development, providing a model for the genetic analysis of proliferation control. This study reports a cell cycle checkpoint at the G1-S transition that initiates the SMW; Notch signaling is required for cells to overcome this checkpoint. Notch triggers the onset of proliferation by multiple pathways, including the activation of dE2F1, a member of the E2F transcription factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp. Notch is also required for the expression of Cyclin A in the SMW (Baonza, 2005).

This work identifies a new cell cycle checkpoint in the second mitotic wave and describes how intercellular signaling overcomes this checkpoint. Delta signaling to Notch triggers a progression from G1 arrest in the morphogenetic furrow into the S phase of the terminal mitosis. Two effectors of this Notch requirement have been identified, dE2F1 transcriptional activity and cyclin A expression. Although the data imply that at least one other target also exists, this is unidentified. The data preclude this additional factor from being Cyclin E. Previous work has identified a later SMW checkpoint, at the G2-M transition. Together, Notch and the EGFR therefore coordinately provide spatial and temporal control of the cell cycle in the SMW (Baonza, 2005).

These results led to a proposal of the following course of events. Notch is activated by the uniform band of Delta in all cells as they emerge from the morphogenetic furrow. Cells that are uncommitted thereby enter S phase, whereas cells that are part of the precluster are blocked from responding and remain in G1. It has been shown that the G1 arrest of precluster cells is dependent on EGFR activation, although the details of the mechanism remain unclear. One of the consequences of EGFR activation in precluster cells is the upregulation of Delta expression. Cells between the preclusters would therefore end up initially receiving low-level uniform Delta, later reinforced by the upregulated Delta in the adjacent preclusters. Together, these provide a robust and modulated activation of Notch in cells that will enter the SMW (Baonza, 2005).

It is emphasized that this work uncovers a normal developmental function for Notch signaling only in the control of a specific terminal mitotic cycle. The fact that clones of Notch and Delta mutant cells can be generated implies that they are not required for the earlier, unpatterned proliferation ahead of the morphogenetic furrow. Similarly, the ability to make clones in other imaginal discs indicates that there is no requirement for Delta/Notch signaling in most cell proliferation in Drosophila. Rather, this signal requirement, and the subsequent EGFR-dependent entry into mitosis, is superimposed upon normal controls in this regulated terminal mitosis. Moreover, the ability of Notch signaling to initiate S phase is restricted to a short period. Notch has other functions later in eye development, and it has been shown that later ectopic signaling does not lead to additional proliferation. Nevertheless, Delta-expressing clones in other tissues also hyperproliferate, suggesting that ectopic Notch activity has a wider ability to trigger inappropriate proliferation (Baonza, 2005).

The EGFR and Notch signal systems play distinct roles in regulating the SMW. After completing its preliminary role in maintaining cells in G1 arrest, EGFR signaling ensures that cells only undergo mitosis if they are adjacent to developing clusters, thereby matching the number of cells born with the number that will be required to complete ommatidial differentiation. In contrast, Notch initiates the whole process by regulating whether cells emerging from the morphogenetic furrow enter the SMW or remain arrested in G1 and start to differentiate (Baonza, 2005).

The secreted protein Hedgehog has a primary role in the forward movement of the morphogenetic furrow. Hedgehog also has an important function in initiating and coordinating the onset of the SMW, specifically the initiation of S phase. Hh and Dpp together lead to the expression of Delta in the furrow. Furthermore, Hh is essential for the expression of cyclin D and cyclin E in the morphogenetic furrow, whereas Cyclin E is the main cyclin that regulates S phase onset. These data imply that Hedgehog signaling activates several independent branches of the pathway that lead to the onset of S phase in the SMW. Incidentally, the observation that Cyclin E accumulates in Notch mutant clones, which lack dE2F1 activity, indicates that, at least in this context, Cyclin E is not sufficient to inhibit RBF and thereby activate dE2F1 activity (Baonza, 2005).

Cyclin A is best characterized as a mitotic cyclin, and its destruction is a key step in the completion of mitosis. An additional function in the onset of S phase in Drosophila remains enigmatic. Mammalian Cyclin A and its associated kinase Cdk2 can drive G1 cell extracts into S phase, and anti-Cyclin A antibodies can block S phase in injected cells. But, in Drosophila, S phase can proceed normally in the absence of Cyclin A and Cyclin A does not bind Cdk2. Nevertheless, when overexpressed, Cyclin A can overcome the lack of Cyclin E and allow cells to enter S phase. Furthermore, overexpression of the Cyclin A inhibitor Roughex blocks entry into S phase in embryos, and roughex mutants show precocious S phase entry in the SMW. Ectopic BrdU incorporation is observed in the eye disc when Cyclin A is misexpressed. The data indicating that Cyclin A is one of the targets of Notch signaling further support the idea that Cyclin A is part of the machinery that controls the onset of S phase in the SMW (Baonza, 2005).

Notch signaling in mammals, as in flies, is pleiotropic and context dependent. This is highlighted in human cancer, where Notch is oncogenic in a number of cases, particularly in hematopoietic neoplasms, but in other contexts has tumor suppressor functions. Moreover, although the current work highlights a proliferative function, it has been shown that Notch inhibits proliferation in the wing disc. Notwithstanding this caveat, it is striking that Notch activity can be hyperproliferative in humans and in Drosophila, and little is known about this proliferative response. It has recently been shown in the developing Drosophila central nervous system that Notch activity can maintain cells in a proliferative state by antagonizing the p21/p27 homolog Dacapo, thereby maintaining Cyclin E expression. Similarly, the dacapo gene is downregulated in response to Notch in the mitotic-to-endocycle transition in Drosophila follicle cells. This work describes a different mechanism: Notch signaling overcomes a G1-S checkpoint via the activation of universally conserved cell cycle components, RBF1, dE2F1, and possibly Cyclin A. Although tempting to speculate that these data may provide some insight into oncogenic mechanisms, it will be important to ascertain whether the particular relationships between Notch and the core machinery that triggers S phase is indeed conserved. In fact, the data imply that Notch probably also influences the mitotic cycle at other points. If the only role of Notch were to advance cells into S phase, they would simply arrest at the next checkpoint, G2-M. The fact that Notch activity leads to overgrowth therefore implies that Notch can also, directly or indirectly, drive cells through the subsequent G2-M checkpoint (Baonza, 2005).

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. This study describes a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. Roles for Jak-Stat and Notch signaling were also characterized. These studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma (Reddy, 2010).

Both normal development and homeostasis require that cells transition from proliferating undifferentiated cells to quiescent differentiated cells. Failure to undergo this transition results in tumor formation, whereas premature differentiation results in hypotrophy. Some tissues balance proliferation and differentiation by employing stem cells that divide asymmetrically to yield both a stem cell and a progenitor cell, which will then give rise to differentiated cells. Most of the Drosophila central nervous system develops in this way: individual cells within the embryonic ectoderm become specified as neural stem cells called neuroblasts (NBs), which divide asymmetrically to yield a neuroblast and a progenitor cell called a ganglion mother cell (GMC). By contrast, much of the vertebrate central nervous system initially develops from neuroepithelia (NE), sheets of epithelial neural progenitor cells that function as symmetrically dividing neural stem cells. This provides for rapid expansion of neural tissue, and then, as development proceeds, asymmetrically dividing progenitor cells arise, although the mechanisms that govern their appearance are not well understood. The optic lobe of Drosophila is unlike the rest of the Drosophila nervous system in that, akin to the vertebrate nervous system, it develops from NE. The optic lobe may thus serve as a model in which the powerful experimental approaches available in Drosophila can be used to investigate mechanisms that control the growth and differentiation of NE (Reddy, 2010).

