To identify an SCFAgo ubiquitin ligase substrate that could explain the accelerated growth of ago mutant cells, two different interaction screens were conducted by using the Ago F box/WD domain. By mass spectrometric analysis of proteins that coprecipitate with Ago, peptides derived from a number of different SCF components were identified, including Cullins and Skp proteins. At a lower frequency, peptides derived were also recovered from putative SCFAgo substrates, including the Drosophila ortholog of the Myc transcription factor (dMyc). In addition to multiple SCF components, a single clone of dMyc was also recovered in a yeast two-hybrid screen for proteins that physically interact with the F box/WD repeat region of Ago (Moberg, 2004).
dMyc was identified as a candidate Ago binding protein, so whether the ability of ago to regulate dMyc involves a direct interaction between Ago and dMyc was examined. In protein extracts from Drosophila S2 cells transfected with epitope-tagged versions of Ago and dMyc (HAAgo and FLAGdMyc), FLAGdMyc was readily detected in anti-HA immunoprecipitates, and in the reciprocal procedure, HAAgo was readily detected in anti-FLAG immunoprecipitates. These experiments indicate that Ago and dMyc interact physically in S2 cells. Significantly, two mutant versions of Ago, Ago1 and Ago3, that correspond to mutations that deregulate dMyc levels and increase growth in vivo, are dramatically impaired in their ability to interact with dMyc in cells despite being expressed at approximately the same level as wild-type Ago protein. Thus, as is the case with the other known SCFAgo substrate, Cyclin E (Moberg, 2001), the ability of Archipelago to bind dMyc protein correlates with its ability to downregulate dMyc levels in vivo (Moberg, 2004).
Coexpression of dMyc also seems to promote Ago accumulation in cells. This increase seems more evident in forms of Ago that bind strongly to dMyc and does not appear to be a general effect of dMyc on all coexpressed proteins. However, the precise mechanism underlying this effect has not been established. It may involve direct dMyc-Ago binding, but it may also be an indirect consequence of Myc's ability to regulate cell metabolism and translation rates (Moberg, 2004).
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 give homozygous mutant cells even a subtle proliferative advantage over their wild-type neighbors. Clones of homozygous mutant tissue 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 show 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).
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 then examined 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; this lack of pattern 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; this condition 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).
archipelago (ago) mutations lead to overproliferation of mutant tissue in the developing Drosophila eye, and ago mutant cells express elevated levels of Cyclin E protein and are delayed in their exit from the cell cycle (Moberg, 2001). The Ago protein, as well as its human ortholog Fbw7/hCDC4, is the F box component of an SCF E3-ubiquitin ligase, and Ago binds Cyclin E and targets it for ubiquitination and subsequent degradation. The overgrowth of ago mutant tissue implies that the ago mutant cells collectively grow (i.e., accumulate mass) at an accelerated rate in comparison to wild-type cells. Because Drosophila Cyclin E has been shown to promote S phase entry but not growth, the increased growth of ago mutant cells suggests that there are other Ago substrates that promote cell growth (Moberg, 2004).
To examine the growth properties of ago mutant cells, marked pairs of ago mutant clones and wild-type sister clones (twin spots) were generated in the developing larval wing imaginal disc. ago mutant clones in the wing and the eye are consistently larger and contain more cells than their respective twin spots (labeled control). The increased cell number in ago clones indicates that ago mutant cells divide more frequently over a fixed period of time than do control cells and thus have a shortened cell cycle duration. Indeed, the calculated length of an average cell cycle in ago cells is approximately 15% shorter than in control cells. However, flow-cytometric analysis indicates that ago cells are not decreased in size. These observations indicate that ago cells coordinately accelerate rates of cell growth and cell division such that normal cell size is maintained (Moberg, 2004).
Because dMyc has been shown to promote growth in imaginal-disc cells, the role of Ago in regulating dMyc levels was examined further. Eye imaginal discs containing ago mutant clones were stained with an anti-dMyc monoclonal antibody. dMyc protein is strongly elevated in ago mutant cells, both in third-larval-instar eye discs and in early pupal-phase eye discs. Increased dMyc staining is observed in ago cells found throughout the larval eye disc and antennal discs. In the pupal eye disc, dMyc levels are especially high in the clusters of nuclei of the bristle cell complex. Immunoblot analysis also indicates that dMyc levels are highly elevated in extracts of ago mutant discs. In this experiment, the twinspots carry two copies of a strong Minute mutation [M(3)] that dramatically impairs their growth such that more than 95% of the disc cells are ago mutant. The dMyc protein detected in ago/M(3) discs also appears to have reduced mobility in SDS-PAGE, suggesting that in the absence of Ago, dMyc accumulates in a modified form. Significantly, overexpression of cyclin E in larval eye discs did not detectably alter dMyc levels, suggesting that accumulation of dMyc protein in ago mutant cells is not an indirect effect of the concurrent deregulation of Cyclin E levels (Moberg, 2004).
