Cyclin E



Cyclin E is an essential regulator of S phase entry. Transcriptional regulation of Drosophila Cyclin E plays an important role in the control of the G1 to S phase transition during development. The first comprehensive analysis of the transcriptional regulation of a G1 phase cell cycle regulatory gene during embryogenesis is presented here. Analysis of deficiencies, a genomic transformant and reporter gene constructs all reveal that Cyclin E transcription is controlled by a large and complex cis-regulatory region containing tissue- and stage-specific components. Separate regulatory elements for transcription in epidermal cells during cell cycles 14-16, central nervous system cells and peripheral nervous system cells were found. An additional cis-regulatory element drives transcription in thoracic epidermal cells that undergo a 17th cell cycle when other epidermal cells have arrested in G1 phase prior to terminal differentiation. The complexity of Cyclin E transcriptional regulation argues against a model in which Cyclin E transcription is regulated simply and solely by G1 to S phase transcription regulators such as RB, E2F and DP. Rather, this study demonstrates that tissue-specific transcriptional regulatory mechanisms are important components of the control of cyclin E transcription and thus of cell proliferation in metazoans (Jones, 2000).

This analysis has revealed the presence of multiple independent tissue-specific regulatory elements acting to control DmcycE transcription during embryogenesis. These include: (1) at least two different elements required for expression in different cells of the peripheral nervous system; (2) an element required for expression in the epidermal cells and central nervous system, and (3) an element required for expression in patches of thoracic epidermal cells that undergo a 17th G1 phase-regulated cycle, in the PNS of the labial and maxillary segments and in the posterior spiracle primordia. These elements were identified by the presence or absence of tissue-specific expression in a genomic transformant, in animals carrying small regulatory region deletions generated by P-element excisions and in genomic fragment-reporter gene constructs. All these regions are detected upstream of the zygotic transcript, within the large region coding for three 5' exons and large introns of the maternal transcript. The results obtained with each of these different approaches are consistent with the others in terms of the location of the different elements. Members of one notable group of elements that were not fully defined in this study are those responsible for Cyclin E expression in endoreplicating tissues. Only one element was identified that is responsible for driving Cyclin E transcription in the central midgut. Presumably elements responsible for regulating the remaining patterns of DmcycE in embryonic endoreplicating tissues lie further 5' or 3' of the genomic regions examined in this study. The failure to define more than the one endoreplicative regulatory sequence is surprising. It is possible that other regulatory sequences lie outside the region studied, but it is also possible that identification of these domains may be hampered by the negative autoregulatory nature of Cyclin E expression in these endoreplication domains. If the elements carrying the endoreplication regulatory sequences also carry the autorepression sequences, expression may be inhibited by the endogenous Cyclin E. It should also be noted that additional complexity is likely to exist in the regulation of Cyclin E transcription. For example, Cyclin E was expressed in a subset of the epidermal thoracic patch cells in Cyclin E 19L deficient embryos, indicating that these patches may themselves be complex domains of Cyclin E transcriptional regulation (Jones, 2000).

Although these analyses have identified separable tissue-specific cis-acting elements in the Cyclin E regulatory region, the factors that operate on these elements to drive Cyclin E transcription remain to be elucidated. Some clues to the trans-acting factors that could be regulating Cyclin E transcription come from studies of cell cycle control mechanisms in mammalian cells, where cyclin E transcriptional regulation also plays an important role in control of the G1 to S phase transition. A well-established model of G1 phase transcriptional regulation in mammalian cells postulates a cascade of events initiated by extracellular growth factor signaling that leads to activation of the Cyclin D/Cdk4 complex, which in turn phosphorylates the tumor suppressor Retinoblastoma (Rb), disassociating it from the S phase-specific transcription factors E2F and DP and allowing transcription of S phase-specific genes, such as cyclin E (Jones, 2000).

In Drosophila, where G1 phase-regulatory events can be studied within a developmental context, E2F and DP are required for embryonic Cyclin E transcription in endoreplicating tissues, but are dispensable for Cyclin E transcription in the CNS divisions that lack G1 and G2 phases. It is not known whether E2F and DP are required for Cyclin E expression in the early embryonic PNS and epidermal divisions, because maternal products of these genes may mask any requirement. Nonetheless, the fact that these transcription factors are dispensable for Cyclin E transcription in the CNS cells argues against the universality of the mammalian model of cyclin E regulation by E2F. In addition, the mammalian model predicts that E2F-dependent transcriptional regulation of Cyclin E in endoreplicating tissues should be mediated through an E2F/DP responsive element. In the experiments described here, this model is shown to be an oversimplification of Cyclin E regulation in Drosophila, as only one element in the 16 kb analysed was found to drive Cyclin E expression in a subset of endoreplicating tissues. Thus a minimum of two regulatory elements are required for driving Cyclin E expression in endoreplicating tissues. The fact that expression of Cyclin E in these tissues has been shown to be dependent on the E2F/DP complex appears at first sight to be paradoxical. The paradox would be resolved if expression requires activation by both E2F/DP and tissue-specific activators. An absolute requirement for a developmental signal cannot however exist, as ectopic expression of E2F and DP together induces Cyclin E expression in all G1-arrested epidermal cells. It remains possible that the high levels of E2F and DP expressed following heat shock induction of the respective transgenes override the tissue-specific regulatory component (Jones, 2000).

Downregulation of Cyclin E transcription during cycle 16 is essential for cycle 17 G1 arrest in epidermal cells prior to differentiation. Significantly, the regulatory element that drives constitutive Cyclin E expression in the epidermal cells during cycles 14-16 shows transcriptional downregulation, characteristic of wild-type Cyclin E expression. If this downregulation requires active repression of Cyclin E transcription, then the regulatory sequences necessary for this repression must also be located in the 1.0 kb regulatory element defined in this study. Alternatively, the downregulation could be a consequence of the loss of activation of Cyclin E transcription in the epidermis. Cyclin E is known to be ectopically expressed in a subset of normally G1-arrested epidermal cells in embryos that are deficient for RBF (the Drosophila Rb homolog). However, Cyclin E transcription is initially downregulated normally and a G1 cycle 17 arrest is established in the absence of RBF. These data suggest that Cyclin E is actively repressed in G1-arrested epidermal cells and that RBF is required to maintain this repression. Although dE2F or dDP do not result in ectopic Cyclin E transcription in epidermal cells, a RBF/E2F/DP complex may still mediate this repression if maternal E2F and DP are not depleted in the respective mutant embryos at this stage. A transcriptional repression mechanism of Cyclin E at this stage may also be mediated by the second Drosophila E2F, E2F2, which has been shown to act as a transcriptional repressor of S phase genes in tissue culture cells. Alternatively, E2F and/or DP may be needed to activate ectopic Cyclin E expression in the absence of RBF. The initial mechanism acting to downregulate Cyclin E transcription remains to be determined. Further dissection of this regulatory element may identify separate activation and repression sequences (Jones, 2000).

The regulators of DmcycE transcription in other developmental contexts may be more difficult to identify, since many candidate genes may be involved. In some cases it is possible to suggest the involvement of particular regulatory genes. For example, the discrete expression of DmcycE in the epidermal thoracic patches, which undergo a G1-regulated 17th cell cycle, is much stronger in the first thoracic segment and is absent in the abdominal segments. The products of the homeotic genes of the Bithorax and Antennapedia Complexes, which are key components of anterior/posterior patterning are therefore candidate upstream regulators of Cyclin E transcription in this tissue (Jones, 2000).

Understanding of cell cycle regulation has primarily derived from the single cell yeast, from cultured cells and from oocytes or very early embryos in which the complex patterning of embryogenesis has not yet begun. In contrast, cell cycles in the embryos of a multicellular organism respond to a variety of developmental cues that give different tissue types and different cell cycle kinetics. The significance of the relationship between embryo patterning and cell cycle control is evident from the pioneering work that has shown that regulation of the cycles that occur during gastrulation and germ band extension in Drosophila embryos is mediated by the transcriptional regulation of the string mitotic activator gene. This transcriptional regulation is mediated by the patterning genes active during these stages. The stg mitotic inducer gene exhibits a remarkably complex transcriptional regulatory region that responds to a variety of patterning genes to control cell cycle progression during early embryogenesis. The analysis of Cyclin E regulation offers an interesting parallel with stg in that both genes exhibit unexpected complexity in transcriptional regulation. It is striking that both Cyclin E and stg, the first cell cycle regulators to have their transcriptional regulation examined in a developmental context, contain tissue-specific regulatory elements. This indicates that transcriptional regulation is mediated differently in distinct developmental contexts, such as during the complex events of gastrulation when cycles 14-16 occur or during the 17th cycle in cells of the epidermal thoracic patches (Jones, 2000).

Why is DmcycE transcriptional regulation so complex? One clue comes from the observation that the G1 regulators cyclin D, cyclin E, Rb and E2F/DP are highly conserved between insects and mammals, but have no specific orthologs in yeast. Perhaps these genes represent a specialized mechanism that evolved to deal with G1 phase regulation in multicellular organisms. Complex transcriptional regulation of cell cycle genes such as Cyclin E may be part of such a mechanism, which permitted the universal eukaryotic cell cycle regulatory genes to be brought under the influence of the more recently evolved transcriptional regulatory mechanisms operating in different tissues and at different developmental stages (Jones, 2000).

Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. This study shows that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. A model is proposed whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes (Anderson, 2010).

The initiation of mRNA transcription involves the assembly of a transcription preinitiation complex (PIC), which as a minimum includes RNA polymerase II (RNAP II), Mediator, and six general transcription factors (TFIIA, -B, -D, -E, -F, and -H) at the core promoter DNA region. PIC assembly is initiated by the binding of the TATA box binding protein (TBP) subunit of TFIID to the promoter, which is stabilized in the presence of TFIIA and Mediator. Subsequently, TFIIB binds to and stabilizes the TFIIA-TFIIB-Mediator-DNA complex and functions as an adaptor that recruits the preformed RNAP II-TFIIF complex to the promoter. TFIIE and TFIIH then join to form the complete PIC (Anderson, 2010).

Once the PIC has been assembled on the promoter, transcription initiation occurs in several steps, which involve extensive phosphorylation of the C-terminal domain (CTD) of RNAP II. Early on in the transition from preinitiation to elongation, phosphorylation of Ser5s in the CTD heptapeptide repeats takes place, and this depends on the activity of the TFIIH-associated kinase cyclin-dependent kinase 7 (Cdk7; mammals)/Kin28 (yeast). Subsequently, Ser2s are phosphorylated by the elongation phase kinase Cdk9 (mammals)/CTDK-1 (yeast) to generate elongation-proficient RNAP II complexes. Another Cdk, Cdk8, can negatively regulate RNAP II transcription, partially via its inhibitory effect on Cdk7 activity. More recently, it has been suggested that Cdk11p110 regulates RNAP II transcription in humans. Thus, Cdk11p110 binds to hypo- and hyper-phosphorylated RNAP II, and antibody-mediated repression of Cdk11p110 activity results in inhibition of RNAP II transcription (Anderson, 2010 and references therein).

In addition to the phosphorylation events that control RNAP II activity, modification of the chromatin structure represents an important mechanism for regulating gene expression. When the chromatin is in its repressed state, the DNA is wrapped tightly around the histones, creating a barrier to the assembly of the RNAP II PIC at the promoter region. Activation of gene expression is associated with a number of histone modifications that loosen the chromatin structure, including acetylation, methylation, ubiquitylation, and phosphorylation. Histone H3 and H4 acetylations are particularly frequent toward the 5' ends of actively transcribed genes and presumably facilitate the initial assembly of the PICs at the promoter region. MOF (males absent on first) is a histone H4 lysine 16 (H4K16)-specific histone acetyltransferase (HAT) in both mammals and Drosophila. MOF is part of several complexes, including the Drosophila male-specific lethal (MSL) complex, which is required for X chromosome dosage compensation, the mammalian counterpart of the MSL complex, and the MOF-MSL1v1 complex, which mediates p53 acetylation at K120. In addition, MOF copurifies with a number of other proteins, such as the forkhead-associated (FHA) domain-containing protein MCRS2, NSL1-3 (for nonspecific lethal 1 to 3), and MBD-R2, as part of the NSL complex (Anderson, 2010).

This study focuses on the function of Drosophila MCRS2 (dMCRS2), the Drosophila ortholog of human MCRS2 (also known as MSP58). Whereas human MCRS1 and -2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 is largely uncharacterized. In addition to the recent observation that human and Drosophila MCRS2s form complexes with MOF (Cai, 2010; Prestel, 2010; Mendjan, 2006; Raja, 2010), several other reports suggest that MCRS1 and -2 proteins could function in transcription regulation via interactions with the transcriptional repressor Daxx or the basic region leucine zipper factor Nrf1 (Anderson, 2010).

Drosophila MCRS2 and its human homologue, MCRS2, are 59% identical, with the highest level of homology being in the FHA domain. Whereas dMCRS2 is largely uncharacterized, MCRS1 and -2 have been linked with a variety of cellular processes, RNAP I-dependent transcription, transcriptional repression, and cell cycle control, though these functions remain poorly understood (Anderson, 2010).

This study shows that dMCRS2 is an essential nuclear protein required for cell cycle progression and growth during development. The data show that dMCRS2 physically associates with Cdk11 and RNAP II and colocalizes with RNAP II PICs on polytene chromosomes in vivo. Consistent with this, dMCRS2 is required for optimal binding of RNAP II components to the cyclin promoter regions and for normal levels of cyclin gene expression (Anderson, 2010).

The demonstration of colocalization of dMCRS2 with RNAP II on numerous sites on polytene chromosomes is in agreement with a recent ChIP-seq analysis, which revealed that dMCRS2 is present on the promoters of over 4,000 genes, correlating with 55% of active genes (Raja, 2010). Furthermore, gene expression profiling studies show that dMCRS2 depletion elicits the downregulation of over 5,000 genes. This essential function as a broad-specificity transcriptional regulator is reflected by the extreme growth defect of dMCRS2-depleted cells both in vivo and in cell culture and in the fact that dMCRS2 has been recovered as a hit in RNAi screens for diverse cellular functions such as centrosome maturation and Hedgehog signaling (Anderson, 2010).

In accordance with its pleiotropic function, dMCRS2 can be purified with a number of proteins, from NSL components to members of the RNAP II machinery. Moreover, dMCRS2 colocalizes with RNAP II PICs on polytene chromosomes in vivo, suggesting that it may regulate an early step in the recruitment and/or assembly of RNAP II PICs. This is consistent with the majority of dMCRS2 binding to the promoter regions of autosomal and X-linked genes and the fact that dMCRS2 is required for the loading of RNAP II components to cyclin gene promoters. Thus, dMCRS2 appears to be an important transcriptional regulator, and the data represent the first evidence for a physical connection between dMCRS2 and the core transcriptional machinery. While the results suggest that dMCRS2 associates with RNAP II complexes via protein-protein interactions, future studies will need to establish the exact molecular nature of this connection (Anderson, 2010).

Interestingly, MCRS2 and dMCRS2 copurify with the MOF HAT independently of the dosage compensation MSL complex. Furthermore, it was observed that dMCRS2 coimmunoprecipitates and colocalizes extensively with MOF on polytene chromosomes. MOF, as well as binding to the 3' ends of MSL targets along the male X chromosome, is also found on numerous promoter regions, both on the X chromosome and on autosomes in both sexes. Since MCRS2 also binds to promoters, it is possible that dMCRS2 and MOF could function in concert in transcriptional regulation. However, despite the evidence that MOF regulates a broad range of both X-linked and autosomal genes, no physical connection between the putative dMCRS2-MOF NSL complex and RNAP II complexes has been established so far. This study shows that both dMCRS2 and MOF associate with core RNAP II complexes in cultured cells (Anderson, 2010).

dMCRS2 may promote transcription by different mechanisms. Through its HAT activity, dMCRS2-associated MOF may create a relaxed chromatin state favorable to PIC assembly, either by inducing the physical weakening of DNA/histone or histone/histone interactions or by promoting the recruitment of bromodomain-containing factors. dMCRS2 may also induce PIC formation by recruiting the preformed RNAP II/TFIIF complex and/or promoting transcription elongation through the recruitment of CK2 and the FACT complex, which facilitates transcription elongation by remodeling chromatin. However, whether these different dMCRS2-containing complexes regulate common target genes or whether they represent distinct transcriptional regulators remains to be investigated. In summary, a model is proposed where dMCRS2 binds to multiple sites along the chromosomes and promotes the recruitment of RNAP II PICs to target genes (Anderson, 2010).

Wingless signaling directly regulates cyclin E expression in proliferating embryonic PNS precursor cells

Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. This study further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and shows that multiple sequence elements are required for the full cyclin E PNS enhancer activity. Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation (Deb, 2008).

The results reveal that cyclin E expression in developing PNS precursor cells is regulated by a large enhancer containing multiple sequence elements, including two TCF-binding sites that mediate the regulation by Wg signaling. While these TCF-binding elements are essential for the activity of the PNS enhancer, proximal and distal elements in the 4.6-PNS sequence appear to be important for full activity. The importance of Wg in the regulation of the PNS expression of cyclin E is supported by the fact that wg mutant embryos displayed decreased cyclin E expression in the developing PNS cells. This reduction in cyclin E expression in wg mutant embryos was accompanied by an inhibition of BrdU incorporation in the developing PNS, and an inhibition of the determination of the Pros and Elav expression cells in the developing PNS. It is possible that the block in differentiation into the Pros and Elav positive cells is a consequence of the inhibition of cyclin E expression or perturbations to the cell proliferation. However it is also possible that the observed differentiation block in PNS is due to a function of Wg that is independent of PNS cell proliferation. Further studies will be needed to resolve this issue (Deb, 2008).

In addition to wg, a number of other mutations such as achaete/scute (ac/sc) complex and da have also been reported to block PNS precursor proliferation and affect the expression of several cell cycle genes. Ac/Sc complex proteins and Da are bHLH proteins that are important in all aspects of es-PNS precursor differentiation while bHLH protein Atonal (ato) and Da are required for all aspects of ch-PNS precursor development. Recent studies of the expression of the Cdk inhibitor Dap during cell type specification revealed that Dap expression is directly regulated by the same developmental mechanisms that control the differentiation of these cell types. Therefore it will be interesting to test if bHLH proteins such as Da also directly regulate cyclin E expression in the developing PNS cells (Deb, 2008).

Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in the Drosophila central nervous system

Homeotic/Hox genes are known to specify a given developmental pathway by regulating the expression of downstream effector genes. During embryonic CNS development of Drosophila, the Hox protein Abdominal-A (AbdA) is required for the specification of the abdominal NB6-4 lineage. It does so by down regulating the expression of the cell cycle regulator gene cyclin E (CycE). CycE is normally expressed in the thoracic NB6-4 lineage to give rise to mixed lineage of neurons and glia, while only glial cells are produced from the abdominal NB6-4 lineage due to the repression of CycE by AbdA. This study investigated how AbdA represses the expression of CycE to define the abdominal fate of a single NB6-4 precursor cell. Both in vitro and in vivo, the regulation was examined of a 1.9 kb CNS-specific CycE enhancer element in the abdominal NB6-4 lineage. CycE was shown to be a direct target of AbdA and it binds to the CNS specific enhancer of CycE to specifically repress the enhancer activity in vivo. These results suggest preferential involvement of a series of multiple AbdA binding sites to selectively enhance the repression of CycE transcription. Furthermore, the data suggest a complex network to regulate CycE expression where AbdA functions as a key regulator (Kannan, 2010).

All progenies of both thoracic and abdominal NB6-4 can be traced using Eagle (Eg) as a lineage marker, and Reversed polarity (Repo) for differentiating glial cells. Thus, Eg-only expression marks neuronal fate. The thoracic variant of NB6-4 (NB6-4t) gives rise to both neuronal and glial cells, whereas the abdominal variant (NB6-4a) gives rise to only glial cells. The Hox gene Antennapedia (Antp) is expressed in the NB6-4t lineage of thoracic segments (T1-T3) whereas abdominal A (abdA) and Abdominal B (AbdB) are expressed in the NB6-4a lineage of abdominal segments A1-A6 and A7-A8, respectively. However, loss of Antp function does not affect the lineage development in contrast to loss of abdA or AbdB, which results in NB6-4a to NB6-4t homeotic transformations. Thus, thoracic identity of NB6-4 lineage acts as a default state without the requirement of any Hox gene input, while abdominal identity of the lineage is imposed by the function of abdA and AbdB. AbdA and AbdB function by suppressing the expression of CycE, a cell cycle molecule necessary for G1-S phase transition. This study has focused on the mechanism by which AbdA regulates CycE expression (Kannan, 2010).

Exd and AbdA cooperatively bind as a heterodimer to a consensus DNA sequence. During development, nuclear localization of Exd is regulated by interaction with another homeodomain protein Homothorax. The expression pattern of the cofactors Exd and Hth was examined in NB6-4 lineages of wild-type embryos. Exd expression was detected in glial precursors of the NB6-4a lineage, Interestingly, it was not found in the NB6-4t lineage, although in the ectoderm expression levels of nuclear Exd are higher in thoracic segments than in the abdominal segments. In the case of Hth, the protein was detected in NB6-4a glial cells only after late stage 11, and also weakly in NB6-4t derived glia. Thus, consistent with their requirement to modulate the function of Hox proteins, Exd was found expressed in the NB6-4a lineage and not in NB6-4t lineage, although weak expression of Hth was detected in glial cells of NB6-4t lineage. Assuming that both are required together to modulate Hox function, modulation of Hox function is predicted only in NB6-4a lineage (where abdA and AbdB are expressed) and not in NB6-4t (where Antp is expressed). Indeed, loss of abdA and AbdB show NB6-4a to NB6-4t transformations, while loss of Antp has no phenotypic consequence (Kannan, 2010).

To gain additional evidence on the relevance of exd and hth expression in the NB6-4a lineage, their loss of function mutations were analyzed. exd mutant embryos showed an increase in the number of NB6-4a progeny. Some of these cells migrated medially in a pattern similar to glial cells of NB6-4t, while others migrated to the dorso-lateral cortex, suggesting neuronal identity. Abdominal hemisegments of hth mutant embryos did not show an increase in glial progeny, but generated ectopic neurons in the dorsal lateral cortex suggesting homeotic transformation of NB6-4a to NB6-4t. The fact that mutations in both the cofactors of AbdA independently induced homeotic transformations, although at much lesser degree (11% in exd mutants and 7% in hth mutants, compared to 100% in abdA mutants), suggests that this observation is a phenocopy of the abdA loss of function phenotype. The mildness of the phenotypes could be an indication of their role as cofactors to enhance the effect of AbdA rather than essential factors to regulate cell-fate specification (Kannan, 2010).

Next the expression pattern of CycE transcripts was investigated in the transformed abdominal NB6-4 lineage in exd mutant embryos. Consistent with the phenotype at the cellular level, CycE mRNA was observed exclusively in neuronal cells of transformed NB6-4a (Eg expressing cells) in exd mutant background as is the case for the thoracic NB6-4. In hemisegments that show no transformation and thus represent the wild-type NB6-4a, absence of CycE mRNA was found (Kannan, 2010).

The complex cis-regulatory region of zygotic CycE comprises of tissue and stage specific activator and repressor elements within an at least 10 kb genomic region including upstream and downstream elements. Based on the expression pattern of a 1.9 kb lacZ reporter gene (CycE-lacZ) in transgenic assays, it is evident that this region includes cis-acting sequences that drive zygotic CycE transcription both in epidermis (mitotic cycles 14-16) and CNS and regulatory elements responsible for CycE down regulation at the end of st11. Similar to CycE transcripts and CycE protein, CycE-lacZ is not expressed in NB6-4a and is activated in abdA, AbdB double mutant embryos. Thus, this 1.9 kb lacZ reporter reliably reflects the CycE expression in the abdominal NB6-4 lineage (Kannan, 2010).