At the end of larval development, the optic lobes comprise the lateral half of each of the two brain hemispheres, and are organized into lamina, medulla and lobula layers. The optic lobes originate from clusters of epithelial cells that invaginate from a small region on the surface of the embryo (the optic placode). During larval development, these cells separate into an inner optic anlagen (IOA), which will give rise to the lobula and inner part of the medulla, and an outer optic anlagen (OOA), which will give rise to the outer part of the medulla and the lamina. Initially, the IOA and OOA are composed entirely of NE cells, but during the third larval instar they begin to differentiate. Along the lateral margin of the OOA, NE cells undergo cell cycle arrest in G1, and then are recruited to differentiate into lamina neurons by signals from the arriving retinal axons. Along the medial margin of the OOA, a wave of differentiation sweeps across the NE from medial to lateral, converting NE cells into medulla NBs. These NBs divide perpendicularly to the plane of the neuroepithelium, and appear to follow a NB developmental program, giving rise to additional self-renewing NBs, and to GMCs, which ultimately give rise to neurons (Reddy, 2010).

The Fat-Hippo signaling pathway encompasses distinct downstream branches that regulate planar cell polarity and gene expression. Transcriptional targets of the pathway include genes that influence cell proliferation and cell survival, and consequently Fat-Hippo signaling is an important regulator of growth from Drosophila to vertebrates. The influence of Fat-Hippo signaling on transcription is mediated by a co-activator protein, called Yorkie (Yki) in Drosophila and Yes-associated protein (YAP) in vertebrates. Warts (Wts)-mediated phosphorylation and binding to cytoplasmic proteins negatively regulate Yki by promoting its retention in the cytoplasm. Wts is regulated in at least two ways: Wts kinase activity is promoted by Hippo; and Wts protein levels are influenced by Dachs. Upstream regulators of the pathway include the large cadherin Fat, and the FERM-domain proteins Merlin (Mer) and Expanded (Ex). Fat acts as a transmembrane receptor, regulated by the cadherin Dachsous (Ds), and the cadherin-domain kinase Four-jointed (Fj). The mechanisms that regulate Ex and Mer are not completely understood, but Ex localization can be influenced by Fat, and, in mammalian cells, Mer mediates an influence of contact inhibition on Hippo signaling. Genetic studies in Drosophila have also revealed that the relative contributions of pathway components can vary among different tissues (Reddy, 2010).

Optic NE cells proliferate during larval development, but aside from a requirement for the transcription factor DVSX1 (Erclik, 2008), how this proliferation is regulated is not understood. The progression of NE cells to medulla NBs in the OOA is antagonized by Jak-Stat signaling (Yasugi, 2008), but, aside from this, the regulation of this differentiation wave is not understood. This study demonstrates that Fat-Hippo signaling regulates the proliferation and differentiation of NE cells in the optic lobe. By contrast, Fat-Hippo signaling does not detectably influence the proliferation or differentiation of NBs or their progeny. A role is identified for Notch signaling in controlling the progression of NE cells to medulla NBs, and relationships are characterized between the Fat-Hippo, Jak-Stat and Notch signaling pathways. The results indicate that a transient pause in the cell cycle is needed for cells to transition from NE cells to NBs, and suggest a model in which a cell cycle arrest modulates Notch signaling by contributing to accumulation of Delta expression. The insights these results provide into the role of Fat-Hippo signaling in NE growth and differentiation in Drosophila are likely to be relevant to recently described roles of YAP in vertebrate neural development and medulloblastoma (Reddy, 2010).

The Fat-Hippo pathway has emerged as an important regulator of growth, but has not previously been implicated in neural development in Drosophila. The observation that expression of an activated form of Yki, or mutation of tumor suppressors in the pathway (i.e. fat, ex or wts), promotes growth, whereas mutation of yki impairs growth, identify a crucial role for Fat-Hippo signaling in regulating the proliferation of optic neural progenitor cells (i.e. NE). Indeed, expression of activated Yki can result in massive overgrowths that are taken up in folded sheets of NE, which push into the central brain, forming tumors of undifferentiated NE cells. Although the influence of Fat-Hippo signaling on NE growth parallels its influence on imaginal discs, the influence of Fat-Hippo signaling on NE differentiation does not, as clones of cells mutant for tumor suppressors in the pathway can differentiate cuticle in the head, thorax and abdomen (Reddy, 2010).

In contrast to the extensive overgrowth and suppressed differentiation of NE, NBs and their more differentiated progeny appear refractory to Fat-Hippo signaling. Developing tissues that are unaffected by Fat-Hippo signaling have not been well characterized. The restriction of Fat-Hippo signaling to the NE is matched by the preferential expression of several pathway components, but even when a constitutively activated form of Yki was expressed outside of the NE, neural development in the central brain was not obviously perturbed. Given the emerging importance of Hippo signaling in cancer, determination of what makes different cell types sensitive or resistant to activated Yki is an important direction for future studies (Reddy, 2010).

The progressive nature of NE to NB differentiation in the optic lobe, with different stages displayed in a spatial pattern, make it a sensitive system for investigating differentiation. The extent of delay associated with Fat-Hippo pathway tumor suppressors varied depending on strength of the mutations, which suggests that progression of NE to NB involves a balance of positive and negative influences. The silencing of Yki expression as cells differentiate further suggests that there is negative feedback of differentiation signals onto Yki, which might normally help to ensure a sharp transition between NE and NBs. When Yki activity is further elevated, by overexpression of activated Yki, a complete block in differentiation could be achieved. The observation that a complete block in differentiation could also be achieved by combining overexpression of wild-type Yki with a mutation that influences Yki phosphorylation (wts) is intriguing in light of observations that several human cancers are associated with an increase in levels of Yki expression, rather than a simple change in its localization or phosphorylation. Thus, it is suggested that the two-hit scenario observed in the optic lobe, in which both Yki activity and Yki levels need to be affected in order to transform cells permanently, could also be relevant to human tumors (Reddy, 2010).

This analysis of optic lobe development and the influence of Fat-Hippo signaling implies that a transient pause in the cell cycle is required for cells to transition from NE to medulla NBs, and that Fat-Hippo signaling influences differentiation via an effect on the cell cycle. This model is supported by several observations: there is normally a cell cycle pause along the edge of the outer optic anlagen NE; inhibition of Fat-Hippo signaling, or activation of Yki, impairs both this cell cycle pause and differentiation; and direct manipulation of multiple cell cycle regulators can delay NE differentiation. Although multiple cell cycle regulators appear to be involved in this cell cycle pause, this analysis implicates E2f1 as a key player. PCNA-GFP is downregulated at the edge of the NE, which indicates that E2f1 activity is low there. As E2f1 activity is negatively regulated by association with Rb, and Rb is negatively regulated by phosphorylation by Cdks, expression of CycD+Cdk4 is expected to increase E2f1 activity. Thus, the significant delay in differentiation observed when CycD+Cdk4 were co-expressed with E2F1+DP could all be due to increased E2f1 activity. Importantly, E2f1 is normally regulated by Fat-Hippo signaling in the optic NE, and E2f1 is functionally important for the influence of Fat-Hippo signaling on NE differentiation, because mutation of E2f1 suppressed the wts-mediated differentiation delay. A cell cycle pause also occurs in conjunction with a wave of differentiation that sweeps across the developing eye imaginal disc; however, direct manipulation of cell cycle progression did not affect the differentiation wave in the eye disc, nor does mutation of wts, hpo or sav affect differentiation of photoreceptor cells, even though it does prevent the normal cell cycle pause in the eye disc (Reddy, 2010).