To determine if dMyc-dependent transcription is also deregulated in ago cells, expression of a dMyc target gene was examined in ago/M(3) and FRT80B discs. In three independent RNA preparations from equal numbers of staged discs, ago mutant discs were observed to contain approximately twice as much total RNA as control discs. Since ago/M(3) and FRT80B discs are approximately the same size and contain similar levels of total protein, this indicates that ago mutant cells contain more RNA than control cells. The increase in the amount of RNA appears to result from a disproportionate increase in rRNA in ago/M(3) discs. As a result, when equal amounts (4 μg) of total RNA are analyzed by Northern blotting, a control RNA (β-Tubulin 56D mRNA) is less abundant in the ago/M(3) sample compared to FRT80B. In contrast, the level of RNA of the dMyc target gene pitchoune is increased. If this change were normalized to the levels of β-Tubulin 56D RNA, this would represent an approximately 3-fold increase of pitchoune RNA in ago/M(3) discs relative to the wild-type. These findings provide evidence for increased expression of a putative dMyc target gene in ago mutant cells (Moberg, 2004).
To begin to examine how ago normally functions to inhibit dMyc levels, in situ analysis of dMyc mRNA on FRT80B and ago/M(3) larval eye discs was performed. In control discs, an anti-sense dMyc RNA probe detects dMyc expression at low levels throughout the eye disc, with a stronger stripe immediately posterior to the morphogenetic furrow, whereas a sense dMyc RNA probe produces no discernable staining in wild-type discs. The pattern and level of dMyc expression is unchanged in ago/M(3) discs. It is possible that subtle increases in dMyc mRNA levels are below the limits of the detection techniques used in this study, but it is clear that the anti-sense dMyc RNA probe easily detects increased dMyc transcripts in pGMR-Gal4;UAS-dMyc eye discs. These data suggest that ago inhibits dMyc accumulation largely through a posttranscriptional mechanism (Moberg, 2004).
Because Ago and dMyc proteins interact, whether perturbing Ago function in S2 cells could modulate dMyc levels and stability was examined. A putative dominant-negative form of Ago that lacks the F box domain (AgoΔF) was constructed. AgoΔF is predicted to bind target proteins via an intact WD repeat domain but to be unable to recruit them into SCFAgo. Expression of AgoΔF is thus predicted to stabilize SCFAgo targets. Coexpression of dMyc and AgoΔF in cells increases the amount of Ago-dMyc complex recovered in coimmunoprecipitation experiments and increases the overall levels of dMyc in these cells. To determine whether AgoΔF expression alters dMyc stability, dMyc levels were assayed in the presence or absence of coexpressed AgoΔF protein after treatment with the translation inhibitor cycloheximide (CHX) . When dMyc is expressed alone, its levels decline rapidly after CHX treatment, indicating that dMyc is normally quite unstable. In contrast, dMyc coexpressed with AgoΔF persists longer in cells after CHX addition, indicating that dMyc is more stable when SCFAgo activity is reduced. In support of this, treatment of cells with double-stranded ago RNA (dsRNA) or with the proteasome inhibitor MG132 was found to increase the amount of transfected dMyc detected in cells. These data indicate that dMyc is degraded via the proteasome in vivo in a manner similar to mammalian c-Myc and that, as the substrate specificity component of an SCFAgo ubiquitin-ligase, Ago is likely to participate in this process (Moberg, 2004).
In addition to Ago, one or more of the 23 other F box proteins encoded by the Drosophila genome might also play a role in regulating dMyc levels in vivo. Of particular note are the Drosophila F box proteins Slmb and CG9772. The Slmb protein, encoded by the supernumerary limbs (slmb) gene, is the Drosophila protein most similar to Ago within the F box and WD repeats. The gene CG9772 may encode the Drosophila ortholog of the human F box protein Skp2 (54% similarity and 31% identity between CG9772-PA and Skp2 across their length), which has recently been shown to regulate c-Myc levels in a transformed mammalian cell line (Moberg, 2004).
To assess the relative roles of these F box family members in regulating endogenous dMyc levels, double-stranded RNA interference (dsRNAi) was used in S2 cells to reduce the RNA levels of ago, slmb, and CG9772. Reducing ago function in S2 cells by ago dsRNAi results in the stabilization and accumulation of dMyc protein. In contrast, despite significant reduction in the levels of slmb and CG9772 RNAs, there is no discernible change in dMyc levels. Whether the CG9772-PA protein (a CG9772 isoform containing the complete F box and leucine-rich repeat domains) could bind dMyc in a manner similar to Skp2, its putative human ortholog, was also tested. Ago and CG9772 accumulate to similar levels in the absence of coexpressed dMyc. However, CG9772-PA displayed very little dMyc binding activity compared to Ago. Although these data do not rule out a role for other F box proteins in regulating dMyc levels and/or activity, they do indicate that at least in S2 cells, and among the Ago, Slmb, and CG9772 proteins, only Ago is able to bind to dMyc and regulate its stability (Moberg, 2004).
