dacapo: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - dacapo

Synonyms -

Cytological map position -

Function - cyclin dependent kinase inhibitor

Keywords - cell cycle, tumor suppressor

Symbol - dap

FlyBase ID: FBgn0010316

Genetic map position -

Classification - Cip/Kip family

Cellular location - nuclear and cytoplasmic

NCBI links: Entrez Gene

dap orthologs: Biolitmine
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.
Otsuki, L. and Brand, A. H. (2019). Dorsal-ventral differences in neural stem cell quiescence are induced by p57(KIP2)/Dacapo. Dev Cell. PubMed ID: 30905769
Quiescent neural stem cells (NSCs) in the adult brain are regenerative cells that could be activated therapeutically to repair damage. It is becoming apparent that quiescent NSCs exhibit heterogeneity in their propensity for activation and in the progeny that they generate. NSCs have been shown to undergo quiescence in either G0 or G2 in the Drosophila brain, challenging the notion that all quiescent stem cells are G0 arrested. G2-quiescent NSCs become activated prior to G0 NSCs. This study shows that the cyclin-dependent kinase inhibitor Dacapo (Dap; ortholog of p57(KIP2)) determines whether NSCs enter G0 or G2 quiescence during embryogenesis. The dorsal patterning factor Muscle segment homeobox (Msh; ortholog of MSX1/2/3) binds directly to the Dap locus and induces Dap expression in dorsal NSCs, resulting in G0 arrest, while more ventral NSCs undergo G2 quiescence. These results reveal region-specific regulation of stem cell quiescence.
Otsuki, L. and Brand, A. H. (2019). Dorsal-ventral differences in neural stem cell quiescence are induced by p57(KIP2)/Dacapo. Dev Cell. PubMed ID: 30905769
Quiescent neural stem cells (NSCs) in the adult brain are regenerative cells that could be activated therapeutically to repair damage. It is becoming apparent that quiescent NSCs exhibit heterogeneity in their propensity for activation and in the progeny that they generate. NSCs have been shown to undergo quiescence in either G0 or G2 in the Drosophila brain, challenging the notion that all quiescent stem cells are G0 arrested. G2-quiescent NSCs become activated prior to G0 NSCs. This study shows that the cyclin-dependent kinase inhibitor Dacapo (Dap; ortholog of p57(KIP2)) determines whether NSCs enter G0 or G2 quiescence during embryogenesis. The dorsal patterning factor Muscle segment homeobox (Msh; ortholog of MSX1/2/3) binds directly to the Dap locus and induces Dap expression in dorsal NSCs, resulting in G0 arrest, while more ventral NSCs undergo G2 quiescence. These results reveal region-specific regulation of stem cell quiescence.
Bivik Stadler, C., Arefin, B., Ekman, H. and Thor, S. (2019). PIP degron-stabilized Dacapo/p21(Cip1) and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels. Development 146(13). PubMed ID: 31289041
During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21(Cip1) and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCF(Fbxw7/Ago) targets phosphorylated CycE, whereas p21(Cip1) and Dap are targeted by the CRL4(Cdt2) complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. This study analyses the role of ago (Fbxw7)-mediated CycE degradation, and of Dap and p21(Cip1) degradation during Drosophila CNS development. ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21(Cip1) transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.
Morita, S., Ota, R., Hayashi, M. and Kobayashi, S. (2020). Repression of G1/S Transition by Transient Inhibition of miR-10404 Expression in Drosophila Primordial Germ Cells. iScience 23(3): 100950. PubMed ID: 32179474
Cell-cycle quiescence is a common feature of early germline development in many animal species. In Drosophila germline progenitors (pole cells), both G2/M and G1/S transitions are blocked. G2/M transition is repressed by maternal Nanos through suppression of Cyclin B production. However, the molecular mechanism underlying blockage of G1/S transition remains elusive. This study found that repression of miR-10404 expression is required to block G1/S transition in pole cells. Expression of miR-10404, a microRNA encoded within the internal transcribed spacer 1 of rDNA, is repressed in early pole cells by maternal polar granule component. This repression delays the degradation of maternal dacapo mRNA, which encodes an inhibitor of G1/S transition. Moreover, derepression of G1/S transition in pole cells causes defects in their maintenance and their migration into the gonads. These observations reveal the mechanism inhibiting G1/S transition in pole cells and its requirement for proper germline development.
Doherty, C. A., Diegmiller, R., Kapasiawala, M., Gavis, E. R. and Shvartsman, S. Y. (2021). Coupled oscillators coordinate collective germline growth. Dev Cell. PubMed ID: 33689691
Developing oocytes need large supplies of macromolecules and organelles. A conserved strategy for accumulating these products is to pool resources of oocyte-associated germline nurse cells. In Drosophila, these cells grow more than 100-fold to boost their biosynthetic capacity. No previously known mechanism explains how nurse cells coordinate growth collectively. This study reports a cell cycle-regulating mechanism that depends on bidirectional communication between the oocyte and nurse cells, revealing the oocyte as a critical regulator of germline cyst growth. Transcripts encoding the cyclin-dependent kinase inhibitor, Dacapo, are synthesized by the nurse cells and actively localized to the oocyte. Retrograde movement of the oocyte-synthesized Dacapo protein to the nurse cells generates a network of coupled oscillators that controls the cell cycle of the nurse cells to regulate cyst growth. It is proposed that bidirectional nurse cell-oocyte communication establishes a growth-sensing feedback mechanism that regulates the quantity of maternal resources loaded into the oocyte.
Kim, M., Delos Santos, K. and Moon, N. S. (2021). Proper CycE-Cdk2 activity in endocycling tissues requires regulation of the cyclin-dependent kinase inhibitor Dacapo by dE2F1b in Drosophila. Genetics 217(1): 1-15. PubMed ID: 33683365
Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)-Cdk2 activity, which is known to be largely regulated at the level of transcription. This study reports an additional level of E2F-dependent control of CycE-Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). Evidence is provided showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE-Cdk2 activity. Furthermore, this study demonstrated that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, this study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.