The transition from NE to NB is regulated by Notch signaling, and the results of this study suggest a model in which high level expression of Dl at the edge of the NE autonomously inhibits Notch activation, resulting in upregulation of L(1)sc, which promotes NB fate. This model is supported by the observations that activation of Notch or mutation of Dl can inhibit NE differentiation. At the same time, high-level expression of Dl should enhance Notch activation in neighboring cells, which, as Dl is upregulated by Notch activation, would contribute to the progressive spread of elevated Dl expression across the NE. This simple model allows for the input of other pathways into NE to NB progression via effects on Dl expression, and indeed this appears to be the point at which Fat-Hippo and Jak-Stat signaling intersect with Notch. As a unifying model, it is proposed that a cell cycle pause facilitates the accumulation of the high levels of Dl expression needed to autonomously block Notch signaling, and thereby to upregulate the expression of proneural genes like L(1)sc. A possible mechanism for this hypothesized effect on Delta is suggested by the recent observation in vertebrate NE that Delta1 transcripts are unstable during S-phase. The hypothesis that the influence of Fat-Hippo signaling on differentiation is due to its effect on Dl expression also provides an explanation for the specificity of this phenotype, as Dl is not generally required for the differentiation of imaginal disc cells (Reddy, 2010).

Studies of homologues of Yki, Sd, Hpo and Wts in the chick neural tube identified influences on proliferation and differentiation (Cao, 2008). These studies identified effects on Sox2-expressing neural progenitor cells, but could not distinguish between effects on NE cells versus other neural progenitor cells. A recent study has also implicated YAP in Hedgehog-associated medulloblastoma. Vertebrate NE cells give rise to progenitor cells (e.g. radial glial cells and basal progenitors) that share with neuroblasts the ability to divide asymmetrically to give rise to both another progenitor cell and a more differentiated cell. Since this analysis of the Drosophila optic lobe indicates that Fat-Hippo signaling functions specifically to regulate the proliferation and differentiation of NE, it is suggested that YAP might also function specifically within NE cells in vertebrates. Notably, the observation that depending on the level of expression, Yki can delay rather than block differentiation, provides for the possibility that YAP-dependent tumors could nonetheless contain a mixture of NE cells and more differentiated cells. In Drosophila, each of the three upstream branches of the pathway (i.e. Fat-dependent, Ex-dependent and Mer-dependent, contribute to Yki regulation in NE. Studies in vertebrates have not addressed how the pathway is normally regulated, but Fat-, Ds- and Fj-related genes are all normally expressed in vertebrate NE, consistent with the possibility that they function there (Reddy, 2010).

Artificially slowing the cell cycle can promote precocious differentiation in the cortex, although in this context increasing cell cycle length was associated with a transition from proliferative to differentiative divisions of basal progenitors, which appear functionally similar to NBs rather than to NE cells. The differentiation of optic lobe NE cells into medulla NBs also differs from the general model of increasing cell cycle length causing differentiation, because NBs proliferate even more rapidly than NE cells, and thus this step is not associated with a general lengthening of the cell cycle, but rather a transient pause. Nonetheless, it is intriguing that, in the spinal cord, overexpression of CyclinD did not block differentiation, but did appear to transiently delay it, reminiscent of the delay in NE to NB progression that this study identified in the optic lobe. Moreover, CyclinD expression is regulated by Hippo signaling in the chick neural tube, and overexpression of CyclinD inhibits differentiation there. Although further studies are required to identify the CyclinD-sensitive mechanism in the vertebrate nervous system, the reported instability of Delta1 transcripts during S phase, together with the role of Notch signaling in maintaining NE progenitors in vertebrates and the analysis of NE differentiation and Dl expression in the Drosophila optic lobe, suggest that the possibility of a general influence of cell cycle progression on Notch signaling warrants further investigation as a contributor to the link between cell cycle progression and differentiation in the nervous system across different phyla (Reddy, 2010).

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

Effects of Mutation or Deletion

Embryos homozygous for null mutations of E2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. Cell cycles in the CNS of E2F mutants gradually decrease DNA synthesis after stage 12, instead of undergoing their normal proliferate during a rapid cycle of about 40 minutes. The CNS neuroblasts lack an obvious G1 or G2. In addition, histoblasts that normally undergo endoreduplication, bypassing mitosis and amplifying their DNA via a sequence of distinct S phases, also cease DNA replication in E2F mutants. Messenger RNAs coded for by DmRNR2, the gene for the small subunit of ribonucleotide reductase, and for PCNA, required for DNA synthesis, disappear in E2F mutants. Thus E2F is required in most cells for a G1-S transcription program and for G1-S progression (Duronio, 1995a).

The co-expression of dE2F and dDP disrupted normal eye development, resulting in abnormal patterns of bristles, cone cells and photoreceptors. dE2F/dDP expression causes ectopic S phases in post-mitotic cells of the eye imaginal disc but does not disrupt the onset of neuronal differentiation. Most S phases are seen in uncommitted cells, although some cells that have initiated potoreceptor differentiation are also driven into the cell cycle. The co-expression of baculovirus p35 protein, an inhibitor of cell death, strongly enhances the dE2F/dDP-dependent phenotype. Thus the elevation of E2F activity causes post-mitotic cells to enter the cell cycle, but these cells fail to proliferate unless rescued from apoptosis (Du, 1996).

A dominant mutation in the E2F gene, correlating with a strong reduction in the amount of transcript, enhances position-effect variegation. Overexpression of the gene in transgenic flies has the opposite effect of suppressing variegation. Thus there is a link between the dose of a transcriptional activator, which controls the cell cycle, and epigenetic silencing of chromosomal domains in Drosophila (Seum, 1996).

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

The E2F transcription factor, a heterodimer of E2F and DP subunits, is capable of driving the G1-S transition of the cell cycle. However, mice in which the E2F-1 gene had been disrupted developed tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that disrupt G1-S transcription late in Drosophila embryogenesis. Five mutations were identified in the dDP gene. The lines were selected on the basis that they fail to express the PCNA transcript in 8- to 15-hour embryos. The Ribonucleotide reductase 2 transcript is also affected in the mutant embryos (Royzman, 1997).

Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase still occurs. In mutant embryos the rate of replication is slowed and the developmental time of onset of the later polytene S phases appears to be delayed. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are necessary for viability; mutations in these genes cause lethality at the late larval/pupal stage. Although dE2F mutant animals survive through larval life, there is a dramatic delay in larval growth, which takes more than twice as long as for normal flies. Mutant larvae are very sluggish and much smaller than their wild-type counterparts. Polytene salivary glands and diploid imaginal discs could not be identified, presumably because they were so small. The brains are greatly reduced in size. These mutants exhibit melanotic pseudotumors, groups of cells within larvae that are recognized by the immune system and encapsulated in melanized cuticles. The eyes of dDP mutants show reduced size, missing and disorganized ommatidia, as well as stunted, missing, and disorganized bristles. Thus, the mutant phenotypes reveal that both genes promote progression of the cell cycle. It is suggested that continued S phases in mutant flies might be due to maternal pools of components of the replication machinery (Royzman, 1997).

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

Drosophila oogenesis makes it possible to examine aspects of DNA replication that are not readily apparent during embryogenesis. Ovarian follicle cells undergo a set of mitotic divisions before switching to an endo cycle (a cycle consisting of only S phase and a gap phase) and becoming polyploid. Genomic replication ceases after four endo cycles, but two genomic regions that contain clusters of chorion genes continue to replicate so that the chorion genes are amplified as much as 80-fold relative to genomic DNA. The chorion genes encode the eggshell proteins. Amplification of the chorion genes is needed to produce sufficient chorion protein for a normal eggshell, and amplification occurs by repeated rounds of initiation of DNA replication and fork movement to produce a gradient of amplified DNA extending ~100 kb. Mutants with reduced amplification have a phenotype of thin eggshells and female sterility. Chorion gene amplification appears to use components that are required normally for initiating DNA replication. Origin recognition complex (ORC) is a complex of six subunits and is required for initiation of replication. Mutations in the Drosophila orc2 gene disrupt amplification. Overexpression and inhibition studies indicate that cyclin E is needed for amplification also (Calvi, 1998). Because the levels of Cyclin E protein oscillate with genomic replication but remain constant in follicle cells undergoing amplification, it has been postulated that the high Cyclin E activity blocks genomic replication and that some mechanism permits the amplicons to escape this block to rereplication (Royzman, 1999 and references).