To elucidate the mechanisms that AbdA specifies in collaboration with Exd/Hth, the 1.9 kb CNS-specific CycE regulatory element for known AbdA, Exd and Hth-binding sites. The element harbours at least 3 binding sites for AbdA, and one each for Exd and Hth in close association. Since strong repression of β-Gal expression from the 1.9 kb CycE-lacZ regulatory element was observed in the NB6-4a lineage, it was wondered whether this regulation could be due to the presence of AbdA and Exd/Hth-binding sites. Therefore, the 1.9 kb CycE-lacZ fragment in more detail both in vitro and in vivo (Kannan, 2010).

Binding of AbdA, Exd and Hth to the regulatory sequences of CycE was tested by electro-mobility shift assays (EMSAs) on three spatially separated AbdA binding sites named AbdA-1, -2 and -3. AbdA-2 is in close association with binding sites of cofactors Exd and Hth, named as 68 bp fragment. EMSA suggested physical association of AbdA protein with all the three putative binding sites (AbdA-1, -2 and -3) when tested independently with corresponding oligosequences. In addition, the association of AbdA, Exd and Hth complex was observed in the 68 bp fragment. To check whether the putative AbdA, Exd and Hth sites identified within the CycE enhancer are responsible for assembling the complex, the core sequences that make critical contact to each of these factors was mutated. Mutations in Hox binding sites resulted in the loss of association of AbdA, Exd and Hth to the core sequence. These results suggest cooperative binding of AbdA and its cofactors Exd and Hth to the CycE enhancer element (Kannan, 2010).

To test the functional relevance of binding of AbdA, Exd and Hth in vivo, reporter gene constructs were constructed, with presence or absence of either of three AbdA binding sites, upstream of a minimal promoter driving β-Gal expression. Transgenic flies were generated by P-element mediated transformation (Kannan, 2010).

Embryos homozygous for lacZ transgenes (two independent insertion lines for each transgene to rule out position variation effects) were stained for Eg, Repo and β-Gal to visualize the regulatory behaviour of the CNS-specific CycE element in the abdominal NB6-4 lineage. The enhancer elements deleted independently for putative binding sites AbdA-1 and AbdA-2 drive β-Gal expression in abdominal NB6-4 cells, suggesting the preferential requirement of both sites for transcriptional repression of CycE. In contrast, the transgene deleted for AbdA-3 showed the wild-type 1.9 kb CycE-lacZ expression pattern i.e. no expression in NB6-4a, suggesting that AbdA-3 may not be a preferred binding site for repressive activity. As expected, regulatory elements deleted for both AbdA-1 and AbdA-3 or AbdA-2 and AbdA-3 drive lacZ expression in abdominal NB6-4 progenies. However, deletion of both the repressor elements abdA1 and abdA2 (CycE-lacZAbdA-1&2) did not result in de-repression of lacZ in NB6-4a. Interestingly, deletion of all the three elements resulted in the activation of lacZ in NB6-4a. This suggests that AbdA-3 may act as a cryptic repressor, functional only in the absence of both AbdA-1 and AbdA-2. In addition, while deletion of AbdA-2 alone resulted in the activation of β-Gal expression in NB6-4a, CycE-lacZ68bp element deleted for Exd/Hth and AbdA-2 mimicked wild-type β-Gal expression pattern suggesting that in its absence, AbdA-1 and AbdA-3 may maintain repression of CycE in NB6-4a (Kannan, 2010).

These results do not rule out the possibility of other sequences in the regulatory region of CycE that contribute to the AbdA-mediated repression. The fact that lacZ is strongly repressed in CycE-lacZAbdA-1&2 and CycE-lacZ68bp embryos, but de-repressed in CycE-lacZAbdA-1,2&3, CycE-lacZAbdA-1&3 and CycE-lacZAbdA-2&3 embryos, suggest that other regulators may function together with this enhancer in vivo. There is a possibility that this regulatory region is between AbdA-1 and AbdA-2, which is required to assemble a repressor complex. Computational screening of the 1.9 kb enhancer fragments revealed the existence of at least 3 Engrailed (En)-binding sites. Two of the En sites are between AbdA-1 and AbdA-2. It is likely that a minimum of two AbdA binding sites along with this regulatory region is required to assemble a repressor complex that also involves Exd/Hth and probably En. When either AbdA-1 or AbdA-2 is deleted, this repressor complex fails to assemble and hence leads to activation of CycE-lacZ in NB6-4a. In the absence of both AbdA-1 and AbdA-2, the putative En-binding region may come closer to AbdA-3 and still be able to assemble a repressor complex. Interestingly, repression of lacZ was observed when the whole 68 bp region is deleted. This could also be due to the fact that AbdA-3 is now much closer to the putative En-binding sites. Unfortunately, this could not be tested in the background of loss of function of en since NB6-4 itself is not born in those embryos. Nevertheless, the above mentioned model appears to be identical to the way Dll expression is repressed in the epithelial cells, which is mediated by Ubx and En. Further investigation in this direction involves Chromatin immunoprecipitation for AbdA or En followed by Western blot analyses for the other protein under different conditions (Kannan, 2010).

To conclude, these results suggest the preferential involvement of a series of multiple AbdA binding sites for enhanced repression of CycE transcription. These data suggests a complex network to regulate CycE expression where AbdA functions as a key regulator. This may have evolved to ensure tight repression of CycE as it is a potent regulator of cell fate in NB6-4 and possibly other CNS lineages (Kannan, 2010).

Transcriptional Regulation

cyclin E expression at G1-S requires E2F, activation of E2F without Cyclin E is not sufficient for the induction of S phase. Early in G1, ectopic expression of Cyclin E alone can bypass E2F and induce S phase. Cyclin E is the downstream gene that couples E2F activity to G1 control. However it appears that S phase has a requirement for Cyclin E that is independent of transcription.

However, not all embryonic cycles are similarly coupled to E2F activation. The rapidly proliferating CNS cells that exhibit no obvious G1 express Cyclin E constitutively and independently of E2F. Instead, Cyclin E expression activates E2F in the CNS. Thus, this tissue-specific E2F-independent transcription of Cyclin E reverses the hierarchical relationship between Cyclin E and E2F. Both hierarchies activate expression of the full complement of replication functions controlled by E2F with this caveat: whereas inactivation of E2F can produce a G1 when Cyclin E is downstream of E2F, an E2F-independent source of Cyclin E eliminates G1 (Duronio, 1995b).

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 has been disrupted develop tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that disrupt G1-S transcription late in Drosophila embryogenesis and five mutations in the dDP gene were identified. Sequencing of dDP reveals the presence of several important motifs, including the DNA-binding region, the DEF box that is predicted to be required for DP/E2F heterodimerization, and three other highly homologous regions named DP-conserved box 1 (DCB1), DCB2, and negatively charged box (NCB). Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The mutant phenotypes reveal that both genes promote progression of the cell cycle (Royzman, 1997).

The striking observation from the Drosophila dDP and dE2F mutants is that although cyclic transcription of cyclin E, PCNA, and ribonucleotide reductase 2 (RNR2) is not detectable, S phase still occurs. Although the possibility that cyclic transcription of these genes occurs at a low level driven by maternal pools of dDP and dE2F cannot be excluded, the bursts of transcription that normally precede S phase are not essential for the G1-S transition. In these mutants the cell cycle may be driven by basal levels of transcripts and post-transcriptional regulation. The maternal pools of components of the replication machinery can persist until late in development, as evidenced by the fact that mutations in PCNA and MCM2 cause late larval lethality (Royzman, 1997 and references).

E2F 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).

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. 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).

Neurons and glia are often derived from common multipotent stem cells. In Drosophila, neural identity appears to be the default fate of these precursors. Stem cells that generate either neurons or glia transiently express neural stem cell-specific markers. Further development as glia requires the activation of glial-specific regulators. However, this must be accompanied by simultaneous repression of the alternate neural fate. The Drosophila transcriptional repressor Tramtrack is a key repressor of neuronal fates. It is expressed at high levels in all mature glia of the embryonic central nervous system. Analysis of the temporal profile of Tramtrack expression in glia shows that it follows that of existing glial markers. When expressed ectopically before neural stem cell formation, Tramtrack represses the neural stem cell-specific genes asense and deadpan. Surprisingly, Tramtrack protein levels oscillate in a cell cycle-dependent manner in proliferating glia, with expression dropping before replication, but re-initiating after S phase. Overexpression of Tramtrack blocks glial development by inhibiting S-phase and repressing expression of the S-phase cyclin, cyclin E. Conversely, in tramtrack mutant embryos, glia are disrupted and undergo additional rounds of replication. It is proposed that Tramtrack ensures stable mature glial identity by both repressing neuroblast-specific genes and controlling glial cell proliferation (Badenhorst, 2001).

The absence of Ttk69 from replicating glia implies that ectopic expression of Ttk69 may block normal glial development by inhibiting cell cycle progression. Whether ectopic expression of Ttk69 blocks entry into S-phase was examined. BrdU incorporation is inhibited by ectopic expression of Ttk69 using the Kr-Gal4 driver. In Ttk69-expressing segments of a Stage 10 embryo, the normal BrdU incorporation in the ventral neuroectoderm is inhibited. In the embryonic nervous system, entry into S phase is driven by bursts of transcription of S-phase cyclins -- specifically cyclin E. Heat-shock induced overexpression of Ttk69 blocks zygotic transcription of cyclin E. At earlier stages, maternally deposited cyclin E transcripts are unaffected by Ttk69 overexpression, indicating that Ttk69 affects transcript synthesis rather than stability, consistent with its role as a transcription repressor. Ectopic expression of Ttk69 was induced by a 1 hour heat-shock, after which embryos were processed immediately for in situ hybridization. The rapidity of repression of cyclin E and the presence of multiple Ttk69 consensus recognition sites in the cyclin E promoters both suggest that repression is direct (Badenhorst, 2001).

It is clear from the current study that, at least in some glial populations, Ttk69 has the ability to regulate proliferation. In the Drosophila glioblasts Ttk69 is expressed at low levels soon after glial specification, blocking neural genes. This expression is not constant, though, but oscillates during the cell cycle. Significantly, Ttk69 can not be detected when glia enter S phase and commence DNA replication. Like neuroblasts, glioblasts appear to delaminate in G2 of the cell cycle. The timing of BrdU incorporation indicates that immediately after mitosis they enter S phase and undergo DNA replication. Although Ttk69 is expressed in glia in G2, Ttk69 is not detected in glia that incorporate BrdU. Since Ttk69 can repress cyclin E expression, the absence of Ttk69 allows replication to occur. Once replication is complete, Ttk69 is expressed again. The BrdU incorporation experiments indicate that the LGB undergoes three synchronous cell divisions to produce eight longitudinal glia. This agrees well with a recent estimate of between 7-9 longitudinal glia obtained by DiI labeling of the longitudinal glioblast. After the third mitosis, longitudinal glia do not undergo replication but instead express higher levels of Ttk. By inhibiting cyclin E, high levels of Ttk69 would keep glia in G1 of the cell cycle. Similarly, differentiation of oligodendrocytes and Müller glia is accompanied by high levels of the cyclin-dependent kinase inhibitor p27, blocking re-entry into the cell cycle (Badenhorst, 2001).

Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. To understand how the proneural and neurogenic genes pattern the response to EGFR activation, the expression of genes involved in transduction of the pathway was analyzed. The orphan nuclear-receptor svp functions downstream of the EGF receptor to promote cell divisions in the tubules. In the absence of Svp function, cycE and stg transcription is abolished, with a consequent reduction in EGFR-driven cell divisions. These late divisions in the tubules of stage 12 wild-type embryos were followed and it was found that BrdU incorporation (and hence, cell division) is confined within the svp-lacZ domain. These results define the svp domain of expression as including those cells which will divide in response to Egfr activation. However, the expression of svp-lacZ is initiated in a group of cells surrounding the tip mother cell, before the birth of the TC. This early onset of svp expression occurs before the late divisions start (cycle 17 onwards), when neither Svp function nor Egfr activation is required for cell proliferation. The pattern of gene expression observed suggests that the Svp-positive cells surrounding the tip mother cell derive from the proneural cluster (Sudarsan, 2002).

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, salvador (sav), a gene that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

Elevated levels of Cyclin E protein are found in the basal nuclei of sav clones posterior to the MF. These are the nuclei of the undifferentiated cells that continue to proliferate in sav clones. Such discs were examined for levels of cyclin E RNA. When sav clones are generated using eyFLP, a large proportion of cells in third instar discs are mutant, and these discs contain large patches of mutant tissue. In wild-type discs, cyclin E RNA is expressed in a narrow stripe immediately posterior to the morphogenetic furrow. In discs containing sav clones, the stripe of expression is broader and more intense, indicating that cyclin E RNA levels are elevated in these discs. Thus, the increased level of Cyclin E protein is likely to result, at least in part, from an increase in cyclin E RNA levels (Tapon, 2002).

Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E

Although mutations that activate the Hedgehog (Hh) signaling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumor formation is not well understood. A mutation in patched (ptc), an inhibitor of Hh signaling, was identified in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Hh signaling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signaling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signaling promotes tumor formation (Duman-Scheel, 2002).

During eye development in Drosophila, initiation of neural differentiation, marked by an indentation referred to as the morphogenetic furrow, begins at the posterior end of the disc and passes anteriorly. Cells within the furrow arrest in G1 phase before differentiating. Cells located just posterior to the furrow exit G1 arrest and enter a synchronous S phase referred to as the second mitotic wave. Overexpression of the Drosophila Retinoblastoma-family gene (Rbf), an inhibitor of the S phase promoting transcription factor E2F, produces a 'rough' adult eye phenotype, characterized by loss of bristles and fusion of ommatidia. This phenotype results from delay of S phase progression in cells of the second mitotic wave as a consequence of inhibited E2F target gene expression. Loss of one copy of the ptc gene suppresses this rough eye phenotype and restores E2F target gene expression. The observed genetic interaction between Rbf and ptc suggests that the Hh signaling pathway might regulate the cell cycle during eye development (Duman-Scheel, 2002).

Hh is secreted from differentiating neurons located just posterior to cells entering S phase in the second mitotic wave. This expression pattern is consistent with the idea that reception of the Hh signal might be required for S phase entry in the second mitotic wave. To test this hypothesis, the effect of blocking Hh signaling during eye development was assessed. Cells with a mutated smoothened (smo) gene cannot respond to the Hh signal and fail to enter S phase in the second mitotic wave. Conversely, when Ci, the transcription factor that mediates Hh signaling, is overexpressed in the furrow, cells normally arrested in G1 enter S phase. Ectopic expression of Ci can also promote S phase in G1-arrested cells located in the wing margin and in the brain. Thus, Ci can induce S phase in a variety of tissues (Duman-Scheel, 2002).

Cyclin E is a principal regulator of S phase during Drosophila development. Although Cyclin E is an inhibitor of Rb, it also has additional Rb/E2F-independent cell-cycle roles. The initiation of high levels of Cyclin E expression in cells of the second mitotic wave that is located just anterior to neurons secreting Hh protein, is consistent with the idea that Hh signaling may regulate Cyclin E expression in the eye. Indeed, loss of smo results in reduction of Cyclin E levels in cells entering the second mitotic wave. Conversely, when Ci is overexpressed in furrow cells, high levels of Cyclin E transcript and protein can be detected within these cells. In the eye, the ability of Ci to induce high levels of Cyclin E expression is limited to the furrow region, where it is capable of inducing S phase. Overexpression of Ci is also capable of inducing high levels of Cyclin E transcript and protein expression in the wing. Although Cyclin E is an E2F target gene, the ability of Ci to promote Cyclin E expression in the presence of RBF-280 indicates that Hh can induce Cyclin E expression independently of E2F. Therefore, in addition to promoting expression of Cyclin D and activating E2F, Hh signaling also promotes expression of Cyclin E independently of E2F. The ability of the Cyclin E-dependent kinase inhibitor Dacapo (Dap) to inhibit the ability of Ci to induce S phase in the furrow and wing margin indicates that Cyclin E is the principal mediator of the ability of Hh to promote S phase (Duman-Scheel, 2002).

To investigate the mechanism by which Hh signaling induces Cyclin E transcription, the Cyclin E promoter was examined. Several sequences with homology to the consensus Ci-binding site were identified within the 5' regulatory region of Cyclin E. Ci was found to bind to three Ci-binding sites (A, B and C in gel shift competition experiments). To examine whether this interaction occurs in vivo, chromatin immunoprecipitation (ChIP) experiments were carried out. These assays demonstrate that Ci-binding sites A-C are occupied by Ci protein in vivo. Several lines of evidence indicate that the observed ability of Ci to bind to the Cyclin E promoter is important for regulation of Cyclin E expression in the developing eye. First, normal upregulation of Cyclin E expression in the second mitotic wave is disrupted in Cyclin EJP mutant flies, which bear a P element inserted adjacent to Ci-binding sites A-C. Also, flies carrying the 16.4 Cyclin E lacZ13 reporter, which contains all three Ci-binding sites, show upregulation of ß-galactosidase (ß-gal) expression in the second mitotic wave; this pattern resembles the endogenous pattern of Cyclin E expression. By contrast, upregulated ß-gal levels in the second mitotic wave are not observed in flies carrying reporter 13.2 Cyclin E lacZ13, which lacks Ci-binding sites A and B. Furthermore, overexpression of Ci in the furrow can drive ectopic ß-gal expression from reporter 16.4, but not from reporter 13.2. These experiments suggest that the presence of sites A and B is required for normal Hh-mediated upregulation of Cyclin E expression in the second mitotic wave. Taken together, these results provide strong evidence that Hh signaling promotes S phase through direct induction of Cyclin E expression by the transcription factor Ci (Duman-Scheel, 2002).

The investigation demonstrates that Hh signaling has a distinct ability to promote cellular growth, which is mediated by Cyclin D. In addition, Hh signaling can induce proliferation during development by promoting expression of Cyclin D and Cyclin E. This study reveals a direct connection between Hh signaling and induction of Cyclin E expression, which is accomplished through binding of Ci to the Cyclin E promoter. Upregulation of murine cyclin D1, D2 and E in response to Hh signaling has been observed. It is therefore likely that the mechanism for Cyclin E induction by Hh described here is conserved in mammals. Furthermore, because both overexpression of Ptc-1 or mutation of cyclin D1 produces a small mouse phenotype, it is likely that the ability of Hh to promote cellular growth through upregulation of D-type cyclins is also conserved in mice. Thus, constitutive Hh signaling (which promotes deregulated expression of G1/S cyclins that have been associated with diverse forms of human cancer) would promote both cell proliferation and growth in tumors. In contrast, during development, cell growth and proliferation must be carefully regulated and coordinated with the processes of cell patterning and differentiation. These same processes are also regulated by Hh signaling. This delicate balance is probably maintained by tight control of the temporal and spatial expression patterns of Hh targets and the molecules that regulate them (Duman-Scheel, 2002).

scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila

Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).

A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).

The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).

scrib- clones ectopically express cyclin E and undergo ectopic S phases and mitoses. Since cyclin E is rate limiting for cell cycle progression in the developing eye, it is likely that upregulation of cyclin E in scrib- clones is critical for the ectopic cell proliferation. Indeed, alleles of scrib and lgl were originally isolated as dominant suppressors of a hypomorphic cyclin E allele, DmcycEJP, suggesting that these cell polarity genes normally play a critical role in limiting cyclin E expression. Currently being investigated is which signaling pathways are altered in scrib, dlg or lgl mutants that could be responsible for cyclin E upregulation. A recent study in human lung epithelial cells shows that disrupting cell polarity allows mixing of the heregulin-alpha ligand and the erbB2-4 receptor, which are normally physically separated, resulting in activation of the pathway and cell proliferation. Further studies are required to determine whether the ectopic expression of cyclin E observed in the absence of Scrib is simply a consequence of the tissue disorganization induced by disrupting cell polarity, or if Scrib has a direct role in limiting cell proliferation independent of cell polarity. Interestingly, the rounding up of cells in the absence of Scrib appears to be predominantly a cell-autonomous effect, yet clearly non-cell-autonomous defects are also apparent, including the upregulation of cyclin E. This would suggest that altered cell-cell interactions between wild-type and mutant cells can also alter signaling pathways within wild-type cells, and that the loss of apical-basal polarity and collapse of the columnar epithelium is not intrinsically responsible for the deregulated expression of cyclin E. A deeper understanding of the relationship between epithelial cell polarization and cell proliferation is clearly important for understanding the development of cancer, since a loss of cell polarity often accompanies tumor progression and metastasis (Brumby, 2003).

In Drosophila, activated Ras exerts its oncogenic effects through Raf and the MAPK pathway. Downstream targets of MAPK in the eye disc promote differentiation, cell survival and cell proliferation. This work also demonstrates that Ras can increase cyclin E protein levels in the eye disc. In combination with scrib-, the differentiation output of RasACT signaling appears to be attenuated, and the proliferative and anti-apoptotic responses prevail (Brumby, 2003).

This study has described a novel multi-hit model of tumorigenesis in Drosophila. Furthermore, although it has been suspected that disruptions to cell polarity could potentiate tumor progression and metastasis, this work demonstrates for the first time how the oncogenic effects of activated Ras and Notch are unleashed in the absence of epithelial polarity regulators. It is predicted that in mammals also, defects in apical-basal polarity could cooperate with oncogenes during neoplastic development. This approach in Drosophila can now be used to screen for novel oncogenes that, when specifically overexpressed in scrib- clones, are capable of inducing cooperative tumorigenesis, and can also be extended to identify cooperative interactions between other tumor suppressors and oncogenes within a whole animal context (Brumby, 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).

Control of Drosophila endocycles by E2F and CRL4CDT2

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. This study used genetic tests in parallel with computational modelling to show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the PCNA/replication fork-associated E3 ubiquitin ligase CRL4CDT2 (Cul-4), and this in turn mediates the destruction of E2F1 (Shibutani, 2008). It is proposed that the transient loss of E2F1 during S phases creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, it was found that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles (Zielke, 2011).

Altogether these results indicate that periodic E2F1 degradation is necessary for endocycling for three reasons: (1) it creates a window of low CYCE/CDK2 activity; (2) it promotes high APCFzr/Cdh1 activity and thereby suppresses geminin accumulation; and (3) it allows E2F2 to maintain repression of CDK1 and its cyclins. Each of these conditions is required for preRC assembly and endocycle progression. This cell cycle mechanism is fundamentally different from that used in mitotic cycles, wherein destruction of the M-phase cyclins by APCCdc20/Fzy, rather than of E2F1 by the CRL4CDT2, throws the switch that allows preRC assembly. Indeed it is noteworthy that the periodic degradation of E2F1 and depletion of CYCE are not required for mitotic cell cycles in Drosophila. CRL4CDT2 is required for endocycling in plants, indicating that this element of the endocycle oscillator is conserved (Zielke, 2011).

Finally, it was asked what factors control E2F production to regulate endocycle rates. Endocycle speed and number can be manipulated by altering cell growth through changes in dietary protein or growth-regulatory genes including Myc and insulin/PI3K/TOR signalling components. Hence larvae were starved of protein to suppress insulin/TOR signalling, reduce protein synthesis, and block cell growth. Starvation arrested the salivary endocycles within 24h and strongly depleted E2F1. E2f1 and Dp mRNA levels were not affected, but the E2F targets CycE, pcna and rnrS were reduced. To test whether this was responsible for starvation-induced endocycle arrest E2F1 was overexpressed in the salivary glands of starved animals. Although these glands failed to grow their nuclei incorporated BrdU and accrued approximately sevenfold more DNA than controls. Overexpression of RHEB, which activates the Target of rapamycin (TOR) kinase and increases ribosome biogenesis and cap-dependent translation, also restored cell growth, E2F1 protein, and endocycle progression in starved animals. Thus E2F1 appears to act as a 'growth sensor' that couples rates of endocycle progression to rates of cell growth. A likely mechanism for this, corroborated by modelling, involves increased translation of E2F1 in rapidly growing cells. Indeed, it was found that the association of E2F1 mRNA with polyribosomes was greatly reduced in protein-starved animals. Translational control of E2F is an attractive mechanism for coupling growth to G1/S progression not only in endocycling cells, but also in growth-dependent mitotic cells with extended G1 periods (Zielke, 2011).

