Gene name - dacapo
Cytological map position -
Function - cyclin dependent kinase inhibitor
Symbol - dap
FlyBase ID: FBgn0010316
Genetic map position -
Classification - Cip/Kip family
Cellular location - nuclear and cytoplasmic
|Recent literature||Swanson, C. I., Meserve, J. H., McCarter, P. C., Thieme, A., Mathew, T., Elston, T. C. and Duronio, R. J. (2015). Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication. Development [Epub ahead of print]. PubMed ID: 26493402
In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E/Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E/Cdk2 activity are not fully understood. This study shows that the Drosophila Cyclin E/Cdk2 inhibitor Dacapo is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. It is proposed from this model and the in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E/Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy.
|Shaikh, M. N., Gutierrez-Avino, F., Colonques, J., Ceron, J., Hammerle, B. and Tejedor, F. J. (2016). Minibrain drives the Dacapo dependent cell cycle exit of neurons in the Drosophila brain by promoting asense and prospero expression. Development [Epub ahead of print]. PubMed ID: 27510975
A key issue in neurodevelopment is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. This study shows that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene MNB/DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27kip1. On the one hand, Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. On the other, Mnb induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo. In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons.
Cyclins and their partners, the cyclin dependent kinases (cdks), regulate progression of the cell cycle. For example Cyclin E can induce S phase in the absence of protein synthesis (Richardson, 1995). In vertebrates, up-regulation (activation) of inhibitors of cyclin dependent kinases accompanies, indeed, is integral to the switch from proliferation to differentiation. There are two families of vertebrate cdk inhibitiors: Ink and Cip/Kip. The characteristic feature of the Kip family is an N-terminal domain that mediates binding to the complex formed by cyclin dependent kinases with cyclins (Lane, 1996 and references). Dacapo, the subject of this overview, is also a member of the Cip/Kip family of cdk inhibitors.
Evidence for the existence of inhibitor regulation of cell cycle progression in Drosophila predates the cloning of dacapo. Human cdk inhibitor p21 was expressed in the Drosophila eye to discover its effect on eye differentiation. Expression of p21 in all cells posterior to the morphogenetic furrow can block the wave of S phase that immediately follows the furrow. When mitosis is blocked in mitotic eye cells by human p21, cell differentiation takes place according to each cell's predetermined fate. Nevertheless, there is a deficit of cell types that are determined last in temporal order: the pigment cells and bristle cells. This results in a rough eye phenotype. P21 expression does not alter cell fate, but it is able to block cell cycle progression. This suggests that the mechanism of inhibition of cell cycle progression by p21 is general and conserved across species, and that similar mechanisms act to promote differentiation in the fly (de Nooij, 1995).
With the cloning of dacapo (similar to the musical term, da capo, cueing the performer to repeat a passage "from the beginning"), its effects on cell cycle progression in the eye could be tested directly. In flies bearing up to two copies of a daptransgene, eyes appear to be wild-type, but in flies bearing four copies of a dap transgene, the eyes are slightly rough (de Nooij, 1996).
The effects of dap overexpression are dramatically enhanced by overexpression of the Drosophila Retinoblastoma-family protein) (Rbf) (Du, 1996). Discussion of the relation of DAP and RBF is incomplete without mentioning E2F, a transcription factor responsible for activation of S phase gene. It is unclear what the relationship is between cyclin E and dE2F in Drosophila. In Drosophila, cyclin E transcription appears to be downstream of dE2F during some phases of development (in endocycling cells of the midgut); during other phases, cyclin E is required for dE2F-dependent transcription and appears to act upstream of dE2F (in the CNS) (Duronio, 1995 and Sauer, 1995). In mammals RB and related proteins associate with E2F and repress E2F dependent transcription.
While flies carrying either two copies of dap or Rbf transgenes have wild-type eyes, the combination results in extremely rough eyes. Many pigment cells are missing consistent with a marked deficit of precursor cells, and the stripe of DNA synthesizing cells posterior to the morphogenetic furrow is completely blocked. Thus dap and Rbf exhibit significant synergy in arresting cell cycle entry in vivo. This synergy is likely to involve binding of Rbf to E2F and interference of cyclin E/cdk involvement with E2F (de Nooij, 1996).
dap expression during embryogenesis is sufficient to arrest cell proliferation. dap was expressed in seven epidermal stripes using a paired promoter. DNA synthetic phase 16 is largely inhibited in the prd-expressing regions, and subsequent mitosis 16 (early in embryogenesis) is also inhibited. Epidermal cell counts during stage 14 (late in embryogenesis), by which time epidermal cell proliferation is long over, reveals a reduction in cell density in these regions. Mitosis 15 is refractory to inhibition by DAP (Lane, 1996).
DAP is not the only protein to limit cell cycle progression in developing embryos. The G1 arrest observed after the terminal division of the epidermal cells is dependent on the inactivation of cyclin E/cdk2 activity. In addition to the up-regulation of DAP, down-regulation of cyclin E transcription appears to contribute to the timely inactivation of cyclin E/cdk2 activity (Knoblich, 1994). This cyclin E down-regulation also occurs normally in dap mutants. In addition to cyclin E, other cell cycle regulators that have been analyzed (cyclin A, cyclin B, cyclin B3, cdc2 and string) are also transcriptionally down regulated, when epidermal cells become postmitotic (Lane, 1996 and references). Thus cell cycle exit is subject to tight regulation by means of multiple regulatory events. This makes sense when considering how crucial cell quiescence is, to avoid unwanted cell proliferation.
In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).
Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG-SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG-SD dimer activity, thereby providing a feedback control (Legent, 2006).
Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG-SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG-SD dimers. Therefore, sd induction may efficiently restrain VG-SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG-SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).
The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG-SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG-SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG-SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG-SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG-SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).
The results also demonstrate that dE2F1-DP regulates VG-SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).
Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG-SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).
In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).
Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG-SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG-SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG-SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).
Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).
Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG-SD, but also via additive impairment of VG-SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG-SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG-SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).
At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG-SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG-SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).
Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG-SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).
Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG-SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).
The DAP protein shares 20-27% amino acid identity with vertebrate Kip inhibitors of the p21/p27 family and a 28% identity to a C. elegans gene. The few N-terminal motifs that are identical in all these Kip family members are required for cyclin/cdk binding in the analyzed inhibitors. With the C-terminal region, DAP contains basic clusters characteristic of bipartite nuclear targeting signals that are also present in the vertebrate inhibitors. Two additional regions have been shown to be necessary for cdk inhibition (Lane, 1996 and deNooij, 1996).
date revised: 5 JAN 96
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