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

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

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

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

The fact that the levels and activity of Cyclin E are not affected in dDP or dE2F mutant follicle cells suggests that the role of Cyclin E in amplification is either parallel to or upstream of E2F. In evaluating the mechanism by which dDP and dE2F affect ORC localization and DNA replication it is useful to consider each of the three alleles and the distinct effects separately. There are two aspects of ORC localization: clearing of ORC uniformly present in the follicle cell nuclei and subsequent specific localization of ORC to the amplicons. The dDPa1 mutation has the most severe effect in reducing BrdU incorporation and produces eggs with the thinnest shells. In addition, in some egg chambers continued follicle cell polyploidization occurs in place of amplification. The fact that in all the dDP mutant egg chambers nuclear localization of ORC2 persists, and ORC2 is not detectable specifically at amplifying foci could indicate that amplification requires that ORC be cleared from genomic chromatin and assembled at amplification origins. There are two outcomes from persistence of genomic ORC localization. It either blocks amplification or in a minority of egg chambers permits continued genomic replication. The clearing of ORC from genomic origins may be linked to a global change that permits rereplication and amplification of those loci that retain ORC binding (Royzman, 1999).

The dE2Ffi1 mutants have less severe phenotypic effects. ORC is cleared from genomic origins but is not localized to amplification origins. The outcome of this appears to be that genomic polyploidization appropriately stops, but amplification is reduced. These effects also support the idea that ORC concentration at amplifying foci is needed for rereplication. It is proposed that the dE2Ffi1 defect is less severe than that of dDPa1 because a second dE2F gene exists that is able to compensate partially for the dE2F mutant protein (Royzman, 1999).

The absence of an effect of the dE2Ffi2 mutation on ORC localization is consistent with the fact that in this mutant, genomic replication ceases and amplification occurs. It is striking that amplification occurs earlier and has increased levels in mutant flies with a predicted truncated form of dE2F lacking the RB-binding domain. Thus restriction of the onset and extent of origin amplification may be regulated by E2F complexed with RB. It has been demonstrated that RB, when complexed to E2F, is capable of recruiting histone deacetylase and thereby converting chromatin to a compacted state. This state is correlated with inaccessibility to transcription factors, and it is reasonable to propose that it would also hinder binding of replication factors. Thus in this model, E2F in complex with RB would cause histone deacetylation in the vicinity of replication origins, leading to inhibition of amplification until stage 10B. The inability of dE2Fi2 protein to bind RB would prevent inhibition and result in premature amplification (Royzman, 1999).

The differences between the three mutations in the E2F subunits provides insights into the mechanism by which E2F may influence ORC localization. This effect could be direct or indirect. Both the dDPa1 and dE2Fi1 mutations are predicted to weaken E2F DNA binding. Thus the known E2F activities should be present but at reduced levels. For example, these two mutant proteins should retain transactivation activity and the ability to bind RB, repress transcription, and alter chromatin structure. Despite these activities, ORC foci are not detected, implying that the ability of E2F to bind DNA is crucial for its ability to influence ORC localization. This conclusion is supported by the fact that ORC is localized properly in the dE2Fi2 mutant in which the protein has a normal DNA-binding motif and is predicted to lack the transactivation and RB-binding domains (Royzman, 1999).

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

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

A genetic screen for modifiers of E2F in Drosophila

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16ink4a/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was use to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty three enhancer mutations of this phenotype were isolated by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma (brm), moira (mor), osa, pointed (pnt), and polycephalon (poc). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21CIP1 expression, indicating an important role for Polycephalon in cell-cycle control (Staehling-Hampton, 1999).

The molecular basis of the interaction between E2F and a BRM/MOR/OSA chromatin-remodeling complex is not yet clear and a range of possibilities exists. The genetic interaction may result from a direct physical interaction between RBF/E2F complexes and chromatin-remodeling machinery. In support of this idea the human homologs of BRM, hBRM, and BRG1 have been found to physically associate with pRB. This raises the possibility that BRM/MOR/OSA may help E2F/RBF repressor complexes bind to their target sites. This interpretation is supported by experiments from Trouche and co-workers who used transient transfection of mammalian cells to demonstrate that BRG1 can cooperate with pRB to repress E2F-dependent transcription (Trouche, 1997). Consistent with this model, the introduction of two copies of GMR-RBF into a GMR-dE2FdDPp35/+; brm-/+ background suppresses the enhancement by brm. Thus the effect caused by low levels of brm can be overcome by increasing the dosage of RBF. Additional evidence has been sought that would be predicted by this model; to date, however, these experiments have been inconclusive. BRM lacks the LXCXE motifs found in hBRM and BRG1, which have been suggested to mediate the interaction with pRB. To date no physical interaction between BRM and RBF or between BRM and dE2F has been detected. The interaction between endogenous pRB and hBRM or BRG1 proteins is hard to detect even in mammalian cells, and the failure to find BRM/RBF complexes may simply reflect difficulty in extracting chromatin-associated proteins under conditions that maintain the interaction (Staehling-Hampton, 1999).

An alternative possibility is that the BRM/MOR/OSA chromatin-remodeling complex is an important regulator of the expression of some key E2F-target genes, but this complex does not interact directly with either RBF or E2F. In this case the functional interaction occurs because these proteins converge on overlapping sets of promoters. This model is difficult to test because it is not yet clear which, and how many, E2F target genes are functionally significant. RNR2, one example of an E2F-dependent gene, is expressed normally in embryos mutant for brm, osa, or mor; no change in the expression of RNR2 in GMR-dE2FdDPp35 eye disks heterozygous for brm, osa, or mor alleles could be detected. While RNR2 expression is often used to provide an in vivo readout of E2F activity, experiments suggest that it is not a critical E2F target. The effects of brm, mor, and osa may only be evident at a subset of E2F-regulated promoters and an extensive screen of E2F targets will be necessary to find the appropriate gene (Staehling-Hampton, 1999).

It is possible that E2F and brm act in distinct pathways that influence cell-cycle progression. In this model the activity of a BRM/MOR/OSA-containing complex may have a function that influences the ability of E2F or RBF to control S-phase entry. Several observations have linked BRM-related proteins to cell-cycle control. brm null clones in the adult cuticle often show duplications of bristle structures, suggesting a possible role for brm in proliferation, and mice lacking the BRM homolog SNF2alpha show evidence of increased cell proliferation. Although brm, mor, and osa have no effect on the GMR-p21 phenotype, both brm and mor mutations have been isolated as suppressors of a hypomorphic cyclin E eye phenotype, demonstrating that brm and mor can affect other cell-cycle phenotypes in the eye. Other studies have shown that the activity of hSWI/SNF complexes is itself cell-cycle regulated. Transformation by activated Ras decreases the expression of the murine ortholog of hBRM in mouse fibroblasts, whereas growth arrest leads to an accumulation of protein. Recently, BRG1 and BAF155, a human ortholog of Moira, have been shown to associate with cyclin E and are suggested to be targets for cyclin E-dependent kinases during S-phase entry (Staehling-Hampton, 1999 and references).