The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis

Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).

Mutations are described in hippo, which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator Cyclin E and apoptosis inhibitor DIAP1. Three alleles of hippo are lethal either when homozygous or in trans to another allele. Eyes containing hpo mutant clones and wild-type clones have an overrepresentation of mutant tissue when compared to eyes containing clones of the wild-type parental chromosome suggesting that the mutant tissue may have a relative growth advantage. Mutant ommatidial facets are slightly larger than wild-type facets and sometimes contain extra interommatidial bristles. When homozygous clones of hpo were generated in other imaginal discs using hsFLP, outgrowths of tissue were observed and portions of wings containing large hpo clones were larger than the corresponding portion of a wild-type wing, indicating a role for hpo in regulating organ size in tissues other than the eye. Retinal sections of adult eyes containing hpo clones reveal that mutant ommatidia appear to have the normal complement and arrangement of photoreceptor cells. However, hpo mutant ommatidia appear to have significantly more tissue between adjacent ommatidia. Cell outlines are visualized more readily in the pupal retina. In contrast to the single layer of interommatidial cells observed in wild-type retinas, mutant ommatidia have several additional interommatidial cells. These phenotypic abnormalities are very similar to those observed in sav or wts mutant clones (Harvey, 2003).

sav or wts mutant cells in the eye imaginal disc fail to exit from the cell cycle at the appropriate time. The additional rounds of cell division generate an excess of interommatidial cells. Elevated levels of cyclin E protein detected in mutant cells may underlie the delayed cell cycle exit (Harvey, 2003).

In a disc mosaic for the wild-type parent chromosome, BrdU-incorporation was evident in the anterior portion of the disc and in a narrow stripe, the second mitotic wave (SMW), but not in the morphogenetic furrow (MF) or posterior to the SMW where cells arrest in the G1 phase of the cell cycle. In hpo mosaic eye discs, the pattern of S phases was normal in the anterior portion of the disc and in the SMW but in mutant portions of the disc, BrdU-incorporating nuclei were observed posterior to the SMW and also in the MF. Thus, hpo cells continue to cycle when surrounding wild-type cells are arrested in G1, indicating that hpo function is essential for timely cell cycle exit. In discs containing clones of the wild-type parental chromosome, mitoses, visualized with anti-phospho histone H3, were observed in the anterior portion of the eye disc and in several rows of developing ommatidia immediately posterior to the SMW. In discs mosaic for hpo however, extra mitoses were seen in mutant clones many ommatidial rows posterior to the SMW, indicating that at least a subset of hpo mutant cells continue to divide when wild-type cells are mitotically quiescent. These abnormalities are similar to, though less severe than, those found in sav and wts imaginal discs (Harvey, 2003).

Elevated levels of cyclin E were found in hpo mutant clones immediately anterior to the MF, in the SMW, and posterior to the SMW. Cyclin E expression appeared normal in hpo clones in the most anterior portions of the third instar larval eye-antennal disc. In contrast, cyclins A, B, and D were expressed at normal levels throughout the disc. In wild-type discs cyclin E RNA is expressed in a narrow stripe corresponding to the SMW. In discs mosaic for hpo, the level of cyclin E RNA was elevated and the expression domain of cyclin E was broader. Additionally, an increase in cyclin E mRNA was detected by semiquantitative RT-PCR performed on eye imaginal discs composed almost entirely of hpo mutant tissue. This indicates that, at least in part, hpo regulates cyclin E at the level of transcription or RNA stability but additional posttranscriptional regulation is also possible. In experiments where hpo function was reduced in S2 cells by RNAi, the levels of cyclin E protein were increased without an obvious change in RNA levels as assessed by Northern blotting. Thus, hpo may be capable of regulating cyclin E levels at both transcriptional and posttranscriptional levels (Harvey, 2003).

The cycling properties of hpo mutant cells were analyzed by generating clones of mutant cells in third instar larval wing discs. Larvae were heat shocked 48 hr after egg deposition (AED) and wing discs were dissected 120 hr AED. Following dissociation with trypsin and staining with Hoechst, cells were subjected to flow cytometry. Cell size, as gauged by forward scatter, and DNA content were measured and found to be almost indistinguishable between wild-type and hpo mutant cells (Harvey, 2003).

The number of cells in hpo mutant clones is consistently larger than in wild-type sister clones. The median population doubling time in hpo clones was 13.1 hr, which is significantly shorter than that of the GFP-bearing tester chromosome, which was 14.7 hr. By comparison, clones derived from the parent FRT42D chromosome had a population doubling time that was not significantly different from the same tester chromosome. It is unlikely that the increased cell numbers in hpo clones can be explained by a block in apoptosis, since overexpression of the caspase inhibitor p35 in wing disc cells at this stage of development does not appreciably alter the population doubling time. Thus, cells appear to divide faster in hpo clones. Since cell size is essentially unchanged, this would imply that hpo cells have an increased rate of growth (mass accumulation) and a commensurate increase in the rate of cell division (Harvey, 2003).

In wild-type pupal retinas, excess interommatidial cells are eliminated in a wave of apoptosis during the midpupal stage. There is a defect in apoptosis in sav and wts mutant tissue. As a result, the additional cells generated by excess cell division in sav and wts tissue are not eliminated and account for the increased number of interommatidial cells (Harvey, 2003).

The mob as tumor suppressor gene is essential for early development and regulates tissue growth in Drosophila

Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. The Mob family protein Mats functions as a coactivator of Wts kinase. This study shows that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, this study found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function (Shimizu, 2008).

Mats is essential for normal development; mats mutants stop their growth at the second instar larval stage and eventually die. In fact, this growth retardation phenotype facilitated identification of matsroo and matse235 mutant larvae for DNA sequence analysis. Using matse235 allele and the P-element-induced allele matsPB, it has been shown that mats homozygotes and hemizygotes grow slowly and their imaginal discs are much smaller than that of wild-type larvae at the same age. mats mutant cells in mosaic tissues acquire growth advantage likely through comparison and competition with neighboring wild-type cells. In contrast, the absence of wild-type cells in homozygous mats mutant animals renders no competitive growth advantage to mutant cells. The mechanism by which mats mutants acquire growth advantage in the context of mosaic tissue still needs to be investigated. mats mutant embryos missing both maternal and zygotic mats functions failed to hatch, indicating that mats is essential for embryonic development. By analyzing mitotic cells, it was found that maternally mats-depleted embryos show aberrant DNA segregation such that uneven amounts of DNA are segregated toward opposing centrosomes. However, this does not appear to be due to the compromised function of mitotic spindle checkpoint, since mats mutant tissue still accumulate M-phase cells in response to inhibition of mitotic spindle formation by colcemid treatment. Thus, mats is not required for mitotic spindle checkpoint, unlike mps1 (Shimizu, 2008).

Cyclin E is a critical cell cycle regulator. Through a Cdk2-dependent mechanism, cyclin E-Cdk2 plays a critical role in accelerating G1-S transition in the cell cycle. As a general rule, cyclin E is tightly regulated during the cell cycle by Cdk2 and GSK-mediated phosphorylation and subsequent degradation. A nondegradable cyclin E mutant can cause extra rounds of DNA synthesis and polyploidy, and overexpression of cyclin E is frequently detected in tumor cells exhibiting polyploidy. Intriguingly, cyclin E is a centrosomal protein that functions to promote S-phase entry and DNA synthesis in a Cdk2-independent manner (Matsumoto, 2004). Loss of cyclin E expression in the centrosome inhibits DNA synthesis, whereas ectopic expression of cyclin E in the centrosome accelerates S-phase entry. Thus, the centrosome is an important subcellular organelle for cyclin E to regulate cell proliferation, and the level and activity of cyclin E in centrosomes must be tightly controlled. The fact that Mats and Wts colocalize with cyclin E at the centrosome raises the possibility that Mats may function together with Wts kinase to regulate cyclin E function in the centrosome for mitotic control. In support of this hypothesis, loss-of-function mutations in mats increase the levels of cyclin E protein and both gain- and loss-of-function mutant alleles of cyclin E modulate the eye phenotypes caused by Wts overexpression. Although Mats/Wts-mediated inhibition of cyclin E could occur through Yki to regulate cyclin E transcription, a direct control of cyclin E at the protein level would allow a rapid response to an upstream signal (Shimizu, 2008).

The fact that both Mats and Wts show a intracellular localization pattern very similar to that of their respective yeast relatives Mob1 and Dbf2 suggests that their function is conserved. This conservation may extend to mammals; human LATS1, LATS2, and MOB1A (MATS2) also localize at the centrosome. In addition, localization at the bud neck/midbody appears to be conserved in humans. Interestingly, such centrosomal localization of Mats and Wts does not seem to rely on Wts kinase activity as kinase-inactive Wts and Mats can be still localized at the centrosome. To examine whether endogenous Mats protein localizes at the centrosome, embryo immunostaining was done with Mats antibodies. As in larval tissues, expression of Mats protein in developing embryos does not exhibit any obvious pattern and Mats expression level is low and ubiquitous. Although centrosomal localization of endogenous Mats protein has not been shown, likely due to some technical problems, Mats (CG13852/Mob4) has been recently reported to be a centrosomal protein (Shimizu, 2008 and references therein).

Both loss- and gain-of-function analysis supports a model in which cyclin E and diap1 are critical downstream targets of Hpo/Wts signaling. Evidence in this report suggests that Hpo/Wts signaling may also target cyclin A and cyclin B. Consistent with this notion, elevated levels of cyclin B were found in ex mutant cells. In addition, wts has been shown to be required for a negative control of cyclin A but not cyclin B expression. In humans, LATS1 was shown to be a negative regulator of Cdc2/cyclin A and to function at the G2/M-phase transition, while LATS2 affects cyclin E/Cdk2 activity and regulates G1/S phase passage. Thus, the ability of Hpo/Wts signaling to target cyclin genes important for cell cycle progression appears to be evolutionarily conserved (Shimizu, 2008).

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G1 arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G1 arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G1-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G1-S progression. Thus, G1 arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G1 progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).

The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G1-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G1-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G1-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G1 arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G1 arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G1 progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G1 progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G1 arrest in the ZNC. Still, it is possible that Wg promotes G1 arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).

It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).

Upregulation of Mitimere and Nubbin acts through Cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. These results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to understanding of maintenance and loss of pluripotential stem cell identity (Bhat, 2004).

Maintenance of a self-renewing fate can be viewed as a state where activities of certain genes maintain that state. Once the activity of such genes is switched off, the cells become committed to a differentiation pathway. The results reported in this study indeed support this type of mechanism. That POU genes might be a class of genes that maintain a self-renewing capacity is indicated by the fact that the Oct4 POU gene (Pou5f1 -- Mouse Genome Informatics), which is expressed in pluripotent stem cells of the mouse early embryo, is turned off when these cells begin to differentiate (Rosner, 1990). Similarly, SCIP is expressed in the progenitors of oligodendrocytes, but it is downregulated when these cells are induced to differentiate (Collarini, 1992). The current results provide direct evidence that these genes can induce a cell that is committed to a differentiation pathway to acquire a self-renewing capability in a lineage specific manner. Moreover, studies undertaken in the past several years using the Drosophila nervous system as a paradigm have revealed how asymmetry can be generated during cell division to produce two distinct postmitotic cells. However, there is very little information on how an asymmetric self-renewing division pattern is determined. In this paper, results are presented that provide insight into this particular process. (Bhat, 2004).

The strongest evidence that a GMC-1 undergoes a self-renewing type of asymmetric division in embryos overexpressing miti/nub or CycE, and in embryos mutant for ago, comes from the presence of hemisegments with two sibs and one RP2. There are two ways the second sib cell can be generated: (1) a self-renewed GMC-1 generates another sib when it divides, and (2) some other cell is transformed into a sib. The following set of evidence indicates the former scenario: (1) the second sib cell always appears later in development, i.e. at ~8.5 hours of age (as opposed to in wild type where the GMC-1 terminally divides by ~7.5 hours of age into an RP2 and a sib); (2) the dynamics of Eve expression itself in the sib -- expression of eve is switched off in a sib during the asymmetric division of GMC-1 and there is no de novo synthesis of Eve thereafter. If a postmitotic cell from an Eve-negative lineage transforms into a sib, it would be negative for Eve and would not be detected. The development of the other Eve-positive neuronal lineages is normal in these embryos, thus it is unlikely that a cell from those Eve-positive lineages is transformed into a sib. (3) The Eve and Spectrin staining of UAS-nub; ftz-GAL4 embryos provides more direct evidence for the self-renewal of GMC-1. In ~8. 5-hour-old UAS-nub; ftz GAL4 embryos, the larger GMC-1 (this Eve-positive cell is Zfh1 negative, indicating that it is indeed a GMC-1) can be observed undergoing asymmetric cytokinesis for the second time. From the heat-shock induction experiments of nub or miti mutant embryos, it can be argued that higher levels of these proteins in the parental NB4-2 cause later born GMCs to adopt a GMC-1 fate. However, the GMC-1 self-renewing phenotype observed following targeted expression of nub using the ftz-GAL driver makes this scenario unlikely. (4) The results obtained with the mitiP; insc and mitiP; nb double mutant embryos (P referring to prolonged expression), and the mis-localization of Insc in GMC-1 of these embryos, are also consistent with this conclusion. (Bhat, 2004).

These results indicate that the level, timing and duration of presence of Miti or Nub proteins determine the dynamics of the GMC-1 division pattern. For example, the asymmetric divisions (which generate the 3-cell phenotypes) and the symmetric divisions (which generate the 4-cell phenotype) were observed when the transgenes were induced for 20-25 minutes. However, the multiple cell-phenotype was observed only when the transgenes were induced for 90 minutes. Once the induction was stopped and the levels returned to normal, the two GMC-1s appeared to exit from the cell cycle to generate postmitotic cells. Similarly, when the transgene was induced with ftz-GAL4, only the 3-cell phenotypes, and not the 4-cell or multi-cell phenotypes were observed. Thus, the following picture emerges from these results. Although high levels of Miti and Nub proteins are required for the specification of GMC-1 identity, their level must be downregulated in order for the GMC-1 to divide asymmetrically into postmitotic RP2 and sib. Maintaining a high level of these proteins in GMC-1 commits that cell to adopt a self-renewing stem cell type of division pattern. The results described here also show that Miti and Nub prevent GMC-1 from exiting the cell cycle by upregulation of CycE (Bhat, 2004).

The results clearly show that upregulation of CycE in late GMC-1 is the cause for the adoption of a self-renewing asymmetric division pattern. In other words, presence of high levels of CycE in late GMC-1 and its unequal distribution to one of the two daughter cells prevents this cell from exiting the cell cycle. Since this daughter cell still maintains the GMC-1 identity and has sufficient CycE to divide again, a further asymmetric division(s) is ensured. The cell that has lower amounts of CycE becomes committed to a differentiation pathway (RP2 or sib) (Bhat, 2004).

What lines of evidence support this conclusion? (1) In contrast with wild type, there is a significant amount of CycE present in a late GMC-1 in embryos overexpressing miti or nub. This CycE preferentially segregates to one of the two daughters of that GMC-1, usually the larger cell. When miti or nub genes are overexpressed only briefly, the level of CycE is downregulated after just one additional round of division, whereas with prolonged induction, the level is maintained at high levels in one or two cells of the multi-cell cluster for a prolonged duration of time (Bhat, 2004).

(2) Upregulation of CycE in a late GMC-1 is also observed in embryos mutant for ago, which is known to regulate CycE levels. In ago mutants, the two daughter cells of such a GMC-1 have unequal CycE levels accompanied by a self-renewing asymmetric division phenotype. The CycE is always downregulated after one additional GMC-1 division, which is consistent with the finding that the self-renewal occurs only once in these embryos. Since penetrance in ago mutants is partial, and CycE is downregulated in this lineage after just one additional division, there must be additional factors that mediate the downregulation of CycE in this lineage (Bhat, 2004).

(3) Embryos expressing high levels of CycE from a CycE transgene exhibit the same GMC-1 phenotypes as embryos expressing high levels of Miti or Nub. Thus, these results indicate that upregulation of CycE alone is sufficient for the GMC-1 to adopt a self-renewing type of division pattern. Finally, mitiP phenotypes are found to be dependent on CycE. That is, no multi-cell clusters were observed in mitiP; CycE double mutant embryos (Bhat, 2004).

In wild type, the downregulation of CycE in GMCs appears to occur through switching off CycE transcription and degradation of the protein by factors such as Ago. At what level does Miti or Nub regulate CycE? Since POU genes are thought to be transcriptional activators, they can regulate transcription of CycE either directly or indirectly. However, this does not seem to be the case since expressing high levels of miti does not have a discernible effect on the levels of CycE mRNA in GMC-1, as assessed by whole-mount RNA in situ hybridization. In addition, the putative promoter/enhancer region of CycE gene does not contain any consensus POU protein-binding sites. Therefore, it seems likely that Miti and Nub regulate factors that are involved in the degradation of CycE in late GMC-1 (Bhat, 2004).

The question arises as to how only one cell has a high level of CycE. There are several ways this can happen. There might be an asymmetric degradation of CycE. This scenario seems unlikely since there is only one of two daughter cells with high levels of CycE in ago mutants. Given that Ago downregulates CycE via a protein degradation mechanism, if there was an asymmetric degradation, in those hemisegments where the levels of CycE was elevated in GMC-1, it would initially be expected that both the daughter cells would have high CycE levels. However, this was not the case. An asymmetric transcription of the CycE gene also seems unlikely since the transcription of CycE ceases prior to GMC-1 division, as judged by whole-mount RNA in situ hybridization. The most likely possibility is that CycE is unequally distributed between the two daughter cells of GMC-1. The unequal distribution of CycE could be a passive process due to the size difference between daughter cells, especially in the GMC-1-->RP2/sib lineage. Moreover, no cytoplasmic crescent of CycE was observed during mitosis. By contrast, it could also be an active process. For instance, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process (Bhat, 2004).

Finally, the results indicate that while a GMC that does not normally express Miti or Nub is insensitive to its ectopic expression (e.g., GMC1-1a of NB1-1; this GMC produces an aCC/pCC pair of neurons), a brief induction of CycE in the same GMC causes it to undergo self-renewing asymmetric division. Therefore, CycE can confer a stem cell type of division potential to more than one GMC. Another important conclusion one can draw from this result is that the segregation of CycE may be an active process. In the case of GMC1-->RP2/sib lineage, the cytokinesis of GMC-1 is asymmetric, and the size difference between an RP2 and a sib is significant. Thus, CycE can be asymmetrically segregated because of this size difference. However, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process. It is possible that the difference between the levels of CycE needed to retain a cell within the cell cycle and the levels that do not support maintaining the cell within the cell cycle are quite small. Thus, even a minor change in the amount that a cell receives during division might be sufficient to make a difference. Thus, the segregation of CycE can still be a passive process. Nonetheless, these results reveal how a cell can adopt a self-renewing asymmetric division potential through CycE. (Bhat, 2004).

Pros has been implicated in inhibiting the ability of GMCs to divide more than once by preventing continued expression of cell-cycle genes. The caveat of this study, however, is that none of the GMC lineage was examined using cell-specific markers to determine whether GMCs continue to divide in embryos mutant for pros. The conclusion that Pros inhibits GMC division was mainly based on the presence of additional BrdU-positive cells in late stage (post 15-hours-old) pros mutant embryos. Pros is expressed in GMC-1 of the RP2/sib lineage and, in null alleles, this GMC-1 identity is not specified. In pros17, a loss-of-function allele, ~5% of the hemisegments had an RP2/sib lineage specified. In these hemisegments, the GMC-1 divides only once to generate an RP2 and a sib cell as in wild type. Moreover, specification of U and CQ lineages was observed in ~20% and ~13% of the hemisegments, respectively, and no additional cell division appeared to occur in these lineages. A previous study found that the aCC/pCC neurons (from GMC1-1a) have an abnormal axon morphology, but it did not find any additional neurons in this lineage. Similarly, NB6-4 of the thoracic segment produced the normal number of progeny in pros mutant embryos. These results suggest that Pros does not regulate cell division in RP2/sib, U and CQ lineages, and possibly not in many other neuronal lineages, and therefore it is unlikely to function in the miti/nub pathway (Bhat, 2004).

How is the specification of identity of one of the two progeny, either as an RP2 or as a sib, from a self-renewing asymmetric division of GMC-1 regulated? (Specification of the other progeny as GMC-1 is by high levels of CycE.) The results indicate that specification of an RP2 versus a sib identity to this differentiating cell is through a combination of low levels of CycE and localization of Insc. This is indicated by the finding that overexpression of Miti and Nub causes localization of Insc to be non-asymmetric. Non-asymmetric Insc also causes non-asymmetric localization of Numb. The cell that has lower levels of CycE and also has Numb becomes an RP2. Whenever the cell with lower levels of CycE fails to inherit Numb (the effect of overexpression of Miti or Nub on the localization of Insc is partially penetrant) that cell will become a sib. That the generation of an RP2 during the asymmetric division of GMC-1 is tied to Numb is also indicated by the analysis of mitiP; numb embryos. Although the self-renewal of GMC-1 in mitiP embryos is numb-independent, the commitment of a progeny to become a sib is numb-dependent. Thus, in ~13-hour-old mitiP; numb embryos, multiple cells are observed adopting a sib fate. An often overlooked fact is that in insc mutants the GMC-1 division is normal in ~30% of the hemisegments despite having no insc. Similarly, the penetrance of the symmetrical division of GMC-1 in pins (where Insc localization is affected as in mitiP embryos) is also partial, indicating the presence of additional (partially redundant) pathways for Insc that mediate asymmetric fate specification. These very same additional pathways must also influence the choice between a sib and an RP2 when the GMC-1 in mitiP embryos undergoes a self-renewing type of asymmetric division (Bhat, 2004).

CycE and Ago are part of a mechanism that converts a normal cell into a cancer cell. In ago mutants, CycE protein is not degraded and a number of cancer cell lines carry a mutation in ago. The current results showing that these genes are also involved in a stem cell type of division suggest a commonality between stem cells and cancer cells. These results also provide a molecular mechanism of how self-renewing asymmetric divisions are possible (Bhat, 2004).

Prospero, through regulation of CycE, maintains the mitotic potential of glial precursors enabling them to respond to neurons

During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. To a large extent, the molecular mechanisms controlling glial precursor proliferative potential are unknown. This study shows that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development (Griffiths, 2004).

The longitudinal glia (LG) of the Drosophila CNS share some features with vertebrate oligodendrocyte precursors. Like oligodendrocytes, LG are also produced in excess and the excess cells are eliminated through apoptosis. The survival of both oligodendrocytes and at least some of the LG depends on contact with axons and on Neuregulin/Vein. There is also suggestive evidence that LG proliferation may be under non-autonomous control. The LG originate from the segmentally repeated longitudinal glioblasts (LGBs). DiI labelling of the LGB produces a clone of variable number of progeny cells, resulting in between 7 and 10 progeny cells. There is apoptosis in up to three cells in this lineage in normal embryos, meaning that the resulting progeny of the LG lineage if they were all to survive may be around 12 cells. This suggests that the mitotic profile of the LGB lineage is not simply symmetrical and/or perhaps LG precursor divisions are under non-autonomous control (Griffiths, 2004).

This study analyzed the mechanisms that regulate proliferation of the LG as they interact with pioneer axons. Proliferation of the LG is shown to be regulated by neurons and prospero (pros) is shown to play a key role in linking glial proliferation and axon guidance. Early on, Pros enables glial proliferation in response to pioneer neurons. Once the axonal bundles are formed, Pros maintains glial precursors in an arrested, immature state, enabling pros-expressing cells alone to divide further upon elimination of neurons (Griffiths, 2004).