Consistent with the observed physical interaction between the Ago and dMyc proteins, it was found that an ago mutation could modify dMyc mutant phenotypes. The dMyc allele diminutive1 (dm1) is a hypomorphic viable mutation caused by a gypsy element insertion into the first intron of the dMyc genomic locus. dm1 homozygous females and dm1 hemizygous males are smaller than wild-type flies, and the females are sterile. dm1 flies that are heterozygous for ago (dm1;ago1/+) are larger than dm1 flies. Quantitation of this effect shows that heterozygosity for a mutation in ago increases dm1 female body length by approximately 12%. The wings of these dm1;ago1/+ adults are also approximately 15% larger than those of dm1 adults. To determine whether these effects are due to an increase in cell number, cell size, or both, wing hair density was determined in the relevant genotypes. Because each cell in the wing generates a single hair, hair density varies inversely with cell size. The hair density in dm1, dm1;ago/+, and wild-type wings is the same, indicating that the cells are of comparable size. Thus, dm1 wings are small because they contain fewer cells, and a 2-fold reduction in wild-type ago gene dosage increases the size of these mutant wings by increasing the number of normally sized cells. However, ago mutations do not rescue size defects associated with the dMyc alleles dmP0 and dmP1. These are stronger loss-of-function mutations than dm1 and reduce organism size by reducing cell size, with little effect on cell number. dm1 may therefore represent a weaker dMyc allele whose body-size phenotype remains sensitive to ago gene dosage (Moberg, 2004).
In addition to restoring cell number in the dm1 mutant wings, reducing ago function can also ameliorate the female fertility defect of dMyc mutant animals. Unlike dm1;FRT80B/+ females, dm1;ago1/+ females lay eggs that can give rise to viable larvae. In addition, dm1/dmPG45;ago/+ females are approximately 10-fold more fertile than dm1/dmPG45;FRT80B/+ females, which normally show a 2%-3% egg hatching rate. These data indicate that, in addition to modifying dMyc organ and organism size phenotypes, ago is also an antagonist of dMyc in the female germline (Moberg, 2004).
Consistent with a role for Ago in inhibiting dMyc, ago expression retards organ growth. Expression of an N-terminally truncated ago cDNA, that contains an intact F box domain and WD repeat region, in the posterior compartment of the wing decreases posterior compartment size by approximately 35%. Hair density measurements indicate that cells in the ago-expressing compartment are approximately 32% smaller than controls, suggesting that the reduction in compartment size is largely an effect of reduced cell size. Thus, overproduction of an N-terminally truncated version of Ago in the developing wing mimics the effect of dMyc mutations and inhibits growth (Moberg, 2004).
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).
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).
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).
The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (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).
Why is the function of Ago/hCdc4/Fbw7 critical to endocycles but not to mitotic cycles in follicle epithelial cells? A potential answer might reside in Dacapo, a CIP/KIP-type inhibitor of Cyclin E/Cdk2 complexes that is regulated in the mitotic to endocycle transition by activation of Notch pathway. dacapo is downregulated at mitotic-to-endocycle transition because of Notch activation and ectopic expression of dacapo represses endocycle progression. It is plausible that during mitotic phases Ago and Dacapo share a redundant role for regulating the Cyclin E activity level, however, dacapo is downregulated by Notch pathway at the time of mitotic-to-endocycle transition and at that point Ago gains the critical role of sole regulator of Cyclin E protein activity level. However, downregulation of Dacapo does not readily explain the reduction of CycE levels observed in mitotic-to-endocycle transition. Elevation of CycE protein level is detected in response to Dacapo overexpression, pointing out that this CKI may stabilize CycE in an inactive form. One possibility therefore is that less CycE protein is observed after the Dacapo downregulation because Dacapo is no longer stabilizing it (Shcherbata, 2004).
Why is Dacapo downregulated at the time of endocycle transition? Expression of Dacapo is important for proper cell cycle regulation. For example, during vertebrate development, members of the CIP/KIP family of CKIs are often upregulated as cells exit the mitotic cycle and begin to terminally differentiate. Also, reduced expression of p27Kip1 is frequently shown to correlate with a poor prognosis in various cancers, and in the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S-phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Also, p21 elimination causes centriole overduplication and polyploidy in human hematopoietic cells. In the Drosophila germ line Dap is differentially regulated in the nurse cells versus the oocyte. High Dap levels in the oocyte are critical to the maintenance of the prophase I meiotic arrest and ultimately to later events of oocyte differentiation, and in the nurse cells the oscillations of Dap drive the endocycle. In contrast to all these examples, in endocycling follicle cells reduction of p21/Dacapo is a requirement for normal endocycle progression. Similarly, in a megakaryocytic cell line, differentiation is correlated with a downregulation of p27. It is proposed that the downregulation of Dacapo is a reasonable strategy to bypass the G1/S transition and to enter endocycling when mitosis is not completed, however, how these endocycling cells escape possible centrosome amplification and apoptosis that could be consequences of the lack of Dacapo/p21-activity is not clear. This diversity in the processes, that allow cells to exit from mitotic cell cycle, is generating or representing regulatory multiplicity that might be reflected in the ways eukaryotic cells acquire tumor formation capacity (Shcherbata, 2004).
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date revised: 14 October 2004
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