During this study it was observed that GMR-dE2FdDP p35/+; brm-/+ eyes develop necrotic patches that increase in severity with the age of the adult fly. This raised the possibility that brm mutations might enhance the phenotype by promoting E2F-induced apoptosis. However, further experiments have failed to support this hypothesis. brm mutations fail to enhance the GMR-dE2FdDP phenotype, which has elevated levels of apoptosis, or to modify a GMR-rpr phenotype. In addition, brm mutations have no effect on the phenotype of animals in which GMR-rpr and GMR-hid-induced apoptosis is blocked by GMR-p35. No increase in the number of apoptotic cells is detected when GMR-dE2FdDPp35/+; brm-/+ third instar eye disks are stained with acridine orange (Staehling-Hampton, 1999).

The identification of pnt, Ras1, and rolled mutations as enhancers of an E2F overexpression phenotype suggests that there might be cross-talk between the signaling pathways downstream of receptor tyrosine kinases and the E2F pathway. The basis for this interaction is not clear and may be complex because both loss-of-function mutations (in Ras1 and pnt) and a gain-of-function mutation (in the rolled MAP kinase) enhance the E2F-dependent phenotype (Staehling-Hampton, 1999).

Of all the complementation groups isolated in this screen, polycephalon (poc) shows the most extensive effects on cell-cycle phenotypes. poc has yet to be cloned, but it was initially isolated as a modifier of Egfr pathway signaling. Poc mutations enhance the GMR-dE2FdDP phenotype in the absence of p35 and suppress the GMR-p21 phenotype. Poc is also the only enhancer mutation that can be clearly shown to alter the pattern of S phases in the eye imaginal disc. Partial restoration of the second wave of S phases in GMR-p21, +/poc- eye discs indicates that the level of poc is important for p21CIP1-mediated cell cycle arrest. Mutations in poc also altered the pattern of S phases in the second mitotic wave in eye discs of GMR-dE2FdDP larvae. Unlike wild-type and GMR-dE2FdDP/+ eye discs, in GMR-dE2FdDP, +/poc- eye discs, the intensity of BrdU staining does not drop off at the posterior of the second wave but instead remains elevated throughout. Other mutations have been reported to alter the second mitotic wave. In roughex (rux) mutants cells enter S phase prematurely and as a result the domain of S phases is expanded anteriorly toward the furrow. This is thought to be the first report of a mutation that affects the posterior domain of the second wave. A detailed investigation of the effect of poc on the cell cycle awaits the cloning of this gene (Staehling-Hampton, 1999).

This study demonstrates the use of a genetic screen in Drosophila to isolate genes that functionally interact with the E2F transcription factor. Four of six genes identified are known to regulate gene expression, and three encode components of a chromatin-remodeling complex. These results add to the emerging view that chromatin conformation is a key feature of E2F regulation and suggest that SWI/SNF complexes play an important role either in E2F regulation or in the control of S-phase entry. It is noted that this screen is not saturated and that many additional E2F interactors remain to be identified. Future studies will also be needed to identify the precise molecular basis for these genetic interactions and to determine whether the human counterparts of osa, moira, pnt, and poc also regulate E2F activity in mammalian cells (Staehling-Hampton, 1999).

E2F1 was identified in a screen for modifiers of Cyclin E function in Drosophila melanogaster

In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, a screen was carried out for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that were identified fall into four complementation groups corresponding to the genes split ends (spen), dacapo, dE2F1, and Cdk2(Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19) to be addressed. The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, the results presented here demonstrate that the equivalent sites in Cdk2 play no essential role. The demonstration that phosphorylation of Cdk2 on Thr 18 and Tyr 19 has no essential role during normal development does not exclude its involvement in subtle or stress regulation. Moreover, vertebrate cells, in which Cdk2 phosphorylation on Thr 18 and Tyr 19 has been demonstrated to occur, express A-type cdc25 phosphatases that have been implicated in Cdk2 dephosphorylation and that do not appear to exist in Drosophila (Lane, 2000).

The identification of mutations in Drosophila dE2F1 in this screen was expected on the basis of the large body of evidence demonstrating the tight functional relationship between Cyclin E and E2F/DP transcription factors. However, the fact that dE2F1 mutations result in enhancement rather than suppression of the Sev-CycE phenotype would not necessarily have been predicted since the results of genetic analysis in Drosophila so far have suggested that E2F/DP activity has a positive role in stimulating the transcription of S phase genes (Cyclin E, RNR2, DNA polalpha, PCNA, and Orc1) and cell proliferation. In contrast, the enhancement of the Sev-CycE phenotype observed with dE2F1 alleles points to a growth-suppressive role of dE2F1. Similarly, the E2F1 knock-out phenotype observed in mice has clearly demonstrated a tumor-suppressing function. Moreover, while vertebrate E2F/DP functions as a transcriptional activator in some promoters, it acts as a corepressor in conjunction with pRB in many other promoters. A decrease in E2F/DP levels, therefore, might also result in derepression of unknown proliferation-stimulating genes and synergy with ectopic Cyclin E expression (Lane, 2000 and references therein).

A role for ebi in neuronal cell cycle control: Mutations in ebi were isolated as enhancers of an over-proliferation phenotype generated by elevated E2F/DP activity in the Drosophila eye

Ebi promotes G1 arrest in certain cell types. It is suggested that, in some cell types, Ebi serves to coordinate cell exit with the onset of differentiation. Mutations in ebi were isolated as enhancers of an over-proliferation phenotype generated by elevated E2F/DP activity in the Drosophila eye. ebi alleles also strongly suppress a phenotype caused by the cyclin-dependent kinase inhibitor p21, restoring S phases in the second mitotic wave of the developing eye disk. Ebi physically interacts with Sina and Phyllopod, and Ebi promotes Ttk88 degradation in vitro and in S2 cells. Ectopic expression of Ttk88 inhibits differentiation in embryos and eye discs; however, this block to differentiation is insufficient to promote S phase entry in either of the situations where ebi mutations give this effect. Thus it is concluded that Ebi function limits S phase entry (Boulton, 2000).

An E2F/DP overexpression phenotype in the Drosophila eye has been used to screen randomly generated EMS and X-ray-induced mutations for alleles that are modifiers of E2F activity. The same GMR-dE2F-dDP-p35 chromosome was also used to screen a P-element collection for additional alleles that are important for this E2F-dependent phenotype. From this screen, the P-element P[w+; LacZ]K16213 was isolated as a dominant enhancer of the GMR-dE2F-dDP-p35 phenotype. K16213 enhances the general roughness of the eye, increasing the irregular arrangement of the ommatidial facets relative to the GMR-dE2F-dDP-p35 phenotype alone, and generating a large number of bristle duplications. K16213 is a lethal insertion that maps to position 21C on the left arm of chromosome II. K16213 was tested against mutant alleles that had been mapped to the 21C region and a complementation group of three alleles was found that failed to complement the P-element (CC1, CC3 and CC4). No escapers were observed from any of the possible trans-heterozygous combinations of K16213, CC1, CC3 and CC4, indicating that the mutated gene is essential for Drosophila development. Each of the three EMS alleles enhances the GMR-dE2F-dDP-p35 to a degree similar to that observed for the P-element. Excision of the P-element from K16213 reverted the lethality associated with this chromosome and eliminates its ability to modify the GMR-dE2F-dDP-p35 phenotype. Database searches using K16213 flanking sequence identified Ebi (Boulton, 2000).