This study has found novel roles of Pros in promoting cell proliferation and preventing cell cycle exit. The glia reach the extending growth cones in clusters of four cells, when cell division halts for some time. Normally, two of these LG then divide, resulting in a total of six, which then divide again, but since some LG die the real final number ranges between 8 and 11 cells. In pros mutants, the glia contact the pioneer neurons in clusters of eight cells rather than the normal four, suggesting that LG divided faster than normally in the presence of maternal CycE, skipping a G1 phase. The division of four LG into six is also missed, thus changing the mitotic pattern from its normal 4-6-12 to 4-8 (Griffiths, 2004).

Loss of pros function causes a reduction in LG proliferation, which is manifested in three ways. (1) In pros mutants, the first division of the two anterior LG with highest Pros levels is missed, because there is no dpERK. (2) LG do not divide at the normal times during axon guidance and fasiculation is not produced in pros mutants, because of the absence of CycE. Thus, although LG divided earlier in pros mutants, these divisions are uncoupled from axon guidance. Thus, Pros changes the mitotic profile in the LG from a simple symmetric pattern to a pattern in which the LG respond to incoming axons. (3) In the absence of Pros, LG do not have the potential to overproliferate when neurons are ablated (Griffiths, 2004).

Pros protein is present in all dividing LG and in LG that retain mitotic potential. During growth cone guidance and axonal fasciculation, Pros promotes LG proliferation of the two LG that are able to respond to Vein and activate the MAP kinase pathway. Vein induces LG cell division as well as cell survival of the two EGFR-positive LG. Knock-down of Vein function with targeted RNAi exclusively directed to the the MP2 neurons is sufficient to cause LG apoptosis. Loss of Vein function in genetic null embryos reduces mitosis, also when apoptosis is blocked. Thus, the EGFR/MAPKinase signalling pathway controls both cell survival and cell proliferation in these two LG. The EGFR also controls both cell survival and cell proliferation in the retina, in response to the ligand Spitz. Later on, when the axonal fascicles are formed, Pros maintains the mitotic potential in the LG by preventing them from exiting the cell cycle. In fact, only Pros-positive LG can enter S phase upon ectopic expression of cycE. In this way, at the end of embryogenesis, the LG are divided into Pros-positive G1-arrested LG and Pros-negative LG, which have exited the cell cycle and are in G0. Pros maintains the LG in the G1-arrested undifferentiated, immature precursor state by positively regulating Notch and by antagonising Dacapo (Griffiths, 2004).

These findings on the roles of Pros in the LG during axon guidance differ from Pros' neuroblast functions. In neuroblast lineages, Pros protein is located in a crescent and it is distributed asymmetrically to the daughter cell upon the division of the neuroblast. In the ganglion mother cell, Pros is internalised into the nucleus, where it determines cell fate and it restricts cell division. However, the progeny of the LGlioblast [from the time in which they contact the pioneer axons (four-cell stage)] divide apparently symmetrically, although asynchronously. During these divisions, Pros is present in the nuclei of all dividing LG, and not in crescents. Upon cell division, Pros is segregated symmetrically to the two daughter cells and it is downregulated after cell division, at the time that the posterior LG migrate with the axons. Finally, during axon guidance, pros mutations cause a reduction in LG proliferation rather than an excess, meaning that pros is necessary for cell division to proceed (Griffiths, 2004).

Pros and its vertebrate homologue Prox1 can inhibit cell proliferation and promote cell cycle exit. In fact, both in pros and Prox1 mutants, cell proliferation and the expression of cyclin increase, and both Prox1 and Pros can promote p27/dap expression. In the LG, Pros promotes cell proliferation and it prevents cell cycle exit by antagonising Dap. Therefore, Pros controls cell cycle genes in different ways in different cellular contexts. Moreover, temporal regulation is crucial and Pros can both promote and antagonise dap expression at different time points. Upon ectopic pros expression the LG divide less and do not express cycE. However, in the LG this may not be due to the promotion of cell cycle exit but to the earlier halt of precursors in cell cycle arrest (Griffiths, 2004).

These findings also contrast with the roles of Pros in mixed neuro-glioblast lineages, where Pros is segregated asymmetrically to the daughter cell that will become a glial cell. The LG is a glial-only lineage. In the LG, Pros may control the fate of the two LG with higher Pros levels, which signal through MAPKinase/dpERK. The results show that during axon guidance Pros plays a primary role in the maintenance of the proliferative and undetermined state (Griffiths, 2004).

The current findings on the non-autonomous regulation of glial proliferation contrast with previous work that envisioned a cell-autonomous proliferation profile determined by lineage identity. Accordingly, the LGB would divide in a straightforward symmetrical fashion, into 2-4-8 cells. This conclusion was based on the finding that BrdU is incorporated in four Repo-positive cells. The data show that the incorporation of BrdU into four LG represents a narrow time window in the LG lineage, and not the final division. In fact, mitosis is detected in up to five LG at the same stage (Griffiths, 2004).

The finding of a different LG profile has important implications. It means that the final number of LG is not fixed at eight cells, but variable between 8 and 11, depending on how many LG die. A final fixed number of eight LG could be achieved faster through simple symmetrical divisions without considerable influence on final glial cell mass. In fact, in Pros mutants a final number of eight cells is achieved at an earlier time point, and these eight cells stretch out to occupy the whole length of the segmental neuropile. However, the sequential increase and adjustment in LG number deploys a restricted number of LG at sequential steps in axonal patterning. This enables glia to be in the correct number at discrete time points to enable axon guidance and fasciculation (Griffiths, 2004).

The first event in growth cone guidance occurs at the four-cell stage, when LG stop dividing for some time and wait for the pioneer growth cones to extend. At this time, the LG are in the first G1 phase in the lineage. The G1 phase is a characteristic time in which cells respond to growth factors to signal through ERK, and in the retina axons approach selectively precursors that are in G1. As the growth cones approach, the two anterior LG (with higher Pros levels) of the four-cell clusters divide in response to Vein. Vein is produced by the MP2 pioneer neurons, which require LG for axon guidance. By regulating both cell survival and cell proliferation, Vein ensures that LG are present in the correct number to enable growth cone guidance. Pros regulates the zygotic expression of CycE in LG, thus introducing the first G1-S transition, and the fate of the EGFR signalling cells. In this way, Pros modulates the timing of the response of glia to a neuronal signal to divide. Subsequently, the LG continue to divide at times in which axons undergo fasciculation and defasciculation. In this way, LG are deployed in restricted numbers to enable sorting out of axons through time (Griffiths, 2004).

Prospero, targeting CycE, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP: Yorkie targets cycE and diap1

Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).

Activation of yki leads to increased transcription of diap1 and CycE. The increased cell proliferation and decreased apoptosis resulting from yki overexpression are strikingly similar to those caused by loss of hpo, sav, or wts, suggesting that Yki functions in the Hpo pathway. To further explore this possibility, the transcription of cell-death inhibitor diap1 and cell-cycle regulator cycE, known targets of the Hpo pathway were examined. Elevated DIAP1 protein is detected in yki-overexpressing clones in the eye discs. This regulation is largely mediated at the level of diap1 transcription since the expression of thj5c8, a P[lacZ] enhancer trap reporter inserted into the diap1 locus, is similarly elevated in yki-overexpressing clones in a cell-autonomous manner. A cycE-lacZ reporter containing 16.4 kb of the 5′ regulatory sequence of cycE is also increased in yki-overexpressing clones, especially those close to the MF, although the effect is less profound than that observed with the diap1 reporter. Thus, like loss of hpo, sav, or wts, overexpression of yki results in increased transcription of diap1 and cycE. It is worth noting that previous analyses of hpo mutant clones also revealed a 'tighter' regulation of diap1: while diap1 transcription is elevated in all hpo mutant cells irrespective of their relative position to the MF, cycE transcription is only elevated in hpo mutant cells close to the MF (Wu, 2003). These observations suggest that diap1 might represent a more direct transcriptional target of the Hpo pathway (Huang, 2005).

Appropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Growth inhibitory functions are described for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. Mats and Wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. Since Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans (Lai, 2005).

Cyclin E, a key regulator for the G1-S transition, is normally expressed in the second mitotic wave (SMW) of larval eye discs. In mats mosaic eye discs, Cyclin E levels are elevated in mutant clones located in the morphogenetic furrow (MF) and SMW regions. Moderate upregulation of Cyclin A and Cyclin B expression is also observed. Thus, an important mechanism for mats to control cell proliferation is to negatively regulate expression of key cell cycle regulators such as Cyclins. Interestingly, Cyclin E expression in mutant cells immediately anterior to the MF is much less elevated than that immediately posterior to the MF, suggesting that mats may use a different mechanism to restrict cell proliferation in cells anterior to the MF. The cell proliferation defects observed in mats mutants are similar to those caused by sav, wts, and hpo mutations (Lai, 2005).

A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase

Mitochondria undergo fission-fusion events that render these organelles highly dynamic in cells. A relationship exists between mitochondrial form and cell cycle control at the G(1)-S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at G(1)-S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early G(1) to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for G(1)-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at G(1)-S is linked to cyclin E buildup for regulation of G(1)-to-S progression (Mitra, 2009).

Targets of Activity

A coordinate program of transcription of S-phase genes (DNA polymerase alpha, Proliferating cell nuclear antigen and the two ribonucleotide reductase subunits) can be induced by Cyclin E (the G1 cyclin). In Drosophila embryos, this program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. This dynamic pattern of expression is not disrupted by a string mutation that blocks the cell cycle. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. It is likely that developmental signals control this transcriptional program and that its activation either directly or indirectly drives transition from G1 to S phase in the stereotyped embryonic pattern (Durano, 1995a).

The onset of cyclin expression in endoreduplicating tissues is not dependent on Cyclin E function, as Cyclin E expression is observed in CycE mutants. However, instead of being terminated rapidly, the expression is maintained in CycE mutants. Thus functional Cyclin E protein is required directly or indirectly to shut off its own expression. Consistent with this notion, it is found that overexpression of Cyclin E down-regulates the endogenous CycE transcription (Sauer, 1995).

To test if the cell cycle transcription of double parked (dup) is dependent on E2F, embryos homozygous for a null allele of the dE2F1 subunit, dE2F91 were collected and hybridized with dup riboprobes. The levels of dup transcript are decreased in the endoreplicating gut and appear to be slightly decreased in the CNS. A similar effect on dup transcript is seen in embryos that are homozygous mutant for the other subunit of the E2F transcription factor, of the genotype dDPa2. Thus, dup is a downstream target of the E2F transcription factor. Interestingly, yeast cdt1 transcription is also cell-cycle regulated. Expression of cdt1 is controlled by the G1-S transcription factor Cdc10 that, like E2F, regulates transcription of many genes required for S phase (Hofmann, 1994). This suggests that cell cycle control of dup may be conserved, and Dup may prove to be an important downstream target of E2F in mammalian cells (Whittaker, 2000).

Cyclin E is required to regulate positively the transcription of S phase genes in the nervous system and to downregulate these transcripts in endo cycling cells. The cyclinEl(2)305 and cyclin EPZ5 mutations and a cyclin E deficiency, Df(2L)TE35D1, have similar effects on double parked transcripts. In these embryos, dup is not downregulated properly in the endoreplicating gut such that dup transcripts persist at higher levels than wild type in the anterior midgut, central midgut, and posterior midgut in later embryonic stages. In cyclin E mutant embryos dup transcripts are reduced in the CNS, although not to as great an extent as other S phase genes. Thus, dup expression also is regulated by cyclin E (Whittaker, 2000).

Expression of the cyclin-dependent kinase inhibitor Dacapo is regulated by Cyclin E

The Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) has been implicated in mediating cell cycle arrest prior to terminal differentiation. In many instances, increased expression of CKIs immediately precedes mitotic arrest. However, the mechanism that activates CKI expression in cells that are about to stop dividing has remained elusive. This issue was addressed by investigating the expression pattern of dacapo, a Cip/Kip CKI in Drosophila. The accumulation of Dacapo RNA and protein requires Cyclin E and ectopic expression of Cyclin E can induce dacapo expression. The oscillation of the Cyclin E and Dacapo proteins is tightly coupled during ovarian endocycles. These results argue for a mechanism where Cyclin E/Cdk activity induces Dacapo expression but only within certain windows that are permissive for dacapo expression (de Nooij, 2000).

In a number of different tissues, expression of Dap occurs precisely during the last mitotic division that cells undergo before they terminally differentiate. For instance, in the embryonic epidermis, a rapid accumulation of DAP mRNA and Dap protein is detected only following S-phase 16, just before these cells arrest in the G1-phase of cycle 17. Exit from the cell cycle also requires alterations in the levels of Cyclins and the activity of Cyclin-Cdk complexes. Is the induction of dap directly regulated by the developmental cues that dictate cell cycle exit? Or alternatively, is the induction of DAP RNA and protein a response to an alteration in the activity of one of the other cell cycle regulators (de Nooij, 2000)?

To help distinguish between these possibilities, whether alterations in the levels of one of the known cell cycle regulators would affect the normal pattern of Dap expression was examined in different embryonic tissues. Embryos that were mutant for the G1-S regulators, cyclin E, E2F1 and DP and for the regulators that mediate the G2-M transition, string (stg), cyclin A, rca1, cyclin B and cdc2 were examined. With the notable exception of cyclin E mutants, no obvious abnormalities in the Dap expression pattern could be observed in any of these mutants. Analysis of cyclin E (cycE) mutant embryos, however, shows significant differences in the expression pattern of Dap. Dap protein levels are severely reduced in the cells of the PNS and are almost absent in the cells of the CNS of embryos homozygous or hemizygous for the cycE null allele cycEAr95. The level of DAP RNA is also reduced in the cells of the PNS and the CNS. This suggests that Cyclin E may regulate dap expression at least in part at the transcriptional level. Immunostaining of cycE mutant embryos with an anti-Elav antibody does not show significant abnormalities in the cellularity of the PNS and CNS, suggesting that the loss of dap expression is not a secondary consequence of a failure to form these tissues (de Nooij, 2000).

Interestingly, no reduction in Dap expression levels is observed in the epidermis of cycE mutant embryos. However, cycE mutants have been shown to complete all 16 epidermal divisions normally, possibly due to the activity of residual maternal supplies of Cyclin E. It is therefore likely that the residual supply of Cyclin E may also be sufficient to trigger Dap expression in the epidermis. Alternatively, the apparently normal expression of Dap in the cycE mutant epidermal cells may reflect a difference between the mechanisms that regulate Dap expression in the epidermis and in the nervous system (de Nooij, 2000).

These results also show that normal Dap expression is not contingent upon progression through the cell cycle. Embryos mutant for string, which encodes a Cdc25-like phosphatase, arrest in the G2 phase of cycle 14. Despite the absence of S-phase progression and cell division in string mutants, the onset of Dap expression occurs at the appropriate developmental stage in both the epidermis and the cells of the nervous system. These observations also show that Dap expression is not normally contingent upon cell cycle progression or on reaching cycle 16 in the epidermis (de Nooij, 2000).

The reduced level of dap RNA in cycE mutant embryos seems to indicate a role for Cyclin E in the transcriptional regulation of dap. A potential mechanism for the transcriptional regulation of dap by Cyclin E could involve the E2F transcription factor. It has been demonstrated previously that E2F can mediate the activation of a 'G1-S transcriptional program' that, in the CNS, is dependent upon cyclin E function. E2F can also activate stg expression during G2. However, while E2F1 and DP mutant embryos clearly show a reduction in the RNA levels of the E2F responsive genes PCNA and RNR2, Dap expression appears to be normal in both E2F and DP mutant embryos (de Nooij, 2000).

Cyclin E is also expressed normally in the CNS in E2F and DP mutant embryos. Thus, a redundant function for E2F in regulating dap expression cannot be ruled out, these experiments do not provide any evidence for E2F as a regulator of dap expression. Taken together, these results show that when Cyclin E activity is abolished in the nervous system, Dap expression is no longer observed thus indicating a requirement for Cyclin E activity in regulating Dap expression. This activity of Cyclin E does not appear to involve the activity of the E2F transcription factor. Moreover, Dap expression in either the epidermis or the nervous system is not contingent upon cell cycle progression (de Nooij, 2000).

In both eye and wing imaginal discs, ectopic Cyclin E can induce ectopic expression of Dap. To determine whether the induction of Dap is due to an increase in DAP RNA levels, RNA levels of DAP were examined in whole-mount preparations by in situ hybridizations in eye and wing discs in which Cyclin E was ectopically expressed. In contrast to the dramatic increase in Dap protein, relatively modest increases in the levels of DAP RNA are observed in both the eye and wing discs. Nonetheless, these results are consistent with the notion that Cyclin E regulates DAP RNA levels, and furthermore, suggest that dap transcription may be responsive to Cyclin E levels (de Nooij, 2000).

To test whether dap regulatory elements are responsive to Cyclin E levels, several lines of transgenic flies were generated that contain a 2.7 kb fragment of the dap promoter region linked to a beta-galactosidase reporter. Immunohistochemical analysis of wing discs from flies that contain the pCasdap2.7kb-lacZ transgene either alone, or in the presence of the enGAL4 driver, shows no significant lacZ staining in the posterior compartment of the wing disc. In the presence of both the enGAL4 driver and a UAScycE transgene, increased beta-galactosidase activity is clearly detected in the region where Cyclin E is expressed. Thus Cyclin E can activate the expression of a reporter gene under the control of dap regulatory elements (de Nooij, 2000).

Since Cyclin E appears to be a requirement for the expression of the Dap protein, Dap expression was examined in the context of oscillating levels of Cyclin E in the nurse cell nuclei in the ovary. Oogenesis normally starts with four mitotic germ cell divisions which generate a 16-cell cyst. Surrounded by somatically-derived follicle cells, each cyst forms an individual egg chamber that ultimately gives rise to a mature egg. One of the 16-germ cell nuclei arrests in the prophase of meiosis I, becomes transcriptionally silent and is specified as the oocyte nucleus. The remaining 15 cells, the nurse cells, proceed through a series of endocycles. In wildtype ovaries, Dap expression is first detected in the germ cell nuclei in the germarium at a time when the four mitotic cyst cell divisions are about to be completed (region 2A of oogenesis). In the maturing egg chambers Dap is detected in the endocycling nurse cell nuclei at levels which, within an individual egg chamber, vary from very high to undetectable. This is likely to reflect an oscillation in the level of Dap protein during the endocycles, as has been postulated for Cyclin E. In contrast to the nurse cell nuclei, high levels of Dap are observed in the oocyte nucleus throughout oogenesis (de Nooij, 2000).

Consistent with the immunostaining, DAP RNA can be observed in the germarium early in oogenesis, and subsequently, high levels of DAP RNA are found in the future oocyte throughout oogenesis. However, the pattern of DAP RNA expression in the nurse cells differs significantly from the pattern of Dap protein. In contrast to the dramatic oscillation of Dap protein levels in the nurse cell nuclei, no oscillation of DAP RNA is evident. Only a uniform low level of RNA can be detected in the endocycling nurse cells. Although relative differences may exist but may be beyond the level of detection, this difference in the expression pattern between DAP RNA and protein suggests that post-transcriptional mechanisms may regulate the levels of Dap protein in the individual nurse cell nuclei (de Nooij, 2000).

The expression pattern of Dap protein is reminiscent of the expression pattern of Cyclin E in the ovary, i.e. a strong oscillation in the nurse cell nuclei, and a persistently high level of protein in the oocyte nucleus. Confocal images of ovaries double labeled with antibodies against both Cyclin E and Dap show that the expression of Cyclin E and Dap are largely overlapping. However, some of the nurse cell nuclei in the individual egg chambers do differ in the relative levels of Cyclin E and Dap protein, suggesting that the oscillations of Cyclin E and Dap are slightly out of phase. To assess the regulatory relationship between Cyclin E and Dap in the nurse cells more directly, Dap expression in ovaries was obtained from females homozygous for the cycEfs(2)01672 allele. This allele of cycE specifically perturbs the oscillation of Cyclin E in the nurse cell nuclei and hence ovaries from homozygous cycEfs(2)01672 females show a severe reduction in the amplitude of Cyclin E oscillation. Interestingly, these mutant ovaries also show a strong reduction in Dap oscillation, and most nurse cell nuclei show intermediate levels of Dap expression; nuclei with high or undetectable levels of Dap were almost never observed in mutant egg chambers. Thus in the nuclei of the endocycling nurse cells, expression of Cyclin E and Dap appears to be tightly linked. Dap protein levels oscillate strongly and these oscillations are dampened by a reduction in the extent of Cyclin E oscillation. Since DAP RNA levels in the nurse cells seem to remain relatively constant during oogenesis, it appears likely, that in these cells, Cyclin E controls the level of Dap protein predominantly by post-transcriptional mechanisms (de Nooij, 2000).

The regulation of dap at the transcriptional level seems unlikely to account fully for the dynamic expression pattern of Dap protein. In the situations where Cyclin E was overexpressed, the increase in Dap protein is much more dramatic than the increase in DAP RNA. Likewise, in the ovary, dramatic oscillations in Dap levels are observed without appreciable fluctuations in the levels of DAP RNA. Thus, in these situations, Cyclin E seems to regulate dap at a post-transcriptional level. This is perhaps reminiscent of the type of regulation observed for mammalian p27. Accumulation of p27 has been shown to depend largely on an increased translation of p27 mRNA. Although mammalian Cyclin E has, so far, not been implicated in the translational regulation of p27 or any of the other CKIs, such a mechanism could potentially operate in mammalian cells. Another mode of post-transcriptional regulation that may involve Cyclin E activity is a regulation at the level of protein stability (de Nooij, 2000).

PRC2 Controls Drosophila Oocyte Cell Fate by Repressing Cell Cycle Genes

The oocyte is a unique cell type that undergoes extensive chromosome changes on its way to fertilization, but the chromatin determinants of its fate are unknown. This study shows that Polycomb group (PcG) proteins of the Polycomb repressive complex 2 (PRC2) determine the fate of the oocyte in Drosophila. Mutation of the enzymatic PRC2 subunit Enhancer of zeste [E(z)] in the germline abolishes spatial and temporal control of the cell cycle and induces sterility via transdetermination of the oocyte into a nurse-like cell. This fate switch depends on loss of silencing of two PRC2 target genes, Cyclin E and the cyclin-dependent kinase inhibitor dacapo. By contrast, the PRC1 component Polycomb (Pc) plays no role in this process. These results demonstrate that PRC2 plays an exquisite role in the determination of the oocyte fate by preventing its switching into an endoreplicative program (Iovino, 2013).

Together, the data show that PRC2 controls oogenesis by direct corepression of CycE and dap in a time window that stretches from region 2b to region 3. This prevents improper endocycling, before the oocyte becomes fully silenced and determined by stage 3. Interestingly, E(z) levels rapidly drop in the germline at stage 4, suggesting that the continuous presence of PRC2 is no longer required after oocyte determination. This observation is consistent with the fact that depletion of E(z) after stage 3 using a late Gal4 driver does not affect oocyte fate (Iovino, 2013).

Although differences between PRC1 and PRC2 have been previously reported in mammals (Lessard, 1999; Sauvageau, 2010) and Drosophila (Richter, 2011), it is crucial to understand whether the molecular differences reflect specific biological roles for each of the two complexes. This study has shown that PRC2 controls oocyte cell fate determination, whereas PRC1 components, such as Pc, had no obvious function in the oocyte, consistent with absence of Pc from the germline. Intriguingly, a different situation is seen in the fly male testis, where PRC1 components are required in the germline (Chen, 2011) while PRC2 is dispensable. Therefore, the production of gametes is a critical biological function that separates the function of these two complexes (Iovino, 2013).

Although a few genes that are responsible for oocyte determination have been identified previously, none is known to act on chromatin. The identification of PRC2 as a chromatin effector complex that is required to fix the oocyte fate offers the possibility of starting to dissect the molecular mechanisms that transduce the early asymmetry between the preoocyte and the surrounding cells into a terminally determined fate (Iovino, 2013).

Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin E during eye development

During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. This study shows that Apontic (Apt), which is an evolutionarily conserved transcription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt (Liu, 2014).

Microarray analyses suggested that the bZIP transcription factor, Apontic, regulates the genes required for the neuron, trachea, oocyte, germ cell, bristle and eye development, apoptosis, and cell-cycle regulation. Among these candidates for Apt target genes, this study shows that Apt directly controls the expression of cyclin E and is required for the G1-to-S phase transition during eye development (Liu, 2014).

In the wild type, Cyclin E protein accumulates in a stripe of cells posterior to the MF. Cyclin E was not induced by N, Hh, or Dpp signal in the eye disk because Cyclin E accumulation occurred in Su(H) mutant cells or Mad1-2 Su(H) ci mutant cells. Ci, Mad, and Su(H) are the transcriptional targets of Hh, Dpp, and N signaling, respectively. A prior work suggested that the expression of cyclin E requires activation by both E2F/DP and tissue-specific activators. This study has shown that Apt and Cyclin E are coexpressed at the posterior cells to MF. The expression of Cyclin E was reduced in the apt mutant clones and induced by overexpression of Apt at the eye disk. Apt directly activated the expression of cyclin E at the posterior cells to MF. These data suggest that Apt and E2F1 function together in the activation of cyclin E in the eye disk (Liu, 2014).

Experiments involving ectopic expression of Apt in a GMR-Gal4/UAS-apt fly demonstrate that apt expression can induce S phase.Ectopic expression of Apt at the eye disk, therefore, has a similar effect as that of ectopic expression of cyclin E from a heat-inducible transgene (hs-cycE), suggesting that Apt and CyclinE work on the same pathway. CyclinE is essential and rate limiting for the G1-to-S phase transition. The current finding indicates that Apt controls G1-to-S phase progression by inducing cyclin E expression in the eye imaginal disk (Liu, 2014).

What is the function of Apt during development? Apt is expressed at trachea, head, heart, and CNS and is required for the development of these tissues. However, how Apt controls the development of these tissues is unknown. In this study, the candidates for Apt target genes were identified using microarray analysis. Many of these genes can be assigned to specific aspects of the development of these tissues (Liu, 2014).

Apt and Cyclin E are evolutionarily conserved from Drosophila to humans. However, the function of Apt in human is unknown. In humans, the expression of cyclin E is related to many cancers. Furthermore, Apt overexpression suppresses cancer metastasis in Drosophila. The role of Apt in the regulation of cyclin E might be a conserved function because its human homolog fibrinogen silencer binding protein (FSBP) is also a cancer-related factor and is also expressed in many tissues, including heart, brain, lung, liver, skeletal muscle, kidney, and pancreas. The discovery of a direct connection between cell differentiation and Cyclin E provides insights into the mechanism by which Apt promotes tumor formation (Liu, 2014).

Protein Interactions

Cdc2c kinase, homologous to vertebrate cdk2 kinase and distinct from Drosophila cdc2 (the cyclin dependent kinase associated with Cyclin A and Cyclin B), co-immunoprecipitates with Cyclin E (Knoblich, 1994).

In mammalian systems cyclin E targets Retinoblastoma protein, a negative regulator of entry into S phase. RB associates with E2F in humans, thus rendering it inactive. Whereas the ectopic expression of cyclin E activates Drosophila E2F-dependent transcription, cyclin E does not act directly on E2F but targets, by phosphorylation, a negative regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins (pRB, p107, and p130) that physically associates with E2F in humans. Drosophila RB family homolog (RBF) combines several of the structural features of pRB, p107, and p130, suggesting that it may have evolved from a common ancestor to the three human genes. RBF associates with Drosophila E2F and DP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF is phosphorylated by a cyclin E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF plays a critical role in the regulation of cell proliferation in Drosophila (Du, 1996).

Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. In yeast, inactivation of mitotic cyclins results in acquisition of compentence to initiate another round of DNA replication. The subsequent reactivation of B-type cyclin at the G1/S transition triggers initiation of DNA replication in parallel with a reorganization of protein complexes at origins of replication. fizzy-related (fzr), a conserved eukaryotic gene, negatively regulates the levels of cyclins A, B, and B3. These mitotic cyclins that bind and activate cdk1(cdc2) are rapidly degraded during exit from M and during G1. While Drosophila fizzy has previously been shown to be required for cyclin destruction during M phase, fzr is required for cyclin removal during G1 when the embryonic epidermal cell proliferation stops and during G2 preceding salivary gland endoreduplication. Loss of fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreduplication in the salivary gland, in addition to failure of cyclin removal. Conversely, premature fzr overexpression down-regulates mitotic cyclins, inhibits mitosis, and transforms mitotic cycles into endoreduplication cycles. The coincidence of mitotic cyclin disappearance and cyclinE/cdk2 inactivation during G1 arrest raises the possibliity that fzr activity might be inhibited by cyclinE/cdk2. fzr and fizzy encode highly similar proteins with seven WD repeats in the C-terminal region. WD repeats are found in budding yeast Cdc4p, which is required for the ubiquitin-dependent proteolysis of several cell cycle regulators. The closest yeast relative of fzr, however, is not CDC4 but HCT1, which is required for proteolysis of Clb2p, a budding yeast B-type cyclin with a characteristic destruction box. However, Drosophila fzr is unable to provide HCT1 function in yeast. Thus, fzr transcripts accumulate when cells become postmitotic and fzr is required in proliferating cells progressing through cell cycles with G1 phases and in G2 before endoreduplication, but not during mitosis (Sigrist, 1997).

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. Three Drosophila MCM proteins have been characterized: DmMCM2, DmMCM4, and DmMCM5. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Cyclin:cdks can prevent an assembly of proteins called the "prereplicative complex" on origins of DNA replication. The prereplicative complexes are thought to contain MCMs. Their absence from chromatin is thought to contribute to the inability of the post S phase nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, a test was carried out as to whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B shows that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. Thus MCM-chromosome association is delayed when mitosis is followed by a prolonged G-1 phase. dacapo mutant embryos lack an inhibitor for cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. It is proposed that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. Thus, it is suggested that cyclins have both positive and negative roles in controlling MCM-chromatin association (Su, 1997).

Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines

During Drosophila development and mammalian embryogenesis, exit from the cell cycle is contingent on tightly controlled downregulation of the activity of Cyclin E-Cdk2 complexes that normally promote the transition from G1 to S phase. Although protein degradation has a crucial role in downregulating levels of Cyclin E, many of the proteins that function in degradation of Cyclin E have not been identified. In a screen for Drosophila mutants that display increased cell proliferation, archipelago, a gene encoding a protein with an F-box and seven tandem WD (tryptophan-aspartic acid) repeats, has been identified. archipelago mutant cells have persistently elevated levels of Cyclin E protein without increased levels of cyclin E RNA. They are under-represented in G1 fractions and continue to proliferate when their wild-type neighbors become quiescent. The Archipelago protein binds directly to Cyclin E and probably targets it for ubiquitin-mediated degradation. A highly conserved human homolog is present and is mutated in four cancer cell lines including three of ten derived from ovarian carcinomas. These findings implicate archipelago in developmentally regulated degradation of Cyclin E and potentially in the pathogenesis of human cancers (Moberg, 2002).

To identify genes that restrict cell numbers and tissue growth during development, a genetic screen was conducted to identify recessive mutations that gave homozygous mutant cells even a subtle proliferative advantage over their wild-type neighbors. Clones of homozygous mutant tissue (marked white) were generated in the eyes of heterozygous flies and their size was compared with the wild-type twin spots generated from the same recombination events. Flies were retained in which there was more mutant than wild-type tissue. Of more than 23 loci identified in the screen, multiple alleles were obtained of homologs of several known human tumor-suppressor genes, including TSC1, TSC2 and PTEN. A previously unknown locus, represented by a lethal complementation group consisting of three alleles, was named archipelago (Moberg, 2002).

Compared with an unmutagenized control, adult eyes mosaic for mutations in ago were composed mostly of mutant tissue. Within ago mutant clones, most ommatidial clusters lacked the wild-type complement of photoreceptor cells, and the distance between adjacent photoreceptor clusters was increased. Staining of apical cell profiles during pupal eye development showed that the enlarged interommatidial spaces in ago mutant clones contain excess cells. TUNEL revealed no significant decrease in the extent of cell death in ago mutant clones. Moreover, co-expression of the baculovirus p35 protein, which blocks caspase-dependent cell death, resulted in a further increase in the number of interommatidial cells. These data suggest that loss of ago leads to increased cell proliferation that is partially offset by apoptosis (Moberg, 2002).

Using meiotic and deletion mapping, ago was localized to position 64B on the left arm of chromosome 3. Sequencing candidate transcription units has demonstrated that all three ago alleles (ago1, ago3, ago4) have mutations in an open reading frame designated CG15010. ago encodes a 1,326-amino-acid protein that contains an F-box and seven WD repeats in its carboxy-terminal portion. F-boxes and WD motifs are found in proteins that function as the substrate-recognition component of SCF-type ubiquitin ligase complexes. Within the C-terminal region, the Ago protein is most similar to that encoded by an amino-terminally truncated human expressed sequence tag (EST) previously designated FLJ11071, which has been renamed human Ago. This protein is referred to as hCdc4. A more distant relative is the Caenorhabditis elegans gene sel-10. Two of the mutations are missense mutations that map to the fourth WD repeat. Each alters a residue that is conserved among the three proteins. The third mutation would result in premature termination of translation early in the sixth WD repeat. As the WD repeats of F-box proteins are thought to be required for recruitment of substrates to the SCF complex, it is possible that the alleles recovered in the screen may impair the interaction of Ago with specific substrates (Moberg, 2002).

Because ago mutant cells proliferate more than wild-type cells, it seemed likely that ago mutations would result in increased levels of a positive regulator of the cell cycle. Clones of homozygous mutant tissue were generated in eye imaginal discs and examine them for changes in cyclin levels. In wild-type third-instar discs, Cyclin E is expressed at varying levels and is unpatterned in cells anterior to the morphogenetic furrow, which is thought to correlate with expression at specific stages of the cell cycle in cells that are proliferating asynchronously. A strong stripe of expression can be found immediately posterior to the furrow, which corresponds to cells of the second mitotic wave. Cells posterior to the second mitotic wave express low levels of Cyclin E. In clones of ago mutant tissue anterior to the furrow, almost all cells express high levels of Cyclin E. Clones posterior to the furrow display mild elevations in Cyclin E levels. In contrast to the increase in Cyclin E protein, no alteration in the expression pattern of cyclin E RNA is observed in discs that contain many large ago mutant clones. In wild-type discs, ago mRNA is also expressed throughout the disc, but is expressed at particularly high levels in the morphogenetic furrow. In contrast to the results obtained with Cyclin E, the levels of Cyclin B, Cyclin A and the SCF substrates Cubitus interruptus, Armadillo and Tramtrack are not appreciably elevated in ago mutant clones in the larval imaginal disc, nor are the levels of the putative substrates Dacapo and the intracellular domain of Notch (Moberg, 2002).

Because Cyclin E promotes S-phase entry, an increase in the level of Cyclin E can perturb regulation of the cell cycle. To examine the proliferative properties of ago mutant cells, ago clones were generated in the wing disc of third-instar larvae and their DNA content was compared with that of wild-type cells from the same imaginal discs. In mutant clones, a smaller fraction of cells (21.4%) is found in G1 when compared with wild-type cells (36.8%). The proportion of cells found in S phase and in G2/M is increased. These alterations are extremely similar to those elicited by the overexpression of Cyclin E (Moberg, 2002).

The effect of ago mutations on the patterns of cell proliferation in vivo was also examined. Cells anterior to the morphogenetic furrow in the larval eye disc proliferate asynchronously. It is therefore difficult to visualize differences in rates of cell proliferation in mutant clones at this stage of eye development. In contrast, very few cells proliferate in the wild-type pupal retina. The bristle precursor cell is the only mitotically active cell type detected during this stage; it divides twice during the mid-pupal phase to generate the four cells of the 'bristle complex'. Levels of Cyclin E rapidly decrease after these divisions. In ago mutant clones, elevated levels of Cyclin E are detected in the four cells of the bristle complex well after the levels in the corresponding cells of adjacent wild-type ommatidia have declined. Some of these ago mutant cells also continue to incorporate 5-bromodeoxyuridine (BrdU), suggesting that they continue to cycle after the corresponding cells in adjacent wild-type tissue have exited from the cell cycle. Such additional divisions are likely to contribute to the increased number of interommatidial cells observed in the pupal retina. Thus, the persistence of Cyclin E in ago mutant cells disrupts exit from the cell cycle in a manner similar to that elicited by Cyclin E overexpression (Moberg, 2002).

The simplest explanation of the role of ago in cell cycle control is that Ago binds to Cyclin E and targets it for ubiquitin-mediated degradation. Genetic and physical interactions between Ago and Cyclin E were therefore sought. A genetic interaction was observed between ago and the cyclin EJP allele, which reduces the levels of cyclin E transcription in the developing eye. The rough-eye phenotype of cyclin EJP flies is suppressed in flies that are also heterozygous for a mutation in ago. In addition, ago mutations dominantly suppress the small-eye phenotype produced by eyGAL4-driven overexpression of the cyclin-dependent kinase inhibitor dacapo, which has been shown to reduce Cyclin E-Cdk2 activity. Thus flies that are heterozygous for mutant alleles of ago are likely to have increased levels of Cyclin E (Moberg, 2002).

To test for a direct physical interaction between Archipelago and Cyclin E, the portion of Archipelago containing the F-box and WD repeats was expressed as a protein fused to glutathione S-transferase (GST: GST-AgoDeltaN) and its ability to bind Cyclin E protein was evaluated in lysates of S2 cells transfected with cyclin E and cdk2. Versions of GST-AgoDeltaN were generated that contained the mutations found in the ago1 and ago3 alleles. Binding was readily detected with the wild-type version of GST-AgoDeltaN and was greatly reduced with both mutant versions. Thus the ability of Archipelago to bind Cyclin E in vitro correlates with its ability to downregulate Cyclin E levels in vivo (Moberg, 2002).

These findings, together with the observation that mutations in the C. elegans genes cul1 and lin-23 (which encode a cullin and an F-box protein respectively) have increased cell divisions, highlight the importance of SCF-mediated degradation in regulating cell proliferation through Cyclin E. Because ago RNA is expressed in a dynamic pattern, these results indicate that degradation of Cyclin E is not constitutive in vivo. Dynamic expression of Ago provides another mechanism by which cyclin/cdk activity and cell proliferation can be regulated during development. Finally, impaired proteolysis of Cyclin E is implicated in the pathogenesis of human cancers (Moberg, 2002).

The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth

The essential and highly conserved role of Myc in organismal growth and development is dependent on the control of Myc protein abundance. It is now well established that Myc levels are in part regulated by ubiquitin-dependent proteasomal degradation. Using a genetic screen for modifiers of Drosophila Myc (dMyc)-induced growth, this study identified and characterized a ubiquitin-specific protease (USP), Puffyeye (Puf), as a novel regulator of dMyc levels and function in vivo. puf genetically and physically interacts with dMyc and the ubiquitin ligase archipelago (ago) to modulate a dMyc-dependent cell growth phenotype, and varying Puf levels in both the eye and wing phenocopies the effects of altered dMyc abundance. Puf containing point mutations within its USP enzymatic domain failed to alter dMyc levels and displayed no detectable phenotype, indicating the importance of deubiquitylating activity for Puf function. dMyc induces Ago, indicating that dMyc triggers a negative-feedback pathway that is modulated by Puf. In addition to its effects on dMyc, Puf regulates both Ago and its cell cycle substrate Cyclin E. Therefore, Puf influences cell growth by controlling the stability of key regulatory proteins (Li, 2013).

The mammalian Myc gene family, comprising Myc, Mycn and Mycl, is known to be crucial for growth and development. Myc proteins control multiple cellular processes, including cell growth, proliferation, metabolism and apoptosis, and deregulation of Myc plays an important role in oncogenesis. Non-mammalian Myc has been most intensively studied in Drosophila where the absolute requirement for Drosophila Myc function during development has been demonstrated by the fact that dMyc-null mutants die at an early larval stage (Li, 2013).

Myc transcript and protein abundance are subject to regulation at multiple levels ranging from transcriptional control by numerous mitogenic signaling pathways to extensive post-translational modifications. Of particular interest, given the relatively short half-life of Myc proteins, is the post-translational modification of Myc by the ubiquitin system. Protein ubiquitylation is a fundamental and versatile post-translational modification that controls multiple cellular events by marking proteins as substrates for either degradation or non-degradative processing. In mammals, distinct ubiquitin E3 ligase complexes, including Skp2 and Fbw7, have been reported to influence Myc protein stability and activity (Li, 2013).

The Drosophila ortholog of Fbw7, Archipelago (Ago) is the only ligase identified thus far as involved in proteasome-mediated ubiquitin-dependent turnover of dMyc proteins (Moberg, 2004). Ago mutant alleles were first identified in a genetic screen for regulators of tissue growth in the eye, where it was initially shown to bind and regulate Cyclin E (CycE) levels. Later work demonstrated that Ago also physically interacts with dMyc, and controls dMyc stability and biological function (Moberg, 2004). Unlike c-Myc, which was shown to have a single Myc BoxI phosphodegron associated with Fbw7 binding, several domains containing putative Ago-interacting motifs were shown in dMyc to mediate Casein kinase 1 (CK1)α-, CK1ε- and GSK3β-dependent protein degradation. Although their link to Ago function has not been precisely established, it is clear that GSK3β plays a key role in Ago-mediated dMyc ubiquitylation and degradation (Li, 2013 and references therein).

Protein ubiquitylation is a reversible process in which removal of ubiquitin chains is mediated by deubiquitylating enzymes (DUBs), and the role of DUBs in controlling various cellular processes has attracted considerable interest. DUBs are classified into five subfamilies based on their deubiquitylating domain. Ubiquitin-specific proteases (USPs), which constitute the largest DUB subfamily, share a structurally conserved USP domain of ~350 to 450 amino acids. The USP domain is the catalytic core that mediates the cleavage of ubiquitin conjugates, whereas domains required for protein-protein interaction and substrate specificity are located within N and/or C termini of the USP protein.Although several ubiquitin E3 ligases have been implicated in modulating c-Myc stability, only one deubiquitylating enzyme, USP28, has been demonstrated to catalyze the deubiquitylation of Myc in mammals. Thus far, no deubiquitylating enzyme has been identified that modulates dMyc function or antagonizes Ago-mediated dMyc degradation. Of the 41 predicted Drosophila DUBs, 21 are predicted to have a mammalian USP ortholog. Interestingly, Drosophila does not encode an USP28 ortholog, suggesting that a distinct USP may be responsible for reversing dMyc ubiquitylation in Drosophila. This study reports the identification and characterization of Puffyeye (Puf), a Drosophila USP that antagonizes Ago function and interacts genetically and physically with dMyc. Evidence is presented that Puf regulates dMyc activity at the level of cell and organ growth (Li, 2013).

Although a great deal has been learned recently concerning ubiquitin ligases that interact with Myc proteins, to date only one DUB has been reported that targets Myc. This study has employed a genetic screen based on the rough eye phenotype induced by dMyc overexpression in the eye (GMM) in Drosophila. This screen led to the identification of a USP-type DUB, which was named Puffyeye (Puf; CG9754), as a novel regulator of dMyc function in vivo. Reduced puf expression suppresses, whereas puf overexpression augments, the GMM phenotype. This phenotype is largely an effect of cell overgrowth, yet overgrowth in the eye due to cyclin D/Cdk4 was not influenced by altered Puf abundance. Moreover, knockdown of four other USPs had no effect on the GMM phenotype. This suggests that puf possesses specificity for dMyc-induced growth in the eye. Indeed, puf itself induced a dose-dependent rough eye phenotype, displaying augmented ommatidial size that can be modulated by altering dMyc levels. In the wing disc, dMyc and Puf also were found to collaborate in cell growth. It was also found that Puf is essential for normal development, consistent with a crucial role for Puf in cell growth (Li, 2013).

dMyc levels markedly increase in cells in which puf is overexpressed, whereas dMyc levels are decreased in Puf hypomorphic mutants. These changes in dMyc are predominantly post-translational. This is consistent with the finding that Puf overexpression results in a dramatic increase in dMyc protein stability. Importantly, all of the biological effects of Puf, as well as its effects on dMyc abundance and turnover, are abolished by point mutations in the highly conserved Puf USP catalytic domain. It is surmised that Puf stabilizes Myc through its function as a deubiquitylating enzyme that antagonizes the activity of the Ago ubiquitin ligase, previously shown to target Myc for ubiquitylation and degradation (Moberg, 2004). Importantly, increased Puf exacerbates, and decreased Puf suppresses, the effect of Ago heterozygotes in enhancing the GMM phenotype. The notion that Puf and Ago act as antagonists receives further support from the findings that Puf protein physically associates with both dMyc and Ago in vivo. Interactions between DUBs and their antagonistic E3 ligases, as well as their substrates have been reported previously. The ability of both the Puf short and long isoforms to modify the dMyc-mediated eye phenotype, and stabilize dMyc and Ago proteins in an ubiquitylation domain-dependent manner suggests that domain(s) required for Puf to interact with dMyc or Ago are located in a region N-terminal to the core catalytic domain (Li, 2013).

It was also found that Puf stabilizes CycE, another known Ago substrate, suggesting that Puf antagonizes Ago function in regulating other targets that are crucial for cell cycle control. Indeed, flies homozygous for puf and ago double mutations do not survive, raising the possibility that, in addition to regulating common substrates, they each possess unique targets, as shown for other ubiquitin ligases and DUBs. Notch would be another potential candidate for Puf activity (Moberg, 2004); however, no significant effect of Puf on Notch levels was found in wing discs. In mammalian cells, the ubiquitin-specific protease USP28 was demonstrated to regulate the turnover of c-Myc by binding and antagonizing the activity of Fbw7α, the vertebrate ortholog of Ago. However, Puf and USP28 are not homologs: they appear to be two very distinct USPs in terms of their overall size and amino acid sequence similarity in both their core enzymatic domains and the protein sequence as a whole. The closest mammalian homolog of Puf is USP34 (3546aa). Puf and USP34 are highly homologous in their core catalytic domains (67% identity; 80% similarity) with the catalytic triad conserved, whereas the overall similarity between the two proteins is ~52% (~37% identity) (Li, 2013).

Previous studies have shown that multiple signaling pathways regulate Ago and Fbw7 expression and activity. This study found that Ago levels are increased upon dMyc, as well as upon Puf overexpression. Although the mechanisms by which dMyc and Puf regulate Ago expression are unclear, dMyc-dependent Ago expression may provide a mechanism for dMyc autoregulation, whereas Puf may stabilize Ago by deubiquitylating it. Indeed, Fbw7 has been shown to be regulated through ubiquitylation. A similar type of dynamic relationship has been reported for the ubiquitin ligase Mdm2 and deubiquitylase HAUSP/USP7 in regulating the stability and function of the tumor suppressor p53. Taken together, these data suggest that Ago and Puf represent a regulatory node that controls degradation of Myc and CycE, and very likely other growth control factors. Further studies will be required to identify additional substrates of Puf and to understand the physiological importance of Puf-mediated regulation of protein degradation in Drosophila (Li, 2013).

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development

Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo, and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Whether Nedd8 affects the protein level of CycE was examined. Levels of CycE are regulated by the F-box protein, Archipelago. CycE accumulates in Nedd8 mutant cells in the eye disc. These results suggest that Nedd8 might affect the stability of a broad range of proteins through F-box proteins in flies (Ou, 2002).