Since ebi alleles has been isolated as enhancers of an E2F-dependent phenotype characterized by increased cell proliferation, an examination was carried out as to whether Ebi gene dosage can suppress eye phenotypes caused by a reduction in cell proliferation. Expression of the human cyclin-dependent kinase inhibitor p21 has been shown to strongly inhibit S phase entry in the developing eye disk, giving a rough eye phenotype characterized by reduced numbers of pigment cells and fused ommatidia. The GMR-p21 phenotype is shown here to be dominantly suppressed by mutations in Ebi. The number of ommatidia in the pGMR-p21/ebi adult eye is similar to wild type and, other than minor bristle defects, the pGMR-p21/ebi adult eye is morphologically normal (Boulton, 2000).

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DNA replication-related element factor or DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, transgenic flies were established in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induces ectopic DNA synthesis in the cells behind the morphogenetic furrow that are normally postmitotic, and abolishes photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype is suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, it was found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppresses and that reduction of Distal-less enhances the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed identification of several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).

Functional antagonism between E2F family members

E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, which have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and E2F transcription factor 2 (de2f2). de2f1 and de2f2 provide different elements of E2F regulation, and they have opposing functions during Drosophila development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. These defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs from de2f1;de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions (Frolov, 2001).

Studies in mammalian cells strongly indicate that several E2F family members share overlapping functions. If the Drosophila E2F proteins perform functions in vivo that are similar and overlapping, then de2f1; de2f2 double mutants would be predicted to be more severely affected than either the de2f1 or de2f2 single mutants. However, if dE2F1 and dE2F2 act antagonistically then this result is unlikely. Although several outcomes are possible, the simplest possibility to consider is that the double mutant might be less severely affected than the single de2f1 or de2f2 mutants (Frolov, 2001).

To answer this question, animals trans-heterozygous for null alleles of dE2F2 (de2f276Q1 and de2f2G5.1) and dE2F1 (de2f191 and de2f1rm729) were generated. Trans-heterozygous combinations of alleles were used to minimize the possible effects of any additional mutations that might be linked in cis. Embryos trans-heterozygous for de2f1 mutations lack the G1/S transcriptional program, show a dramatic reduction in DNA synthesis after stage 13, and hatch to give severely abnormal larvae. Five days after egg laying (AEL) de2f1 mutant larvae are much smaller than wild type. These animals fail to develop to the third larval instar and contain severely underdeveloped imaginal discs and CNS (Frolov, 2001).

Strikingly, the slow growth and abnormal larval development of the de2f1 mutant are almost completely suppressed by removing the dE2F2 activity. Trans-heterozygous double mutants, de2f276Q1/de2f2G5.1; de2f191/de2f1rm729, develop normally without any significant delay in larval growth, reach pupal stage, and finally die as mid- or late-pupae. The imaginal discs and CNS of de2f1;de2f2 double-mutant larvae are similar in development to wild-type primordia (Frolov, 2001).

de2f1 has previously been shown to be required for normal cell proliferation and DNA replication at several stages of Drosophila development. de2f1 mutant clones fail to proliferate in eye and wing imaginal discs. Surprisingly, in eye discs that lack both de2f1 and de2f2, the pattern of DNA synthesis is largely normal. In wild-type or in mutant discs, BrdU staining is localized primarily to the large area of asynchronously dividing cells in the first mitotic wave and in the synchronous second mitotic wave. Similar patterns are observed in de2f2 mutant discs. No BrdU incorporation is detected in the morphogenetic furrow, and even relatively long pulses of BrdU incorporation (2 h) fails to reveal a significant number of ectopic S-phases posterior to the second mitotic wave in the double-mutant discs. Staining for ELAV, a marker of committed neuronal precursors, shows that the onset of neuronal differentiation is not severely perturbed by the absence of E2F proteins (Frolov, 2001).

In examining large numbers of eye discs for these genotypes it was observed that the intensity of label incorporated into the most intensely labeled nuclei during long-term BrdU pulses tended to be slightly reduced in de2f1;de2f2 double mutants relative to wild-type discs. However this reduction is considerably less than the normal variation that occurs between discs of a single genotype. To further examine cell cycle progression in the absence of E2F regulation, de2f2;de2f1 double-mutant discs were double stained with anti-cyclin A and anti-phosphorylated histone H3 (phosH3) antibodies. Cyclin A is commonly used as a G2 marker because it is expressed in cells that have passed the G1/S transition and is degraded when cells enter mitosis. The phosH3 antibody preferentially stains M-phase cells. The patterns of Cyclin A and phosH3 staining in de2f2;de2f1 double-mutant eye discs were indistinguishable from wild type. Thus, cells of the eye imaginal disc can proliferate asynchronously in the first mitotic wave, arrest synchronously in the morphogenetic furrow, re-enter the cell cycle synchronously in the second mitotic wave, permanently exit the cell cycle, and differentiate, all in the absence of both dE2F1 and dE2F2 (Frolov, 2001).

It is concluded that the loss of de2f2 suppresses the larval growth, cell proliferation, and DNA-replication defects that are caused by the mutation of de2f1. This suppression confirms that dE2F1 and dE2F2 have antagonistic functions, at least during larval development. In theory, the effects of dE2F2 that are evident in the de2f1 mutant could be caused by an unusual dE2F2 activity that is independent of dDP. However, the loss of dDP function behaves in a similar way to loss of de2f2 and suppresses the developmental defects of de2f1 mutant larvae. Thus it is most likely that the severe defects seen in de2f1 mutant larvae require the action of dE2F2/dDP heterodimers (Frolov, 2001).

The relatively normal patterns of cell proliferation in de2f1;de2f2 mutants are, at first glance, difficult to reconcile with the idea that E2F is a critical regulator of gene expression and cell proliferation. The expression of de2f1-regulated genes was examined in de2f2 and de2f1; de2f2-mutant animals. Third instar eye discs were chosen for this analysis to avoid the possible contribution of maternally supplied products. Initially expression of PCNA, one of the best-characterized E2F-regulated genes, was monitored by in situ hybridization. In wild-type eye discs, the pattern of PCNA expression is tightly coupled to the pattern of DNA replication. High levels of PCNA transcripts are present anterior to the furrow and within a narrow stripe that overlaps with the second mitotic wave. No PCNA expression is present in the furrow and in the cells just anterior to the furrow. In de2f1;de2f2 double-mutant eye discs, PCNA expression is abnormal. In these animals, a weak staining is observed in mutant eye discs without any specific pattern. PCNA transcripts appear to be present at a low level in the anterior portion of the disc, including the morphogenetic furrow and the second mitotic wave. In de2f2 mutant animals, PCNA expression is not downregulated as cells enter the furrow, and this results in an expanded region of PCNA transcription that encompasses both the first and second mitotic waves and the morphogenetic furrow (Frolov, 2001).

To compare the overall levels of E2F-target genes expression, Northern blot analysis was performed using total RNA from third instar eye imaginal discs dissected from de2f2 single and de2f1;de2f2 double-mutant larvae. The steady state level of PCNA transcripts is elevated in the de2f2 mutant and declines in the double mutant to a level that is lower than the wild-type control. These results suggest that dE2F2 represses PCNA expression and that the elevated level of PCNA expression in the de2f2 mutant is due to the activity of dE2F1. The expression of MCM3, another proposed target of dE2F1, increases in the de2f2 single mutant and this effect is suppressed in the double mutant, but in this case the total amount of MCM3 RNA in the double-mutant discs is indistinguishable from wild type. Even though the pattern of RNR2 expression has been shown to be dependent on dE2F1, little change was seen in the total level of RNR2 transcripts in de2f1 mutant larvae or in de2f2, or de2f1;de2f2 mutant eye discs (Frolov, 2001).

It is concluded that both de2f1 and de2f2 are required for the normal pattern of PCNA expression. de2f2 is needed to repress PCNA expression in the morphogenetic furrow, whereas de2f1 is required for the high levels of expression in the first and second mitotic waves. Although the pattern of PCNA, MCM3, and RNR2 transcripts depends on de2f1 and/or de2f2, significant levels of each of these transcripts can be detected in the absence of both de2f1 and de2f2 (Frolov, 2001).