Drosophila cyclin E interacts with components of the Brahma complex

Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, a genetic screen was carried out using a hypomorphic mutation of Drosophila cyclin E (DmcycEJP), which gives rise to adults with a rough eye phenotype. Among the dominant suppressors of DmcycEJP, brahma (brm) and moira (mor) were identified. These genes encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycEJP eye phenotype. Brm complex mutants suppress the DmcycEJP phenotype by increasing S phases without affecting DmcycE protein levels. DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G1 arrest (Brumby, 2002).

Several recent studies have provided strong connections between metazoan SWI- SNF complexes and regulation of the cell cycle. In yeast, the SWI- SNF complex is not essential for viability, and whole genome analyses of swi/snf mutants have shown roles in activation and repression of transcription. A screen for modifiers of E2F1/DP function in Drosophila identified new alleles of brm and mor as enhancers of the rough eye phenotype associated with ectopic expression of E2F1 and DP in the developing Drosophila eye imaginal disc. In support of this, mammalian homologs of Brm and Mor (hBrm/Brg1 and BAF55, respectively) have been reported to be present in cyclin E complexes and to be phosphorylated by cyclin E- Cdk2. Significantly, human homologs of Brm (hBrm and Brg1) inhibit entry into S phase and achieve this at least in part by cooperation with the tumor suppressor Rb. Furthermore, Rb can bind to Brg1 and hBrm, and the ability of Rb to induce G1 arrest has been shown to depend upon hBrm and Brg1 (Brumby, 2002 and references therein).

The genetic interactions with DmcycE or E2F1/DP and Brm complex genes initially were thought to be due to effects on DmcycE transcription or E2F/DP-dependent transcription, given the role of the Brm complex in transcriptional regulation. Surprisingly, the results of this study suggest that the Brm complex functions downstream of DmcycE transcription and protein accumulation. (1) No significant effect on DmcycE protein levels in DmcycEJP eye discs was observed when the dosage of brm or mor was halved. (2) The rough eye phenotype due to overexpression of DmcycE from the GMR driver is enhanced by halving the dosage of brm and mor, indicating that Brm and Mor act to inhibit S phase entry downstream of DmcycE transcription. (3) DmcycE forms a complex with Brm and Snr1. Taken together, these data provide strong evidence that the Brm complex does not inhibit the G1 to S phase transition by acting to down-regulate DmcycE transcription (Brumby, 2002).

It is also likely that the Brm complex does not act to down-regulate E2F1/DP-dependent gene transcription, since no effect was observed for at least two E2F1/DP targets in brm mutants. Thus, mutations in Brm complex genes suppress the DmcycEJP mutant phenotypes by allowing progression into S phase without increasing either DmcycE protein levels or the expression of E2F1/DP-dependent genes. This suggests that one function of the Drosophila Brm complex is to restrict entry into S phase by inhibiting DmcycE-Cdk2 activity or by acting downstream of DmcycE-Cdk2 function. A function for Brm downstream of DmcycE-Cdk2 is consistent with reports that mammalian cyclin E can bind to and phosphorylate components of the Brm complex and thereby inactivate it. Thus the Brm complex may be acting as a curb to S phase entry that needs to be overcome by phosphorylation and inactivation by cyclin E-Cdk2 (Brumby, 2002).

Consistent with studies in cultured mammalian cells, the Rbf1 protein was found to be present in complexes with Brm or Snr1 in larval and embryonic extracts. However, in embryos, only a small portion of total cellular Rbf1 is present in Snr1 immunoprecipitates, in contrast to a significant fraction of the cellular DmcycE, suggesting that most Brm complexes do not contain Rbf1. The observation that Drosophila Rbf1 and Brm form a complex in vivo is consistent with studies in mammalian cells showing that hBrm and/or Brg1 can bind to and cooperate with Rb in transcriptional repression, and that hBrm and Brg1 are required for Rb-induced G1 arrest. However, in Drosophila, no clear evidence was obtained for cooperation of brm or mor with rbf1 in S phase entry. It is possible that the phenotypes being examining were not sensitive enough for S phase effects to be observed. However, the lack of a strong effect of Brm complex mutants on the rbf1 mutant S phase phenotype, when strong genetic interactions were observed with Brm complex genes and DmcycE, suggests that Rbf1 and Brm primarily function independently in negatively regulating S phase entry. Therefore, the suppression of the S phase defect of DmcycEJP by Brm complex mutants may not involve rbf1. Independent roles for Brm and Rb are also likely in mammalian cells since Rb knockout mice have a different mutant phenotype from that of Brg1 or Brm knockouts (Brumby, 2002).

In mammalian cells, Rb can form a complex containing both Brg1 and Hdac1, which is required to repress DmcycE transcription and may also have a role at replication origins. However, reducing the dose of the Drosophila Hdac gene, rpd3, did not suppress the DmcycEJP rough eye phenotype. It is possible that no interaction was observed for rpd3 and DmcycE, because there are a least three other Hdacs in flies that may perform overlapping functions with rpd3. However, mutations in sin3A, which encodes a Hdac-interacting protein, enhance the DmcycEJP rough eye phenotype, suggesting that Sin3a functions in opposition to Brm in regulating DmcycE or S phase entry. Further studies using specific mutations in other Drosophila Hdacs, and Hdac-interacting proteins are required to analyze further their role in the G1 to S phase transition (Brumby, 2002).

How does the Brm complex mediate negative regulation of the G1 to S phase transition? The results suggest that the Brm complex is playing a role independent of DmcycE transcription and E2F/DP-dependent transcription in negatively regulating the G1 to S phase transition. One way in which this may occur is by transcriptional regulation of other critical G1/S phase genes. For example, there is evidence that in Drosophila, the Brm complex is important in negatively regulating Armadillo-dTCF target genes in the Wingless signaling pathway. Although as yet there have been no studies showing directly that G1/S phase-inducing genes are targets of the Wingless signaling pathway in Drosophila, this is possible based on studies in mammalian cells. Furthermore, the Wingless pathway clearly has a role in cell proliferation in some Drosophila tissues. Whether this is the mechanism by which the Brm complex mediates negative regulation of cell cycle entry requires further investigation (Brumby, 2002).

Another way in which the Brm complex may function is by restricting or regulating access to chromosomal origins of replication. Several studies have shown that ATP-dependent chromatin remodeling is important for modulating the initiation of chromosomal DNA replication. The data are consistent with the view that the Brm complex may play a role in this process, possibly functioning to restrict entry into S phase by acting directly to remodel nucleosomes at replication origins. In this scenario, DmcycE-Cdk2 may then act to phosphorylate and inactivate the Brm complex, allowing assembly or function of the pre-replication complex and replication origin firing. Indeed, cyclin E-Cdk2 has been shown to be recruited by the Cdc6 pre-replication complex protein to replication origins at the G1 to S phase transition (Brumby, 2002).

Intriguingly, recent studies have shown that the E2F/DP complex also acts directly at replication origins. In the amplification of the chorion gene clusters during the ovarian follicle cell endoreplicative cycles, it has been shown that E2F1/DP is important in localizing the origin of replication complex specifically to the chorion gene origins and activating replication, and that Rbf1 is important in limiting DNA replication. This mechanism is not limited to these specialized cycles, since transcription-independent roles for E2F1 in inducing S phase have also been documented in the eye imaginal disc. Taken together, these studies suggest that the E2F1/DP-Rbf1 complex plays a non-transcriptional role in S phase by acting directly at DNA replication origins. In mammalian cells, a similar non-transcriptional role for Rb in DNA replication inhibition has been demonstrated, possibly through its functional association with the pre-replication complex protein Mcm7 and its localization to replication foci (Brumby, 2002).

Given the data for a role for Rb-E2F/DP directly at replication origins and the evidence that chromatin remodeling is important in replication initiation, it is possible that Brm and Rbf1 may both have a role at replication origins to prevent premature origin firing in G1. However, the failure to detect a genetic interaction between brm complex genes and rbf1 suggests that they also have other important roles, independent of each other, in the G1 to S phase transition (Brumby, 2002).

In summary, these results have shown that mutations in genes encoding components of the Brm chromatin remodeling complex can dominantly suppress a DmcycE hypomorphic allele by increasing the number of S phase cells without affecting cyclin E protein levels. Consistent with this view, DmcycE physically interacts with Brm and Snr1. Although a complex was also observed between the Brm complex and Rbf1, no genetic interactions have been detected between Brm complex genes and rbf1, suggesting that Rbf1 and Brm function largely independently in negatively regulating the G1 to S phase transition. Taken together, these data suggest that the Brm complex negatively regulates entry into S phase, possibly in partial collaboration with Rbf1, and that this negative regulation can be abrogated by the action of cyclin E at the G1 to S phase transition (Brumby, 2002).

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The exit from mitosis and/or progression through G1 requires the inactivation of cyclin-dependent kinases, mediated by the APC/C-dependent destruction of cyclins. APC/C is regulated by multiple mechanisms, such as phosphorylation and by spindle checkpoints. Key factors for APC/C function and regulation are the WD proteins Cdc20 and Hec1/Cdh. These proteins seem to bind directly to substrates and recruit them to the APC/C core complex. Importantly, Cdc20 and Hec1/Cdh bind and activate APC/C in a sequential manner during mitosis. APC/C-Cdc20 is activated at the metaphase/anaphase transition, and gets replaced by APC/C-Hec1/Cdh in telophase. This second complex remains active in the subsequent G1 phase. In Drosophila the homolog of Hec1/Cdh, Fzr, also induces the APC/C-complex-dependent proteolysis of CycA and B and is required for the G1-phase progression. Fzr is required for cyclin removal during G1 when the embryonic epidermal cell or follicle epithelial proliferation stops and the cells enter endocycles. Premature Hec1/CdhFzr transcription in follicle cells is sufficient to block mitosis and initiate precocious endocycling. This suggests that Fzr is a powerful player in the mitotic-to-endocycle switch, yet regulation of other components is also required for the efficiency of this process. Regulators of G1-S transition, such as Dacapo/CIP/KIP, which also turns out to be a Notch-regulated component, possibly abort premature attempts by follicle cells to enter the endocycle (Shcherbata, 2004).

In addition to Myc and Cyclin D, Cyclin E also plays an important role in the regulation of the G1/S-transition. Cyclin E binds to and activates the cyclin-dependent kinase Cdk2, and thereby promotes the transition from G1 to S. Oscillation of Cyclin E activity is a mechanism responsible for the timely inactivation of this G1 cyclin/Cdk complex and an arrest in cell proliferation. The oscillation of Cyclin E level is controlled partly by a SCF-ubiquitin-dependent proteolysis. Fluctuations of Cyclin E are critical for multiple rounds of endocycles. Cyclin E is critical for endocycles in follicle cells as well, and this analysis shows that the CycE level is controlled by an SCF-regulator, F-box protein, Ago/hCdc4/Fbw7. Fbw7 (Ago) associates specifically with phosphorylated Cyclin E, and catalyzes Cyclin E ubiquitination in vitro. Depletion of Ago leads to accumulation and stabilization of Cyclin E in vivo in human and D. melanogaster. This leads to increased mitosis in certain mammalian and Drosophila cell types. In addition, ago loss-of-function clones in the germ line will cause extra mitotic divisions or, in contrast, cell cycle arrest and polyploidy. However, increased Cyclin E levels observed in ago loss-of-function mutant clones do not affect the mitotic cycles in follicle cells but do halt the transition to endocycles that normally occurs at stage 6 (Shcherbata, 2004).

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 in both Drosophila and human cells was analyzed. Loss of CUL4 in Drosophila cells causes G(1) cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G(1) cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27(Kip1) stabilization and G(1) cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G(1) cell cycle progression in both Drosophila and human cells (Higa, 2006).

The COP9 signalosome promotes degradation of cyclin E during early Drosophila oogenesis

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes defects similar to those caused by mutations in CSN or SCFAgo subunits -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).

To investigate cyst formation and differentiation in CSN5 germaria, wild-type and CSN5 ovaries were stained with anti-Hts antibody to highlight the fusome that connects all the cells of a cyst through the ring canals. Fusome development is essential for germline cyst formation. In CSN5 mutant germaria the fusome was often less branched, and sometimes there were more individual fusomes than in wild-type germaria. Furthermore, spherical spectrosomes (fusome precursors) are frequently found in more posterior regions of the germaria, probably indicating retarded fusome development. CSN5 null mutant clones eventually cease mitotic divisions and often become enormously polyploid. Along with the increase in DNA, these cells often contain oversized spectrosomes or structures similar to a fragmented fusome, indicating dramatic changes in fusome development. Some mutant clones lacked spectrosomes/fusomes. Usually, these clones were found a significant time after heat shock and were localized in ovarioles with no subsequent germline development. CSN4N mutant clones show similar undifferentiated cysts with enlarged cell nuclei and defective fusome development. These data suggest that the intact CSN complex is required for proper cyst divisions and fusome development. The polyploid, nondividing germ cells may be the germline stem cells. More than three of these large polyploid cells are never seen in a particular germarium, and they retain contact with somatic cells that probably correspond to the basal and terminal filament cells of normal germaria (Doronkin, 2003).

The Drosophila F box protein Archipelago (Ago) has been proposed to target Cyclin E for ubiquitin-mediated degradation in imaginal discs. The hypomorphic alleles ago1, ago3, and ago4 were used to test for a similar role in Cyclin E degradation during oogenesis. Immunostaining shows that ago mutant clones marked by lack of GFP persistently accumulate Cyclin E at high levels. With one addition, these clones showed a similar range of phenotypes as those seen in CSN5 or CSN4 mutants or after overexpression of Cyclin E. Some mutant cysts had extra nurse cells and some had fewer than normal, and many were degenerating. Some cysts had been arrested after the stem cell division and some of these single-cell cysts were polyploid. Prominent in the ago clones was a phenotype that had not been previously noticed. Cyclin E accumulation in ago clones correlates with significantly DAPI-bright regions in nurse cell nuclei. Because these regions are likely to include heterochromatic sequences that are usually underreplicated during endoreplication, their enlargement may indicate a more complete replication of both heterochromatic and euchromatic sequences in ago clones. Although enlarged heterochromatin-rich regions are occasionally seen in CSN5 mutants and after Cyclin E overexpression, this phenotype is stronger in ago mutants, possibly suggesting a more specific role for ago in regulation of late replication (Doronkin, 2003).

In addition to the similar phenotypes between ago mutants and CSN, Nedd8, or cullin1 mutants, dominant interactions were found between ago and CSN mutants. CSN5/ago and CSN4/ago double heterozygotes show familiar ovarian defects: extra cystocyte divisions, fewer divisions but higher ploidy, and apoptotic egg chambers. These defects are very similar to the CSN5 mutant phenotype and to defects in oogenesis induced by Cyclin E overexpression. In addition, these double heterozygotes have enlarged heterochromatic regions in nurse cell nuclei, suggesting mutual CSN-ago control of late replication (Doronkin, 2003).

The regulatory lid of the proteasome is an eight-subunit complex that is closely related to the CSN. It appears to be necessary for the removal of ubiquitin side chains from the target protein as it is fed into the barrel of the proteasome for proteolysis. A mutation in the RPN6 subunit of the regulatory lid was tested for genetic interactions in oogenesis with CSN4 and CSN5 mutations. Both double heterozygotes show a strong interaction and the full range of CSN5-like ovarian defects, including apoptosis, incorrect number of mitotic divisions, and fusions of neighboring egg chambers (Doronkin, 2003).

The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27kip1 (Doronkin, 2003).

The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).

Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).

Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).

One of the important results of the current work is the demonstration that the CSN regulates the cell cycle in ovaries primarily through the turnover of Cyclin E. The apparent perdurance and gradual dilution of wild-type CSN5 protein in genetically null germline clones shows that reduced Cyclin E degradation affects both cell division and DNA replication. Slight reductions cause an extra division of the cystocytes. In contrast, continuous or strong accumulation of Cyclin E in null CSN5 mutants is able to reduce or stop cell divisions though often allowing endoreplication to continue. This switch from overproliferation to inhibition of cell divisions is sometimes visible in a single CSN5 mutant ovariole as the wild-type CSN5 protein is diluted by stem cell divisions. These observations support the view that different Cyclin E levels can lead to distinct and sometimes opposite effects (Doronkin, 2003).

Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).

Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).

In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).

Drosophila skpA, a component of SCF ubiquitin ligases, regulates centrosome duplication independently of cyclin E accumulation

Skp1 proteins function in protein degradation as a component of the SCF (SKP1, cullin/CDC53, F-box protein) complex to link the substrate-recognition subunit (F-box protein) to a cullin (see Drosophila Cullin1) that in turn binds the ubiquitin-conjugating enzyme. Centrosome duplication must be coupled to the main cell cycle to ensure that each cell has precisely two centrosomes at the onset of mitosis. Supernumerary centrosomes are commonly observed in cancer cells, and may contribute to tumorigenesis. Drosophila SkpA, the Skp1 component of Drosophila SCF ubiquitin ligases, regulates the link between the cell and centrosome cycles. Lethal skpA null mutants exhibit dramatic centrosome overduplication and additional defects in chromatin condensation, cell cycle progression and endoreduplication. Surprisingly, many mutant cells are able to organize pseudo-bipolar spindles and execute a normal anaphase in the presence of extra functional centrosomes. SkpA mutant cells accumulate higher levels of cyclin E than wildtype cells during S and G2, suggesting that elevated cdk2/cyclin E activity may account for the supernumerary centrosomes in skpA- cells. However, centrosome overduplication still occurs in skpA-; cycE- mutant animals, demonstrating that high cyclin E levels are not necessary for centrosome overduplication. These data suggest that additional SCF targets regulate the centrosome duplication pathway and that Drosophila SkpA regulates centrosome duplication independently of cyclin E accumulation (Murphy, 2003).

This study has directly tested the role of cyclin E in centrosome overduplication by genetically manipulating cyclin E levels in wildtype and skpA- cells. Strikingly, drastically reducing cyclin E levels with a near-null allele does not suppress centrosome overduplication in cycling skpA- cells. One possibility is that cyclin E is not required for centrosome duplication in Drosophila. This seems unlikely, because Drosophila cdk2 does not associate with cyclin A and lacks in vitro kinase activity when immunoprecipitated from cyclin E-deficient embryos, and other functions of cdk2 are conserved between Drosophila and vertebrates. In any case, centrosome overduplication occurs independently of SCF control of cyclin E accumulation (Murphy, 2003).

How do SCF components regulate centrosome duplication? One possibility is that simply lengthening the cell cycle introduces enough time for multiple cycles of centrosome duplication to occur. Although this model cannot be ruled out, it seems unlikely given that a centrosome must duplicate in as little as 55 minutes in a cycling neuroblast but does not reduplicate in the 12-hour cycle of an imaginal wing disc cell. Furthermore, slowing the cell cycle in abdominal histoblasts by overexpressing the Drosophila retinoblastoma-family protein RBF is not sufficient to induce centrosome overduplication (Murphy, 2003).

Instead, the idea is favored that a target of SCF-mediated degradation acts as a Centrosome Licensing Factor (CLiF) that limits centrosome duplication to once per cell cycle. CLiF would be expressed early in the cell cycle, loaded onto centrosomes, and excess CLiF would be targeted to the proteasome by an SCF complex. One cycle of centrosome duplication could then be triggered by Cdk2-E activity, but the daughter centrosomes would not be relicensed until the next cell cycle. SCF mutants would fail to degrade excess CLiF, allowing duplicated centrosomes to relicense and reduplicate in the course of a single cell cycle. One candidate CLiF is nucleophosmin/B23, which is phosphorylated by Cdk2-E and associates specifically with unduplicated centrosomes (Okuda, 2000). Future experiments will need to determine if nucleophosmin/B23 or other candidate CLiFs are targeted for degradation by an SCF complex (Murphy, 2003).

Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis: Encore physically interacts with cyclin E and Cul1

In Drosophila, egg development starts at the anterior tip of the ovary, in the germarium, where the germline stem cells divide to produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of each egg chamber are connected by intercellular bridges called ring canals. Exit from the cell cycle at the end of these four mitotic divisions requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit this division program (Ohlmeyer, 2003).

In encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the ubiquitin-protease pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of three factors, Cul1, phosphorylated Cyclin E, and the proteasome 19S-RP subunit S1, with the fusome is affected in encore mutant germaria. It is proposed that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis (Ohlmeyer, 2003).

Overexpression or loss-of-function mutations in a third group of genes such as Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32 or 8 cells. These genes do not affect fusome integrity and thus timing and spatial characteristics of cell division appear to be intact. The encore gene belongs to this group of genes: its product is necessary for exit from mitosis. Loss of Encore activity results in egg chambers containing 32 rather than 16 cells. Mutations in the encore gene produce additional phenotypes, which show differential temperature sensitivity. encore mutant females raised at 18°C produce egg chambers with 16 cells, but they give rise to ventralized eggs. The extra cell division phenotype is only observed when encore mutant females are raised at high temperatures (25°-29°C). The encore gene encodes a 200 kDa protein with no homolog of a defined biochemical function. The mechanism by which Encore promotes exit from the cell cycle after four germline mitoses has been investigated (Ohlmeyer, 2003).

Cell cycle progression is controlled by a series of cell cycle dependent kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin subunits, by Cdk inhibitors (CKI) and by post-translational modification of the Cdk subunit through both activating and inactivating phosphorylation. Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo and eye imaginal disc. In addition, exit from the cell cycle requires destruction of the cyclins by the ubiquitin-proteasome system (UPS). The addition of ubiquitin requires three different activities; the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is presented to the proteasome, isopeptidase activities in the 19S-recognition particle (RP) of the proteasome cleave the ubiquitin tail, the protein is unfolded and finally destroyed by the proteasome 20S-core particle (CP) (Ohlmeyer, 2003).

There are two E3 enzyme complexes that regulate the cell cycle progression. The first, the APC/cyclosome, regulates progression from G2 to M phase transition. The second, the SCF complex regulates the G1 to S phase transition. The SCF complex is composed of Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via an interaction between the F-box component and the phosphorylated target protein. In Drosophila and mammalian systems, mutations in the Cul3 and Ago genes have been shown to cause the accumulation of Cyclin E, entry to S-phase and doubling of cell number. Thus, proper regulation of the destruction machinery is important for maintaining normal levels of Cyclin E and assuring proper cell cycle progression (Ohlmeyer, 2003).

The work presented in this study demonstrates that the encore gene product associates with the SCF-ubiquitin-proteasome system and is required for proper exit from germline mitosis. The failure to downregulate Cyclin E after four cell divisions in conjunction with an accumulation of Cyclin A protein provide the conditions to promote an extra cell division. Encore can bind to Cul1, Cyclin E-Ub(n) and the proteasome. Cul1 and the proteasome 19S-RP subunit S1 are associated with the fusome and these associations are very much attenuated in encore mutant ovaries. It is proposed that as a direct consequence, Cyclin E is not degraded properly, its activity is misregulated and the cyst undergoes one extra cell division (Ohlmeyer, 2003).

This study shows that the fusome is a regulator of cell division during early oogenesis. Some of the functions ascribed to the fusome are to synchronize cyst mitosis and to provide the scaffold for the transport system necessary for oocyte determination. Limiting the number of cell divisions in the germarium could be achieved by regulating the association of proteins such as the cyclins and/or other cell cycle regulators with the fusome. The expression pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium supports the idea that the fusome plays an important role in the regulation of mitosis. Indeed, Cyclin A association with the fusome is transient and occurs only during cyst division. In encore mutant germaria, Cyclin A remains associated with the fusome after cell division has stopped. Cul1 localization to the fusome suggests that the rest of the SCF complex also associates with the fusome and that substrate ubiquitination may happen at the fusome. The SCF component Cul1 is mainly associated with the fusome in the wild-type germaria. In encore mutant germaria, Cul1 localization to the fusome is very poor, leading to a proposal that this may be one reason why Cyclin E is not degraded properly. This also suggests that the degradation of Cyclin E and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome. The association of P-Cyclin E supports this idea. The localization of P-Cyclin E in the wild type seems to be dynamic, consistent with the idea that the phosphorylated substrate is localized to the fusome, and then rapidly degraded via the SCF-UPS. In encore mutant germaria, the poor localization of Cul1 may result in an inefficient assembly of SCF complexes at the fusome. P-Cyclin E is localized to the fusome, but its degradation is compromised and as a result a consistent expression of P-Cyclin E is observed at the fusome. The partial association of the proteasome 19S-RP subunit S1 to the fusome supports the idea that proteolysis may occur at the fusome. The proteasome 19S-RP would recognize the polyubiquitinated substrate and recruit the rest of the proteasome to the fusome (Ohlmeyer, 2003).

The results suggest that Encore can associate with the SCF ubiquitin-proteasome system machinery and assists with the degradation of Cyclin E and perhaps other SCF substrates. Since the mutant Encore protein can still interact with SCF-UPS components, the mutant protein may form complexes but these might be inactive and/or the mutant protein poisons the degradation machinery. Consistent with such a hypothesis, the encore extra cell division phenotype is milder in hemizygous versus homozygous females at 25°C (Hawkins, 1996). Encore is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably the rest of the proteolysis complex to the fusome. This localization may be more crucial at 29°C, whereas at lower temperatures a less efficient degradation system may have enough time for normal cell cycle regulation. encore mutations do not affect the 20S-Core Particle activity as measured by the rate of degradation of a fluorogenic peptide. It is not known whether Encore retains Cul1 at the fusome or whether Encore directly or indirectly modifies Cul1 in order to promote its localization at the fusome. Cul1 is known to be modified by the addition of Nedd8; however, Cul1 seems to be equally neddylated in encore and wild-type ovary extracts (Ohlmeyer, 2003).

In summary, the results suggest that the Encore protein assists with proper cell cycle progression in the Drosophila germarium by ensuring that Cul1 and the proteolysis machinery is localized at the mitosis coordination center, the fusome (Ohlmeyer, 2003).

Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. The developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms, has been studied. Phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, these results indicate that stringent regulation of Dup protein is critical to protect genome integrity (Thomer, 2004).

To determine whether oscillation of Dup protein levels during cell cycles is due to Dup protein degradation at G1/S, Dup expression within the synchronized cell cycles of the larval eye primordium was examined. Late in third instar, a wave of differentiation sweeps across the eye imaginal disc, which is visible as a morphogenetic furrow (MF). Cells are synchronized in G1 upon entering the furrow. Specific cells posterior to the furrow then enter a synchronous S phase, which is visible as a stripe of BrdU labeling. Labeling with affinity-purified rabbit polyclonal Dup antibody indicates that the protein is abundant in nuclei of late G1 cells, but is undetectable in S phase cells incorporating BrdU. Labeling with a guinea pig anti-Dup antibody gave identical results suggesting that immunolabeling reflects Dup protein in vivo. Double labeling for Dup and cyclin E indicates that both are abundant in nuclei of cells in late G1, but then Dup rapidly declines while cyclin E persists into S phase. Labeling for the G2 and M phase marker cyclin B also indicates that Dup levels decline significantly before cells enter G2. Similar results were obtained for the non-synchronized cell cycles in the eye and other imaginal discs. This rapid decline in protein is primarily due to post-transcriptional regulation because in situ hybridization indicates that dup mRNA persists after G1. Moreover, expression of a dup transgene from the strong hsp70 promoter does not result in detectable Dup protein during S phase. The data suggest that, similar to Cdt1 in humans and other organisms, Dup protein is abundant in G1 when origins are licensed, but is then rapidly degraded when cyclin E appears at G1/S (Thomer, 2004).

Beginning in late mitosis, origins of replication are prepared for replication by binding of a pre-replicative complex (pre-RC), which is subsequently activated to initiate replication at the onset of S phase. The building of the pre-RC onto origins in late mitosis/early G1 is a stepwise process. The origin recognition complex (ORC) serves as a scaffold for subsequent association of Cdc6 and Cdt1, both of which are required to load the Minichromosome Maintenance (MCM) complex, the replicative helicase. Once MCMs are loaded, the origin is considered to be licensed for subsequent replication. Cdc7 kinase, with its activating subunit Dbf4, and CDK2 kinase, activated by cyclin E or cyclin A, are then required for initiation of replication. Initiation is associated with departure of Cdc6, Cdt1, MCMs, and, in multicellular eukaryotes, certain ORC subunits from the origin. Continued CDK activity in S, G2, and early M phases inhibits reassembly of the pre-RC to block origin refiring. Unique to multicellular eukaryotes is another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex. It is only after Geminin and cyclins are degraded at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes (Thomer, 2004 and references therein).

It is likely that part of the cyclin E/CDK2 dependent regulation is direct because Dup associates with CDK2 protein and activity in embryos. The results of the mutagenesis show that the N terminus of Dup is necessary and sufficient for degradation at G1/S. Mutation of the CDK sites in the N terminus, however, only partially stabilize the protein, suggesting the existence of other CDK2-dependent mechanisms for degradation. It is crucial to tightly regulate the abundance of Dup protein because its over-expression is sufficient to induce a full genome reduplication and cell death in the ovary and imaginal discs. The different effects on amplification and genomic replication suggest that phosphorylation of the N terminus of Dup protein may be required for replication fork elongation during amplification and provides insight into the mechanism of this developmentally specific replication program (Thomer, 2004).

The results suggest that cyclin E/CDK2 phosphorylates the Dup N terminus contributing to its instability at G1/S. Dup was degraded during periodic endocycle S phases that are solely regulated by oscillating cyclin E/CDK2, further supporting a link between this kinase and Dup degradation. Although the N terminus was necessary and sufficient for degradation, mutation of the ten N-terminal CDK sites within Dup 10(A) only partially stabilized the protein. This suggests that there are other cyclin E/CDK2-dependent mechanisms that trigger Dup degradation independent of these ten sites during S phase. It has been noted that the C terminus of Dup contains a PEST sequence, and there are several serines and threonines in the C terminus that are potential targets of phosphorylation. Although the requirement for these sites has not been directly tested, the stability of C-Dup indicates that they are not sufficient for degradation at G1/S. To explain these results, a bi-phasic degradation model is suggested where cyclin E/CDK2 phosphorylation promotes Dup degradation in late G1, whereas other fail-safe mechanisms become operative only during S phase. This would explain why inhibiting CDK2 and S phase entry with GMRp21 completely blocked Dup degradation (Thomer, 2004).

A number of recent publications describe results for Cdt1 in human cells that are similar to those in flies. These results suggest that cyclin A/CDK2 phophorylates the human Cdt1 N terminus, which enhances its binding to the Skp2 subunit of the SCF ubiquitin ligase. Like Dup, non-phosphorylatable Cdt1 mutants are only partially stabilized, but simultaneously inhibiting CDK2 and S phase entry with p21 completely blocks degradation. Previous evidence in C. elegans, human and Drosophila cells have suggested that destruction of Cdt1 may be mediated by two ubiquitin ligases, an SCF complex containing Skp2, and an SCF-like complex based on Cul4. For many substrates of the SCF, prior phosphorylation is required for their subsequent recognition and ubiquitinylation, including substrates phosphorylated by CDK2 at G1/S. It is not known whether prior phosphorylation is required for substrate recognition by Cul4-based ubiquitin ligases. It is tempting to speculate, therefore, that the bi-phasic degradation of Cdt1 that may reflect its modification by two distinct ubiquitin ligases: a phosphorylation-dependent ubiquitinylation by the SCF complex, and a phosphorylation-independent ubiquitinylation by a Cul4-based complex. Clearly, more experiments are needed to sort out the complexity of this regulation. Nonetheless, the similar results from flies and humans suggest that tight regulation of Cdt1 abundance is a generally conserved and important mechanism to protect genome integrity in eukaryotes (Thomer, 2004).

Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

Cyclin A expression is only required for particular cell divisions during Drosophila embryogenesis. In the epidermis, Cyclin A is strictly required for progression through mitosis 16 in cells that become post-mitotic after this division. By contrast, Cyclin A is not absolutely required in epidermal cells that are developmentally programmed for continuation of cell cycle progression after mitosis 16. These analyses suggest the following explanation for the special Cyclin A requirement during terminal division cycles. Cyclin E is known to be downregulated during terminal division cycles to allow a timely cell cycle exit after the final mitosis. Cyclin E is therefore no longer available before terminal mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence, Cyclin A, which can also function as a negative regulator of Fizzy-related/Cdh1, becomes essential to provide this inhibition before terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1 activity results in the premature degradation of the Cdk1 activators Cyclin B and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without these activators, entry into terminal mitoses is not possible. However, entry into terminal mitoses can be restored by the simultaneous expression of versions of Cyclin B and Cyclin B3 without destruction boxes, along with a Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover, terminal mitoses are also restored in Cyclin A mutants by either the elimination of Fizzy-related/Cdh1 function or Cyclin E overexpression (Reber, 2006).

Mitotic cyclins accumulate during the S and G2 phases of the cell cycle. Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1 (Cdk1). Their rapid degradation during late M and G1 phase depends on the D- and KEN-boxes in their N-terminal domains. These degradation signals are recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during M and G1, respectively. The ubiquitin ligase activity of the APC/C allows cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).

Metazoan species express three different types of mitotic cyclins: A, B and B3. The specific functions of these different cyclins are not understood in detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their functional specificity in Drosophila melanogaster. In this organism, development to the adult stage requires the zygotic function of CycA, but not of CycB or CycB3. Initial analysis of the embryonic cell proliferation program in CycA mutants revealed that epidermal cells fail to progress through the sixteenth round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer dependent upon completion of the preceding S phase. The failure of mitosis 16 in CycA mutants therefore does not simply result from the activation of a DNA replication or damage checkpoint -- a possibility suggested by evidence obtained in vertebrate cells in which Cyclin A binds not only to Cdk1 but also to Cdk2, and provides crucial functions during S phase (Reber, 2006 and references therein).

The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also occurs in CycA mutants, complicates the explanation of why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A. Conversely, mitosis is clearly inhibited in cultured human cells after the microinjection of antibodies against cyclin A. Cyclin A-Cdk1 complexes are thought to have special properties, important for starting up a positive-feedback loop that confers a switch-like behavior on the Cdk1 activation process. In this feedback loop, Cdk1 activity results in phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in phosphorylation and activation of the String/Cdc25 phosphatase, which removes the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the sixteenth round of mitosis during embryogenesis is the last cell division for the great majority of the epidermal cells (Reber, 2006).

After mitosis 16, most epidermal cells enter a G1 phase and become mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses 1-15) are followed by an immediate onset of S phase. The G1 phase after mitosis 16 is therefore the first G1 phase during development. Entry into this G1 phase is dependent upon a complete, developmentally controlled inactivation of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry into S phase. Cyclin E-Cdk2 inactivation results from transcriptional CycE downregulation and concomitant upregulation of dacapo, which encodes the single Drosophila CIP/KIP-type inhibitor specific for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).

Although cycle 16 is the final division cycle for most epidermal cells, some defined regions do not activate the cell cycle exit program during cycle 16. Instead, they maintain CycE expression, enter S phase immediately after mitosis 16 and complete an additional division cycle 17. In these regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A is therefore especially important for terminal mitoses preceding G1 and cell cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle regulator Cyclin A therefore depends on the developmental context (Reber, 2006).

The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality. This study has clarified the molecular basis of the distinct importance of Cyclin A. The results indicate that the crucial role of Cyclin A is linked to its ability to inhibit Fzr-APC/C-mediated degradation. Moreover, this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation pathway is of particular importance for progression through the very last mitotic division preceding cell cycle exit and the proliferative quiescence of epidermal cells during embryogenesis. This particular Cyclin A requirement during terminal divisions is caused by a cell cycle exit program that is initiated already before the terminal mitosis. The cell cycle exit program includes downregulation of Cyclin E-Cdk2, which has a comparable ability to inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an indispensable inhibitor of the premature degradation of mitotic cyclins and String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).

The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated degradation complicates the interpretation of the results. Two findings, however, strongly suggest that Cyclin A functions not just downstream of Fzr, but also upstream as a negative regulator. The observed premature loss of B-type cyclins in CycA mutants is readily explained by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression of the UAS-fzr overexpression phenotype by co-expression of UAS-CycA, which is described here, includes the re-accumulation of B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).

Work in mammalian cells has clearly established that Cyclin A functions as a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1. Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzrpsm variant no longer contains canonical Cdk consensus phosphorylation sites (S/T P) and yet its activity is still suppressed by CycA overexpression. Fzr inhibition by CyclinA-dependent phosphorylation of non-consensus sites remains a possibility in Drosophila. However, it is pointed out that, apart from a potential control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like Drosophila protein Rca1. Rca1 overexpression has been shown to prevent premature Cyclin B degradation and restore mitosis 16 in the epidermis of CycA mutant embryos. Based on these observations, the failure of mitosis 16 in CycA mutants was proposed to reflect premature Fzr activation, a suggestion fully confirmed by the current work. It is conceivable, therefore, that the Cyclin A-mediated suppression of Fzrpsm activity involves Rca1 or other unknown targets. The fact that not only Cyclin A, but also Cyclin E, effectively suppresses Drosophila Fzr and Fzrpsm provides further support of additional regulatory complexity. In vertebrate systems, only Cyclin A and not Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).

The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila embryogenesis, which precedes cell cycle exit and proliferative quiescence. A prominent Cyclin A requirement for terminal mitoses appears to exist in neuroblast lineages during development of the embryonic CNS, although definitive proof will require further work. On the basis of this analysis in epidermal cells, a high Cyclin A requirement for entry into mitosis is expected whenever Fzr levels are high and Cyclin E levels low. During the comparatively slow postembryonic cell cycles of imaginal cells, the periodicity of Cyclin E expression is presumably far more pronounced than during the rapid embryonic cycles in which the persistent presence of maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles, which have a G1 phase, Cyclin E expression might therefore be low before each mitosis, and not just before terminal divisions. In combination with Fzr expression, every imaginal mitosis might therefore be strongly dependent upon Cyclin A. By contrast, in the absence of Fzr, progression through mitosis appears to be almost completely independent of Cyclin A, as is evidenced by the observation that the epidermal cells in fzr CycA double mutant embryos not only progress successfully through mitosis 16, but also complete an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures in these double mutants displayed lagging chromosomes, indicating that cell cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).

The cell cycle exit program, which is activated during the final division cycle in the embryonic epidermis, includes the strong transcriptional upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from the downregulation of Cyclin E and the upregulation of Fzr. Accordingly, genetic elimination of dacapo function should also restore progression through terminal mitosis 16 in CycA mutants. However, mitosis 16 was not observed in the epidermis of dacapo CycA double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears to be insignificant before mitosis 16. After the stage of mitosis 16, however, the epidermal cells in these double mutants entered an endoreduplication cycle, a behavior that is also displayed by some cells in the prospective anterior spiracle region of CycA single mutants. This region does not downregulate Cyclin E during cycle 16 in the wild type, it does not upregulate Dacapo, and it progresses through an additional cycle 17 instead of becoming postmitotic after mitosis 16, in contrast to the great majority of the other epidermal cells. The premature activation of Fzr in CycA mutants, therefore, appears to result in DNA replication origin re-licensing, perhaps as a result of B-type cyclin and geminin degradation. Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells in which it is not effectively eliminated by both Cyclin E downregulation and Dacapo upregulation. Importantly, not all cells in the anterior spiracle region of CycA mutants endoreduplicate, some of the cells still manage to divide. This variability could reflect minor differences in the onset and strength of the zygotic Cyclin E expression. The outcome of insufficient Cyclin A levels appears to be highly dependent on the levels of Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in particular during cell cycle exit. The significance of basic cell cycle regulators in vivo is therefore different in various tissues and developmental stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).

Involvement of CUL4 ubiquitin E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 was examined in both Drosophila and human cells. Loss of CUL4 in Drosophila cells causes G1 cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G1 cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27Kip1 stabilization and G1 cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, it was found that the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G1 cell cycle progression in both Drosophila and human cells (Higa, 2006).

The CUL1 (cullin 1; see Drosophila Cul1) containing SCF (SKP1, CUL1/CDC53, F-box proteins) ubiquitin E3 ligases are key regulators of cell cycle progression from yeast to human. The SCF E3 ligases use different F-box proteins to bind and target various cell cycle regulators for ubiquitin-dependent proteolysis. In mammalian cells, it has been shown that SKP2, an F-box protein, primarily binds and targets phosphorylated CDK inhibitors p27Kip1 and p21Cip1 for ubiquitin-dependent proteolysis, while another F-box protein, human CDC4/AGO/FBXW7 regulates the proteolysis of phosphorylated cyclin E protein. In mammalian cells, the G1 cell cycle is regulated by the relative abundance of G1 cyclin/CDKs and CDK inhibitors such as p27 and p21. Similarly, the Drosophila G1 cell cycle is regulated by the balance between the CDK inhibitor Dacapo, which shares substantial homology to p27, and cyclin E. While cyclin E is regulated by the conserved Drosophila SCFAgo E3 ligase, it is not clear how the level of Dacapo is regulated in the cell cycle (Higa, 2006).

Like other cullin family members, CUL1 is regulated by the covalent linkage of an ubiquitin like protein, NEDD8, through the neddylation activating enzyme E1 (APPBP1 and UBA3) and the E2 enzyme, UBC12. Neddylation of CUL1 dissociates CAND1, an inhibitor of SCF, from CUL1, and consequently promotes the binding of SKP1 and F-box proteins such as SKP2 to CUL1 and the assembly of the SCF E3 ligase complex. The neddylation of CUL1 is removed (deneddylated) by the peptidase activity of the COP9-signalosome complex (CSN; see Drosophila COP9 complex homolog subunit 5). Many lines of evidence suggest that the activity of cullins is regulated by the elegant balance between the neddylation and deneddylation activities (Higa, 2006).

Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase that also contains the UV-damaged DNA-binding protein 1 (DDB1) and Ring finger protein ROC1 (also called RBX1 or HRT1). Unlike Drosophila or other metazoans, mammals encode two paralogues of CUL4, CUL4A and CUL4B. CUL4A and CUL4B are coexpressed in many cells but the functional differences between them remain unclear. Like other cullin E3 ligases, the CUL4 proteins also bind to CAND1 and CSN, and are regulated by neddylation and deneddylation processes. Previous studies suggest that CUL4-containing E3 ligase complexes and CSN regulate the proteolysis of replication licensing protein CDT1 (see Drosophila Cdt/Double parked) in response to UV or gamma-irradiation. Additional studies suggest that DDB1, a potential SKP1-like adaptor for CUL4 E3 ligase is also involved in UV-induced CDT1 proteolysis. The CUL4ADDB1 complex also regulates the proteolysis of c-Jun and DDB2. However, the roles of CUL4-containing ubiquitin E3 ligases in cell cycle regulation remain uncharacterized. This study has investigated the regulation of cell cycle regulators by neddylation and CAND1 and reports the unexpected finding that CUL4 E3 ligase plays a critical role in regulating G1 cell cycle progression (Higa, 2006).

Loss of CUL4 E3 ligases causes a G1 cell cycle arrest that is dependent on CDK inhibitors Dacapo in Drosophila and p27 in human cells. The regulation of Dacapo and p27 by CUL4 E3 ligases occurs at the post-transcriptional levels of protein stability. Although it has not been demonstrated that p27 can be directly polyubiquitinated by the CUL4 E3 ligase complexes in vitro due to technical difficulties, this study raises the possibility that CUL4 E3 ligases may regulate Dacapo or p27 by directly targeting them for ubiquitin-dependent proteolysis. Several lines of evidence support this hypothesis. Dacapo protein is regulated by CUL4 but not by CUL1 in Drosophila cells. Although in human cells, SCFSKP2 regulates p27, there is no structural and functional evidence that SKP2 is conserved in Drosophila cells. In addition, although Dacapo shares substantial homology to p27 or p21 in the core region that mediates cyclin or CDK binding, it diverges greatly at the carboxy terminal end with p27 in which the critical threonine 187 is located for the SCFSKP2- dependent proteolysis of p27 (this threonine is absent in Dacapo). Furthermore, it was found that there are no significant differences in the SCF-dependent p27 degradation between extracts derived from the control and DDB1 or CUL4A siRNA treated cells, suggesting that reduced levels of DDB1 and CUL4A proteins does not significantly affect SCFSKP2 activity. However, these experiments do not completely rule out the possibility that CUL4A/DDB1 are catalytically involved in SCFSKP2-mediated p27 degradation since small amounts of DDB1 and CUL4A proteins remain in the siRNA treated cells. Moreover, although SKP2 represents a major proteolysis pathway for regulating p27 degradation in S phase of human cells, substantial evidence suggests there are additional pathways that regulate the stability of CDK inhibitors. For example, it was found that the Xenopus p27 homologue p27Xic1 is polyubiquitinated on chromatin only when DNA replication starts in the Xenopus egg extracts. Replication licensing protein CDT1 is proteolyzed by CUL4/ROC1 E3 ligase in response to UV or gamma-irradiation. CDT1 is also degraded in S phase in mammalian cells and such an event can be reproduced in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. In C. elegans, loss of CUL4 stabilizes CDT1 in S phase and causes the accumulation of polyploid nuclei containing 100C DNA content. It is possible that CUL4 may also regulate the proteolysis of Dacapo or p27 in similar processes in Drosophila or human cells (Higa, 2006).

Cyclin E protein accumulates in CUL4 silenced Drosophila and human cells often in the absence of CDK inhibitors Dacapo or p27. Although this effect is more pronounced in Drosophila cells, the CUL4 E3 ligase may represent one of several pathways that regulate cyclin E in response to certain signals in mammalian cells. It has been shown that CUL1- and CUL3-containing E3 ligases regulate cyclin E stability in mammalian cells. Cyclin E expression and its protein stability are also regulated by an E2F/DP-1 dependent process. This study found that cyclin E directly interacts with Drosophila CUL4 and human CUL4B and the isolated CUL4A or CUL4B immunocomplexes can polyubiquitinate the associated cyclin E in vitro. These observations raise the possibility that cyclin E may also be a direct ubiquitination target of CUL4 E3 ligases in vivo. These studies indicated that loss of CAND1, APPBP1, or CSN has differential effects on Armadillo/β-catenin and cyclin E. This effect could be partly explained by the observation that while Armadillo is regulated by CUL1-containing SCF ligase, cyclin E is controlled by both CUL1 and CUL4 E3 ligases. Evidence is also provided that the effects of CAND1, APPBP1 or CSN deficiency on the substrates of various cullin E3 ligases may be different. Further analysis is required to investigate the mechanisms for these observations (Higa, 2006).

These data reveal that CUL4 E3 ligase represents a novel and conserved pathway from Drosophila to human cells in regulating CDK inhibitors and cyclin E. In the G1 cell cycle, the CDK inhibitors Dacapo and p27 appear to be the primary targets of CUL4 E3 ligases, since loss of CUL4 in Drosophila or CUL4A in human leads to the G1 cell cycle arrest rather than enhanced S phase entry. Since the gene encoding CUL4A is amplified in many breast cancers and hepatocellular carcinomas and since low or absent expression of p27 is often associated with malignant cancers, these studies also highlight how altered regulation of CUL4 E3 ligase may contribute to the genesis and progression of human cancers (Higa, 2006).

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).

Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).

How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).

More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).

Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).

In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).

How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).

There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).

The Cyclin-dependent kinase inhibitor Dacapo promotes genomic stability during premeiotic S phase

The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, this study identified the p21Cip1/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap-/- females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, this study found that dap-/- ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap-/- DNA damage phenotype. Importantly, the DNA damage observed in dap-/- ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, these data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. dap-/- ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition (Narbonne-Reveau, 2009).