Because Cyclin E is one of the best-known targets of E2F and is rate limiting for S-phase entry the pattern of Cyclin E transcription was examined in the de2f2;de2f1 double-mutant animals. The normal pattern of Cyclin E expression is not altered in de2f2 mutant discs and a similar pattern is also evident in de2f2;de2f1 double mutants. However in the absence of both dE2F1 and dE2F2, the variations in Cyclin E expression are reduced and the pattern of expression is less distinct. Northern analysis shows that the steady-state level of Cyclin E transcripts is not decreased in the absence of E2F proteins. These results are consistent with evidence that de2f1 contributes to the pattern of Cyclin E expression but is not required for Cyclin E transcription. The finding that E2F target genes are expressed in de2f2;de2f1 double mutants may explain, at least in part, why normal cell proliferation is possible in the absence of E2F proteins (Frolov, 2001).

de2f1 provides an essential function in vivo. de2f1 mutants are defective during embryogenesis, show a significant delay in larval growth, and fail to complete larval development. de2f1 mutant embryos lack a G1/S transcriptional program that normally accompanies S-phase entry and loss of de2f1 leads to an almost complete cessation of DNA synthesis by stage 13 of embryogenesis. Analysis of de2f1 mutant clones in imaginal discs confirms that dE2F1 is required for normal cell proliferation and suggests that E2F also acts in postmitotic cells. Studies of partial loss-of-function alleles in the ovary have implicated E2F in the shut off of DNA synthesis in follicle cells and have shown that de2f1 is required in this cell type for amplification of chorion gene clusters (Frolov, 2001).

dDP mutant embryos resemble de2f1 mutants in lacking a G1/S transcriptional program, but the effects of dDP mutation on the expression of genes that are normally expressed at G1/S varies and depends on the target gene examined. Examination of dDP mutant clones and specific alleles of dDP shows that dDP is required during oogenesis and that it is required for the shut off of DNA synthesis in follicle cells. However, the patterns of DNA synthesis and cell proliferation are not severely affected in dDP mutant embryos or dDP mutant larvae, indicating that the functions of dE2F1 and dDP are not equivalent (Frolov, 2001).

The differences between the de2f1 and dDP mutant phenotypes have led to speculation that dE2F1 might have functions that are independent of dDP, or alternatively that dDP might have functions that are independent of dE2F1. One gene that is likely to have an impact on these phenotypes is de2f2, a de2f1-related gene that was uncovered by direct sequencing of transcription units within the 39B-D cytological region, in a two-hybrid screen using RBF1 as bait, and in the Drosophila genome project. Biochemical experiments have shown that dE2F2 can cooperate with dDP to generate specific DNA-binding activity on a consensus E2F-binding site and that a dE2F2 expression plasmid weakly repressed the transcription of an E2F-reporter construct when transfected into the Drosophila Kc cell line. Nothing is known about the normal function of dE2F2. This study shows that dE2F2 is a physiological partner for dDP and RBF and that dE2F2 acts antagonistically to dE2F1 during Drosophila development. Mutations in de2f2 relieve the block to DNA synthesis and larval development that is caused by mutation of de2f1. These results show that E2F-control of cell proliferation results from the interplay between two types of E2F complexes that have opposing activities and antagonistic functions (Frolov, 2001).

E2F is required for nurse cell DNA replication and apoptosis

During Drosophila oogenesis nurse cells become polyploid, enabling them to provide the developing oocyte with vast amounts of maternal messages and products. The nurse cells then die by apoptosis. In nurse cells, as in many other polyploid or polytene tissues, replication is differentially controlled and the heterochromatin is underreplicated. The nurse cell chromosomes also undergo developmentally induced morphological changes -- from being polytene, with tightly associated sister chromatids, to polyploid, with dispersed sister chromatids. Female-sterile dE2F1 and dDP mutants have been used to assess the role of the E2F cell cycle regulator in oogenesis and the relative contributions of transcriptional activation versus repression during nurse cell development. E2F1 transcriptional activity in nurse cells is essential for the robust synthesis of S-phase transcripts that are deposited into the oocyte. dE2F1 and dDP are needed to limit the replication of heterochromatin in nurse cells. In dE2F1 mutants the nurse cell chromosomes do not properly undergo the transition from polyteny to polyploidy. It is also found that dDP and dE2F1 are needed for nurse cell apoptosis, implicating transcriptional activation of E2F target genes in this process (Royzman, 2002).

Whether the activity of the E2F transcription factor is needed for transcription of known E2F target genes in the nurse cells was tested by examining transcript levels of PCNA, ORC1, and RNR2 by in situ hybridization. All three of these transcripts are present at background levels until stage 9 of egg chamber development. The transcripts are induced at high levels in stage 10. Staining of egg chambers with antibodies against dE2F1 protein show that the level of protein increases at the same time that E2F targets are expressed. The dE2F1i1, dE2F1i2, and dDPa1 mutations all caused a significant reduction, although not elimination, of these transcripts. To compare the transcript levels more accurately in the mutants, the in situ hybridizations and histochemical staining were done for ORC1 with dDPa1/CyO controls and dDPa1/Df mutant ovaries in the same tube. The mutant ovaries were distinguished from sibling controls by their smaller size and by the dumpless phenotype. In this experiment it was clear that the level of ORC1 transcript is significantly reduced in dDPa1 mutants. The mutations in dDP and dE2F1 do not generally block transcription at stages 9 and 10 of egg chamber development, because the level of cyclin E transcript is not detectably altered by these mutations. A reduction in the level of RNR2 transcript is observed in germline clones of dDP mutants (Royzman, 2002).

Reduction of the level of Cyclin E protein can result in inappropriate replication of the heterochromatin in nurse cells. Given that there was no effect of the dDP and dE2F1 mutations on cyclin E transcript levels, it seems unlikely that the E2F1 mutant effects are solely due to decreased Cyclin E. To examine this possibility further, Cyclin E protein level in the nurse cells was examined by antibody staining. Varying levels of Cyclin E protein were found in wild-type nurse cells within individual egg chambers. Nurse cell nuclei were observed with high and low levels of Cyclin E protein. The level of Cyclin E protein in the 16th cell of each egg chamber, the developing oocyte, is always high. In dDP and dE2F1 mutant nurse cell nuclei and oocyte, Cyclin E protein is readily observed and the level of Cyclin E protein is similar to wild-type. Thus, the induction of cyclin E appears not to be affected by dDP and dE2F1 mutations in this developmental context, and the E2F mutant defects are not likely to be solely the consequence of reduced Cyclin E. In addition, dDP and dE2F1 mutant nurse cell nuclei with high and low levels of Cyclin E were observed. It is likely, therefore, that the levels of Cyclin E properly oscillate with the S-G endo cycle in the mutants (Royzman, 2002).

If the dE2F1/dDP complex has a direct role in repressing heterochromatin replication, it could act either by affecting S-phase replication patterns or by altering the chromatin in heterochromatic domains. A weak allele of cyclin E has been shown to permit replication of the heterochromatin, and it has been proposed that reduction in the oscillations of Cyclin E lead to longer S phases in the nurse cells that permits heterochromatin to be replicated. Such a mechanism could account for the dDP and dE2F1 mutant phenotypes. Although no reduction of Cyclin E protein or transcripts was detected in the mutants, it is possible that levels are reduced enough to affect late replication in S phase. Because overexpression of Cyclin E also causes inappropriate heterochromatin replication in the nurse cells it is difficult to test whether the E2F1 effects are the consequence of reductions in Cyclin E . The abundant induction of cyclin E transcripts in dE2F1 and dDP mutant stage 10 egg chambers shows that in the nurse cells there is an E2F1-independent mechanism for activating cyclin E transcription. Evidence has been obtained for such a mechanism in the embryonic nervous system, and the cyclin E promoter has been shown to be complex, with many regulatory elements responding to developmental control (Royzman, 2002).

An alternative to E2F1 affecting the properties of S phase is that the heterodimer is responsible for conferring the proper chromatin configuration onto heterochromatin that blocks its replication after endo cycle six. The mammalian Rb/E2F complexes have been shown to bind to several types of proteins that affect chromatin structure, including histone deacetylases, histone methyltransferases, and chromatin remodeling complexes. One of these associated proteins, HP1, is known to control heterochromatin in Drosophila. Furthermore, mutations in dE2F1 have been shown to affect position effect variegation, a property of heterochromatin structure. In follicle cells the RBF/E2F1/DP complex binds at origins with the Origin Recognition Complex and limits origin firing, so it is conceivable that a similar complex could block origin activity in the heterochromatin in later nurse cell endo cycles (Royzman, 2002).

Mutations in Rbf do not reveal a role for this protein in inhibiting replication of heterochromatin or controlling chromosome morphology. Germline clones of Rbf lay normal mature eggs that develop until late in embryogenesis, and a female-sterile allele with reduced levels of Rbf does not visibly affect nurse cell chromosomes. However, the Rbf protein could be stable or present at sufficient levels in these mutants. In addition, a second Rb homolog exists in Drosophila, and this protein could play essential repressive roles in nurse cell differentiation (Royzman, 2002).

The mechanism by which dE2F1 controls nurse cell chromosome morphology remains to be elucidated. There are three possibilities. (1) dE2F1 may not be directly involved in the polyteny-polyploidy switch, and the defects observed in the mutants could be a relatively nonspecific effect of disrupting nurse cell differentiation. (2) The failure to properly regulate heterochromatin replication could alter chromosome structure and affect the transition to polyploidy. (3) dE2F1 could have a direct role in triggering the change in chromosome morphology, either itself affecting chromatin structure or via one of its transcriptional targets. The transition from polytene to polyploid morphology appears to involve the induction of mitotic activities and must be precisely controlled by activity of the anaphase-promoting complex/cyclosome (APC)Anaphase Promoting Complex or Cyclosome. Given the myriad of cell cycle genes that can be regulated both negatively and positively by mammalian E2F proteins, E2F targets could control these transitions in chromosome structure. Further investigation will be required to distinguish these possibilities (Royzman, 2002).

These experiments extend the previous observations on the role of dDP in nurse cell apoptosis by uncovering a requirement for dE2F1. This is relevant to mammalian work in which the activating E2F1 and E2F3 proteins have been shown to contribute to both p53-dependent and p53-independent apoptosis. It is significant that both dE2F1i1, which blocks DNA binding, and dE2F1i2, which binds DNA normally but cannot transactivate or bind Rbf, prevent apoptosis. In mammalian cells overexpression of transactivation defective E2F-1 mutants but not E2F-1 DNA-binding mutants can induce apoptosis in cell culture. Further, RB overexpression inhibits apoptosis induced by wild-type E2F-1 and RB-binding competent E2F-1 mutants. These observations have led to the notion that DNA binding rather than the transactivation activity of E2F-1 is necessary for its apoptotic function, and that apoptosis is the result of alleviation of RB-E2F-1 repression of apoptotic target genes. The phenotypes exhibited by the dE2F and dDP mutants do not support this model. Rather, the Drosophila mutants strongly implicate transcriptional activation of E2F targets as being crucial for developmentally-induced apoptosis. If repression were critical, then apoptosis should have occurred in the dE2F1i1 mutants in which dE2F2 is expected to repress all E2F targets. The cell death activators reaper and hid are not required for nurse cell apoptosis, so it is not yet clear which E2F targets could activate apoptosis in these cells (Royzman, 2002).

Cell cycle-dependent and cell cycle-independent control of transcription by the E2F/RB pathway

To determine which E2F/RB-family members are functionally important at E2F-dependent promoters, RNA interference (RNAi) was used to selectively remove each component of the dE2F/dDP/RBF pathway, and the genome-wide changes in gene expression that occur when each element is missing. The results reveal a remarkable division of labor between family members. Classic E2F targets, encoding functions needed for cell cycle progression, are expressed in cycling cells and are primarily dependent on dE2F1 and RBF1 for regulation. Unexpectedly, there is a second program of E2F/RBF-dependent transcription, in which E2F2/RBF1 or E2F2/RBF2 complexes repress gene expression in actively proliferating cells. These new E2F target genes encode differentiation factors that are transcribed in developmentally regulated and gender-specific patterns and not in a cell cycle-regulated manner. It is proposed that E2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, E2F/RBF-mediated repression is exerted on genes that encode an assortment of cellular functions, and these effects are reversed on sets of functionally related genes in particular developmental contexts. As a result, dE2F/RBF regulation is used to link gene expression with cell cycle progression at some targets while simultaneously providing stable repression at others (Dimova, 2003).

The results challenge the dogma that E2F-regulated genes have cell cycle-regulated patterns of expression. At Group E promoters, E2F2 and RBF proteins provide a repressor activity that is uncoupled from cell cycle progression, and the loss of E2F-mediated repression results in the inappropriate expression of tissue-specific genes and markers of differentiation (Dimova, 2003).

ChIP experiments illustrate two clear-cut differences between the promoters of Group E genes and the more conventional, cell cycle-regulated E2F targets. The first distinction lies in the recruitment of the activator E2F vs. E2F1. E2F2, DP, RBF1, and RBF2 are readily detected at most E2F-dependent genes, and at each of the different groups of E2F targets uncovered in this study, E2F1 is conspicuously and specifically absent from Group E promoters. This specificity does not, at first glance, appear to be due to a simple distinction in the types of E2F binding sites. Computer searches revealed multiple E2F-like binding sites upstream of Group E genes, but each of these variants could also be found in Group A and Group B promoters. It seems likely therefore that the specific recruitment of E2F proteins is influenced by selective interactions with other factors). The absence of dE2F1 at Group E promoters provides a simple mechanism to explain why these promoters escape the cell cycle-regulated burst of dE2F1-mediated activation that occurs during G1/S progression (Dimova, 2003).

Based on these results, a revised view of E2F regulation in Drosophila is presented. It is suggested that dE2F2-repressor complexes occupy the promoters of a diverse variety of genes. Such dE2F2-mediated repression is relieved at particular subsets of genes in response to cues that may come from developmental signals or from cell cycle signals. At cell cycle-regulated, E2F-controlled promoters, the transcriptional activation is mediated by dE2F1, and this switch from repression to activation is likely to involve Cdk-mediated disruption of the repressor complexes. However, dE2F1 fails to target other dE2F2-repressed genes, and the repressor complexes remain stable. Based on the restricted expression patterns of Group E genes, and the failure to detect dE2F1 at Group E genes even when dE2F2 is removed, it is proposed that dE2F2/RBF-mediated repression is relieved at these targets by developmentally regulated signals, and that gene expression is driven by factors other than dE2F1. The notion that not all E2F-regulated genes are expressed at any one time raises the question of whether the set of targets that are induced by activator E2Fs in cycling cells is fixed or variable. Recent studies of mammalian E2F proteins show that the recruitment of activator E2Fs to a promoter involves synergistic interactions with adjacent transcription factors. It is therefore easy to imagine how the expression of genes that have the potential to be induced by activator E2Fs might also be tailored in different cellular situations to favor different subsets of targets (Dimova, 2003).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Loss of dE2F compromises mitochondrial function

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

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

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

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

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

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

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

E2F: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.