During the meiotic cycle, germ cells complete two divisions to produce haploid gametes. Before the two meiotic divisions, the germ cells duplicate their genomes during the premeiotic S phase. Events unique to the premeiotic S phase, such as the expression of REC8, a member of the kleisin family of structural maintenance of chromosome proteins, are required for the full execution of the downstream meiotic program. How this specialized meiotic S phase is regulated, as well as how similar it is to the mitotic S phase, has long been a question of interest. Studies from yeast indicate that the mitotic cycle and the meiotic cycle use much of the same basic machinery to replicate their genomes. For example, the minichromosome maintenance complex (MCM2-7), which functions as a DNA replication helicase, is essential for the duplication of the genome during both the mitotic and premeiotic S phase. Additionally, both the mitotic and premeiotic S phase require the activity of cyclin-dependent kinases (Cdks). Yet, despite its fundamental importance to both the maintenance of genomic integrity and the downstream events of meiosis, little is known about the regulation of premeiotic S phase metazoa (Narbonne-Reveau, 2009).

Drosophila provides an excellent model to examine the early events of the meiotic cycle, because the entire process of oogenesis takes place continuously within the adult female. In Drosophila, each ovary is composed of 12-16 ovarioles containing linear strings of maturing follicles also called egg chambers. New egg chambers are generated at the anterior of the ovariole in a region called the germarium that contains both germline and somatic stem cells. The germarium is divided into four regions according to the developmental stage of the cyst. Oogenesis starts in region 1 when a cystoblast, the asymmetric daughter of the germline stem cell, undergoes precisely four round of mitosis with incomplete cytokinesis to produce a cyst of 16 interconnected germline cells with an invariant pattern of interconnections (individual cells in the cyst are referred to as cystocytes). Stable actin-rich intercellular bridges called ring canals connect individual cystocytes within the cyst. Germline cyst formation is accompanied by the growth of the fusome, a vesicular and membrane skeletal protein-rich organelle that forms a branched structure extending throughout all the cells of the cyst. After the completion of the mitotic cyst divisions, all 16 cystocytes complete a long premeiotic S phase in region 2a of the germarium. Subsequently, the two cystocytes with four ring canals form long synaptonemal complexes (SCs) and begin to condense their chromatin, suggesting that they are in pachytene of meiotic prophase I. Several of the cells with three ring canals also assemble short SCs, and even cells with only one or two ring canals are occasionally seen to contain traces of SCs. However, as oogenesis proceeds, the SC is restricted to the two pro-oocytes and finally to the single oocyte in region 2b. The other 15 cystocytes lose their meiotic characteristics, enter the endocycle and develop as polyploid nurse cells (Narbonne-Reveau, 2009).

During both the mitotic cycle and the meiotic cycle, it is essential that the entire genome is duplicated precisely once during the S phase. In the mitotic cycle, the licensing of the DNA occurs when Cdc6 and Cdt1/Double Parked (Dup) load the MCM2-7 complex onto the origin recognition complex (ORC) to form the prereplication complex (preRC). PreRC formation occurs in late mitosis and G1 when Cdk activity is low. At the onset of S phase, Cdk activity increases, and the preRC initiates bidirectional DNA replication. PreRC formation must be suppressed after the initiation of S phase to prevent rereplication and thus ensure that each segment of the DNA is replicated exactly once per cell cycle. Cdks play a critical role in this process by preventing reestablishment of the preRC through multiple redundant mechanisms. Thus, during the mitotic cycle the precise regulation of Cdk activity ensures that each segment of DNA is replicated once, and only once, per cell cycle (Narbonne-Reveau, 2009).

The p21cip1/p27kip1/p57kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) specifically inhibits Cyclin E/Cdk2 complexes (de Nooij, 1996; Lane, 1996). In Drosophila, Cyclin E/Cdk2 activity is required for DNA replication during both mitotic cycles and endocycles (Knoblich, 1994; Lilly, 1996). Similar to what is observed with CKIs in other animals, Dap functions to coordinate exit from the cell cycle with terminal differentiation. Indeed, high levels of Dap are observed upon exit from the cell cycle in multiple tissues during both embryonic and larval development. Additionally, in the adult ovary, high levels of Dap prevent oocytes from entering the endocycle with the nurse cells as ovarian cysts exit the germarium in stage 1 of oogenesis (Hong, 2003). However, in addition to its well-established developmental function, recent work indicates that during developmentally programmed endocycles Dap facilitates the licensing of DNA replication origins by reinforcing low Cyclin E/Cdk2 kinase activity during the Gap phase (Hong, 2007). In dap-/- mutants, cells undergoing endocycles have reduced chromatin bound MCM2-7 complex, indicating a reduction in the density of preRCs along the chromatin. Additionally, dap-/- cells accumulate high levels of DNA damage due to the inability to complete genomic replication (Hong, 2007). Thus, during developmentally programmed endocycles Dap functions to reinforce low Cdk activity during the Gap phase (Narbonne-Reveau, 2009).

This study demonstrates that the CKI Dap promotes genomic stability during the premeiotic S phase of the Drosophila oocyte. The data indicate that Dap facilitates the licensing of DNA replication origins for the premeiotic S phase by restricting Cyclin E/Cdk2 activity during the early meiotic cycle. These studies represent the first example of a CKI regulating premeiotic S phase and genomic stability in a multicellular animal. Additionally, Dap was found to influence the timing of the mitotic/meiotic switch in ovarian cysts (Narbonne-Reveau, 2009)..

Cells in the mitotic cycle and the meiotic cycle face a similar challenge. To maintain the integrity of the genome, they must replicate their DNA once, and only once, during the S phase. In mitotic cells, this goal is accomplished, at least in part, through the precise regulation of Cdk activity throughout the cell cycle. During the mitotic cycle, Cdk activity inhibits preRC formation. This inhibitory relationship, restricts the assemble of preRCs to a short window from late mitosis to G1, when Cdk activity is low, and provides an important mechanism by which mitotic cells prevent DNA rereplication. However, the inhibitory effect of Cdk activity on preRC assembly necessitates that cells have a strictly defined period of low Cdk activity before S phase, to assemble preRCs for the next round of DNA replication. In mammals and yeast, compromising this period of low Cdk activity by overexpression G1 cyclins results in decreased replication licensing and genomic instability (Narbonne-Reveau, 2009).

One means by which cells inhibit Cdk activity is the expression of CKIs. In the mitotic cycle of budding yeast, the deletion of the CKI Sic1, which contains a Cdk inhibitor domain that is structurally conserved with the inhibitor domain present in the dap homologue p27Kip1, results in inadequate replication licensing and genomic instability due to the precocious activation of Cdks in G1. The current data strongly suggest that Dap plays a similar role in defining a critical period of low Cdk activity during the early meiotic cycle in Drosophila females (Narbonne-Reveau, 2009).

Based on these results, it is proposed that the Dap facilitates the licensing of DNA replication origins in ovarian cysts by restricting the inhibitory effects of Cyclin E/Cdk2 kinase activity on preRCs formation before premeiotic S phase. The data support the model that in the absence of Dap, ovarian cysts enter premeiotic S phase with a reduced number of licensed origins and thus fail to complete genomic replication. This hypothesis is supported by several observations. First, relative to wild-type, dap-/- ovarian cysts spend an increased proportion of their time in premeiotic S phase, as evidenced by the increased proportion of 16-cell cysts that incorporated EdU. The lengthening of premeiotic S phase is in line with the hypothesis that dap-/- ovarian cysts initiate DNA replication from a reduced number of licensed origins. Second, dap-/- ovarian cysts accumulate DNA damage during the premeiotic S phase. The accumulation of DNA damage during the premeiotic S phase is consistent with decreased preRC assembly resulting in intraorigin distances that are too large to be negotiated by DNA polymerase during a single S phase. Third, dap-/- meiotic cysts have decreased levels of the preRC component Dup/Cdt1. Moreover, genetic analysis indicates that Dup/Cdt1 levels are indeed limiting for premeiotic S phase in the dap-/- background. Specifically, it was found that reducing the dose of dup/cdt1 dramatically increases the levels of DNA damage observed in dap-/- ovarian cysts in region 2a and 2b of the germarium. In Drosophila, the levels of Dup/Cdt1 are negatively regulated by Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).

The use of the CKI Dap to restrict Cdk activity and thus promote the formation of preRCs before S phase is observed in multiple cell types beyond the oocyte. In previous work, it was found that in dap-/- mutants, cells in developmentally programmed endocycles also accumulate DNA damage and have dramatically reduced levels of Dup/Cdt1 (Hong, 2007). Thus, Dap functions to promote the accumulation of Dup/Cdt1 in multiple developmental and cell cycle contexts in Drosophila. Indeed, in select mitotic cycles removing one copy of dup/cdt1 in a dap-/- background results in DNA damage and cell death. However, in most mitotic cycles the requirement for Dap is redundant with other mechanisms that restrict Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).

Why Dap is required for preRC assembly in some cell types but not others remains unclear. However, it is interesting to note that DNA replication that occurs outside the confines of the canonical mitotic cycle, during the meiotic S phase and the S phase of developmentally programmed endocycles, is most dependent on Dap function (Hong, 2007). Thus, the increased reliance on the CKI Dap to establish a period of low Cdk activity before the onset of DNA replication may be explained by the absence of cell cycle programs that are specific to the mitotic cycle. For example, the tight transcriptional control of S phase regulators during the mitotic cycle may make the presence of Dap unnecessary for proper S phase execution. Alternatively, there may be differential regulation of the machinery that controls the regulated destruction of cyclins in the archetypical mitotic cycle versus the variant cell cycles of meiosis and the endocycle. In the future, determining why Dap plays a nonredundant role in the regulation of DNA replication during the meiotic cycle, but not the mitotic cycle, will be an important avenue of study (Narbonne-Reveau, 2009).

In addition to its role in the regulation of premeiotic S phase, this study found that dap influences the number of mitotic cyst divisions that occur before meiotic entry. In dap-/- mutants, ∼25% of ovarian cysts complete a fifth mitotic division to produce ovarian cysts with 32 cells. Similarly, mutations that compromise the degradation of the Cyclin E protein also result in production of 32-cell cysts. In line with these observations, females with reduced levels of Cyclin E produce ovarian cysts that undergo only three mitotic divisions and thus contain eight cells. Why Cyclin E/Cdk2 activity influences the timing of meiotic entry is not fully understood. However, the data suggest that the cyst division phenotype is not a direct result of reducing the number of preRCs assembled for the premeiotic S phase. Specifically, it was found that in dap-/- females reducing the dose of dup/cdt1 does not increase the number of ovarian cysts that undergo an extra division. In contrast, reducing the dose of dup/cdt1 in dap-/- females significantly enhances the meiotic DNA damage phenotype. These data strongly suggest that the extramitotic cyst division observed in dap-/- ovarian cyst is not the direct result of high CyclinE/Cdk2 activity inhibiting preRC formation (Narbonne-Reveau, 2009).

Intriguingly, Cdk2 is not the only Cdk that influences the number of ovarian cyst divisions in Drosophila females. Surprisingly, increasing the activity of the mitotic kinase Cdk1 results in the production of egg chambers with eight-cell cysts. Moreover, decreased Cdk1 activity results in ovarian cysts undergoing five mitotic divisions to produce egg chambers with 32 cells. Thus, Cdk1 and Cdk2 seem to have opposing roles in the regulation of the ovarian cysts divisions and/or meiotic entry. One of several possible explanations for these data, is that the number of ovarian cyst divisions is influenced by the amount of time cystocytes spend in a particular phase (G1, S, G2, and M) of the cell cycle. In the mitotic cycle of the Drosophila wing, there is a compensatory mechanism that ensures that changes in the length of one phase of the cell cycle result in alterations in the other phases of the cell cycle to ensure normal division rates. This compensatory mechanism is likely to be operating in multiple cell types and may account for why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions. Alternatively, Cdk1 and Cdk2 may act on truly independent pathways that have opposing roles in regulating the number of mitotic cyst divisions and/or the timing of meiotic entry. Ultimately, why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions that occur before meiotic entry awaits the identification of essential downstream targets of these kinases (Narbonne-Reveau, 2009).

In summary, this study has defined two novel functions for a p21Cip/p27Kip1/p57Kip2-like CKI during the meiotic cycle, the regulation of the mitotic/meiotic transition and the maintenance of genomic stability during the premeiotic S phase (Narbonne-Reveau, 2009).

Drosophila Minus is required for cell proliferation and influences Cyclin E turnover

Turnover of cyclins plays a major role in oscillatory cyclin-dependent kinase (Cdk) activity and control of cell cycle progression. This study analyses a novel cell cycle regulator, called minus, which influences Cyclin E turnover in Drosophila . minus mutants produce defects in cell proliferation, some of which are attributable to persistence of Cyclin E. Minus protein can interact physically with Cyclin E and the SCF Archipelago/Fbw7/Cdc4 ubiquitin-ligase complex. Minus does not affect dMyc, another known SCFAgo substrate in Drosophila . It is proposed that Minus contributes to cell cycle regulation in part by selectively controlling turnover of Cyclin E (Szuplewski, 2009).

Progression through the cell cycle requires periodic activation of cyclin-dependent kinases (Cdks). Oscillation in Cdk activity is achieved in part through cyclical synthesis and controlled degradation of cyclins, the regulatory subunits of the Cdks. Cyclin E is an evolutionarily conserved nuclear cyclin that controls G1/S transition and S-phase progression in animal cells, predominantly by activating Cdk2. CycE and cdk2 are essential genes in Drosophila . Cyclin E acts as the limiting factor for G1-S-phase transition. Cyclin E turnover is important for cell cycle progression and is regulated by a conserved ubiquitin-ligase complex, called SCF. The SCF complex is built on an elongated scaffold protein, Cullin-1, which recruits the substrate recognition module consisting of Skp1 and an F-box protein, as well as a ring domain-containing ubiquitin-ligase module. Substrate selectivity is mediated by the F-box subunit, in part through recognition of phosphorylated motifs on substrate proteins. The Drosophila F-box protein encoded by archipelago (ago) is the ortholog of Fbw7/Cdc4. Scf-Ago promotes degradation of CycE, dMyc, and Trachealess (Szuplewski, 2009).

This study reports the characterization of the classical Drosophila mutant minus. Minus protein can interact physically with Cyclin E and the SCF-Ago complex. Cells lacking Minus fail to degrade CycE, resulting in persistence of CycE. minus mutants show defects in cell proliferation, attributable in part to excess CycE activity, reflecting that normal regulation of CycE turnover is essential for normal cell proliferation during Drosophila development. It is proposed that Minus acts as a cell cycle regulator by selectively controlling CycE turnover (Szuplewski, 2009).

Flies homozygous mutant for a spontaneous mutation in the minus gene mi1 showed small body size, small bristles, and delayed completion of pupal development. minus alleles were also isolated in a screen for female sterility, and the minus gene has been mapped to the cytogenetic interval 59E on the right arm of the second chromosome. To isolate new minus alleles, P-element insertions in 59E were screened for failure to complement the mi1 bristle phenotype. Flies carrying the l(2)SH0818 P-element insertion in trans to mi1 showed a small bristle phenotype, milder than that produced by the combination of mi1 in trans to a deletion. The stronger mutant combination was also female sterile. Thus, l(2)SH0818 appears to have reduced minus activity. l(2)SH0818 is semilethal, but rare homozygous survivors showed small body size and small bristles. These phenotypes were confirmed to be due to the P-element insertion, since flies from which the P-element was precisely excised were homozygous viable and normal in size. Animals homozygous for a null allele of minus also showed reduced body size in larval and pupal stages (Szuplewski, 2009).

The l(2)SH0818 P-element is inserted in the 5' untranslated region (UTR) of the annotated gene CG5360. Two other transposons inserted in this 5' UTR, EY01258 and l(2)k06908, also produced weak bristle phenotypes in trans to mi1, suggesting that they are weak minus alleles. mi1 was isolated as a spontaneous mutation, which can be caused by transposon insertions. It was not possible to amplify DNA spanning the second intron of CG5360 from mi1 homozygous animals by PCR, consistent with the possibility that an insertion of DNA disrupts the CG5360 transcription unit (other parts of the gene amplified normally). An additional mi allele was generated by imprecise excision of the viable P-element insertion EY01258. miDeltaEY22 is a deletion that removes the two first exons and part of the third exon of CG5360. miDeltaEY22 produced phenotypes equivalent to those of a larger deletion that completely removes the gene, and so behaves genetically like a null allele. Homozygous miDeltaEY22 mutants died mainly during early larval stages. The remaining mutants showed a developmental delay and reduced growth rate. After 5 d, the largest mutant larvae were much smaller than comparably aged control larvae. By 11 d, the surviving mutants that had pupated were also small (Szuplewski, 2009).

Minus protein lacks domains of known function, but was identified as a CycE-interacting protein in a genome-wide yeast two-hybrid screen. Ten cyclin-binding sites are predicted using the Eukaryotic Linear Motif server. Some of the predicted cyclin-binding sites are conserved in other insects—Anopheles gambiae, Tribolium castaneum, and Apis mellifera—but none resides in a region of sequence conservation sufficient to permit identification of an orthologous protein outside of the insects. The interaction between Minus and CycE was confirmed in vitro using GST pull-down assays. The Drosophila Cyclin E gene encodes two proteins that differ in their N termini. A GST-Minus fusion protein was able to bind CycE-I from lysates of S2 cells transfected to express Myc-tagged CycE-I. Similar results were obtained with Myc-tagged Drosophila CycE-II protein and with Myc-tagged human CycE isoform 1 (CCNE1) (Szuplewski, 2009).

This study provides evidence that efficient Cyclin E turnover is essential for normal cell proliferation and endoreplication during Drosophila development. Endoreplicative growth and more typical proliferative diploid cell cycles are impaired as a consequence of the elevated Cyclin E levels in minus mutants. Attempts to suppress the cell proliferation phenotype by reducing CycE activity were only partially successful, perhaps due to incomplete compensation for elevated CycE levels. It is also possible that Minus has other targets, in addition to CycE (Szuplewski, 2009).

Minus was identified in a screen for female sterility. Minus' role in Cyclin E turnover suggests a possible link to the encore mutant. encore encodes a protein of unknown function that has been proposed to promote CycE degradation by localizing the SCF complex to a germline-specific cytoplasmic structure called the fusome. In mammals, different Fbw7/Cdc4 isoforms can target different Myc functions in distinct subcellular compartments. Interestingly, the Drosophila Fbw7/hCdc4 protein Archipelago exists in only one isoform, limiting the possibility for isoform-specific subfunctions. Use of accessory proteins, such as Minus, may be another means to confer target specificity on the core Archipelago/SCF complex (Szuplewski, 2009).

At present no Minus ortholog has been detected outside the insects. But, it is noted that Minus binds to human CCNE1 and influences its expression, much as it does with Drosophila CycE. Although this does not constitute evidence for the existence of a mammalian protein with a function analogous to Minus, it is compatible with this possibility. A vertebrate protein having the motifs required for Minus function but in a different number or arrangement might not be readily identified unless these short motifs were embedded in more extensive blocks of sequence similarity. In view of the importance of Cyclin E turnover in cancer, a functional equivalent of Minus might be a good candidate for a tumor suppressor (Szuplewski, 2009).

The homeodomain Iroquois proteins control cell cycle progression and regulate the size of developmental fields

During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. This study shows that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. Physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, the study provides a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors (Barrios, 2015).

The identification of genes that control cell proliferation is paramount in developmental and cancer biology. The Iroquois proteins play multiple roles in regionalization and patterning during Drosophila development. This study shows that they are also involved in the control of cell proliferation and, interestingly for homeodomain-containing proteins, they appear to do so by a non-transcriptional mechanism. This novel function of Iro genes would help developmental fields to attain their correct size and, if altered by Iro downregulation, could be a critical step for tumour progression (Barrios, 2015).

iro hypomorphic and over-expression conditions were analysed; Iro proteins were found to negatively control the G1-S transition of the cell cycle. caup over-expression impaired the activity of CycE/Cdk2 complex, while simultaneously increasing the level of CycE protein. Still, CycE appears to be a limiting factor since its exogenous administration restores cell proliferation, while its reduction enhances it. The presence of Caup in CycE-containing protein complexes led to a proposal that this physical interaction inhibits CycE/Cdk2 activity thus slowing down cell proliferation. This hypothesis is supported by the observation that Caupcyc* and CaupIRObox* mutant proteins show both impaired ability to co-immunoprecipitate with CycE and to restrict cell cycle progression. Although not experimentally demonstrated, it is speculated that Caup may interact with CycE and Cdk2 containing complexes and inhibit their activity by preventing substrate recognition and/or stabilizing p21 binding. Further work is required to determine more precisely these molecular interactions. Since Caupcyc*IRObox* still retains some ability to repress cell proliferation, it is presumed that either the functionality of these domains was not completely abolished by the mutations generated or the existence of additional unidentified interacting sites (Barrios, 2015).

Although other homeobox proteins (and also some epigenetic regulators) have been shown to modulate the activity of cell cycle regulators by protein-protein interaction, many of them do it through transcriptional regulation. A transcriptional effect of Caup on cell cycle regulation can be ruled out since transcriptionally inactive CaupHD*1 and CaupHD*2 are still able to inhibit cell cycle progression (Barrios, 2015).

Iro proteins play redundant roles in several developmental contexts. The three of them are able to repress cell cycle progression when over-expressed and this effect is abrogated by co-expression of cycE. The presence of putative Cyclin binding motives and the high conservation of the IRObox in the Iro proteins leads to the proposal that Ara and Mirr may also physically interact with CycE containing complexes. Since it was found that the penetrance of the dorsal eye enlargement phenotype increases by reducing the overall amount of Iro proteins, it is suggested that they may act in a redundant manner to modulate CycE/Cdk2 activity. Alternatively, the three Iro proteins may be functioning in a stoichiometric complex, this explaining why depletion of only one of them causes eye enlargement (Barrios, 2015).

The current results suggest a novel role of Iro proteins as cell-autonomous regulators of the growth of the domains of the imaginal discs where they are expressed. Furthermore, the results fit to a current model that suggests that growth of territorial fields modulates the response of cells to morphogens. In the eye discs, the ability of Decapentaplegic (Dpp) to induce retina differentiation is counteracted by Wg emanating from the anterior-most region of the discs until the disc attains a size such that dpp expressing cells are beyond the range of action of Wg. Accordingly, it is suggested that the enhanced cell proliferation found in iro mutant discs, would enlarge the physical separation between Wg- and Dpp-expressing cells in the dorsal domain, thus increasing the efficiency of Dpp signalling and causing dorsal eye enlargement (Barrios, 2015).

In analogy with this model for eye disc development, specification of the wing driven by Wg in the distal part of the wing disc is counteracted by the Vein morphogen, which spreads from the most proximal part of the wing disc. In this scenario, reduction of the size of the distal wing disc by inhibition of cell proliferation prevents wing development (with the concomitant generation of a notum-like tissue), by facilitating the inhibition of Wg by Vein. Interestingly, Vein activates Iro gene expression in the notum region while Wg does so in the dorsal eye disc. Thus, it is proposed that Iro genes could provide a molecular mechanism that allow the ligands Vein (in the notum) and Wg (in the dorsal eye) to regulate the size of the morphogenetic field in which they operate (Barrios, 2015).

The results further suggest that a direct regulation of cell cycle progression by Iro/Irx proteins may be relevant for tumorigenesis. Thus, tumorous-like growth was observed in the eye imaginal discs when iro function was reduced in a sensitized genetic background (such as ey>Dl or ey>Dl eyeful flies). Conversely, the ability of caup over-expression was shown to counteract the overgrowth induced by Yki in imaginal discs, and that this is partially mediated by cycE/cdk2 inactivation. These data suggest a role of Iro genes as TSGs in Drosophila and agree with the association found between loss or reduced expression of members of Irx gene family and certain types of human cancer. Note however that the role of Iro/Irx genes in tumorigenesis may be cell type-dependent since in some cases they appear to act as oncogenes. Considering the presence of the IRO box and of putative Cyclin-binding domains in Irx proteins, it is hypothesized that some Irx mutations may contribute to cancer progression in vertebrates by increasing the activity of the CycE/Cdk2 complex and thus accelerating the G1-S transition, a key step frequently affected in cancer cells (Barrios, 2015).

Cyclin E: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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