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.

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila

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).

Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction

A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. This study describes and characterizes a 'proteostatic checkpoint' in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. These findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis (Rodriguez-Fernandez, 2019).

Protein Homeostasis (Proteostasis) encompasses the balance between protein synthesis, folding, re-folding and degradation, and is essential for the long-term preservation of cell and tissue function. It is achieved and regulated by a network of biological pathways that coordinate protein synthesis with degradation and cellular folding capacity in changing environmental conditions. This balance is perturbed in aging systems, likely as a consequence of elevated oxidative and metabolic stress, changes in protein turnover rates, decline in the protein degradation machinery, and changes in proteostatic control mechanisms. The resulting accumulation of misfolded and aggregated proteins is widely observed in aging tissues, and is characteristic of age-related diseases like Alzheimer's and Parkinson's disease. The age-related decline in proteostasis is especially pertinent in long-lived differentiated cells, which have to balance the turnover and production of long-lived aggregation-prone proteins over a timespan of years or decades. But it also affects the biology of somatic stem cells (SCs), whose unique quality-control mechanisms to preserve proteostasis are important for stemness and pluripotency (Rodriguez-Fernandez, 2019).

Common mechanisms to surveil, protect from, and respond to proteotoxic stress are the heat shock response (HSR) and the organelle-specific unfolded protein response (UPR). When activated, both stress pathways lead to the upregulation of molecular chaperones that are critical for the refolding of damaged proteins and for avoiding the accumulation of toxic aggregates. If changes to the proteome are irreversible, misfolded proteins are degraded by the proteasome or by autophagy. While all cells are capable of activating these stress response pathways, SCs deal with proteotoxic stress in a specific and state-dependent manner. Embryonic SCs (ESCs) exhibit a unique pattern of chaperone expression and elevated 19S proteasome activity, characteristics that decline upon differentiation. ESCs share elevated expression of specific chaperones (e.g., HspA5, HspA8) and co-chaperones (e.g., Hop) with mesenchymal SCs (MSCs) and neuronal SCs (NSCs), and elevated macroautophagy (hereafter referred to as autophagy) with hematopoietic SCs (HSCs), MSCs, dermal, and epidermal SCs. Defective autophagy contributes to HSC aging. It has further been proposed that SCs can resolve proteostatic stress by asymmetric segregation of damaged proteins, a concept first described in yeast (Rodriguez-Fernandez, 2019).

While these studies reveal unique proteostatic capacity and regulation in SCs, how the proteostatic machinery is linked to SC activity and regenerative capacity, and how specific proteostatic mechanisms in somatic SCs ensure that tissue homeostasis is preserved in the long term, remains to be established. Drosophila intestinal stem cells (ISCs) are an excellent model system to address these questions. ISCs constitute the vast majority of mitotically competent cells in the intestinal epithelium of the fly, regenerating all differentiated cell types in response to tissue damage. Advances made by numerous groups have uncovered many of the signaling pathways regulating ISC proliferation and self-renewal. In aging flies, the intestinal epithelium becomes dysfunctional, exhibiting hyperplasia and mis-differentiation of ISCs and daughter cells. This age-related loss of homeostasis is associated with inflammatory conditions that are characterized by commensal dysbiosis, chronic innate immune activation, and increased oxidative stress. It further seems to be associated with a loss of proteostatic capacity in ISCs, as illustrated by the constitutive activation of the unfolded protein response of the endoplasmic reticulum (UPR-ER), which results in elevated oxidative stress, and constitutive activation of JNK and PERK kinases. Accordingly, reducing PERK expression in ISCs is sufficient to promote homeostasis and extend lifespan (Rodriguez-Fernandez, 2019).

ISCs of old flies also exhibit chronic inactivation of the Nrf2 homologue CncC. CncC and Nrf2 are considered master regulators of the antioxidant response, and are negatively regulated by the ubiquitin ligase Keap1. In both flies and mice, this pathway controls SC proliferation and epithelial homeostasis. It is regulated in a complex and cell-type specific manner. Canonically, Nrf2 dissociates from Keap1 in response to oxidative stress and accumulates in the nucleus, inducing the expression of antioxidant genes. Drosophila ISCs, in turn, exhibit a 'reverse stress response' that results in CncC inactivation in response to oxidative stress. This response is required for stress-induced ISC proliferation, including in response to excessive ER stress, and is likely mediated by a JNK/Fos/Keap1 pathway (Rodriguez-Fernandez, 2019).

The Nrf2 pathway has also been linked to proteostatic control: 'Non-canonical' activation of Nrf2 by proteostatic stress as a consequence of an association between Keap1 and the autophagy scaffold protein p62 has been described in mammals. A similar non-canonical activation of Nrf2 has been described in Drosophila, where CncC activation is a consequence of the interaction of Keap1 with Atg8a, the fly homologue of the autophagy protein LC3. Nrf2/CncC activation induces proteostatic gene expression, including of p62 in mammalian cells and of p62/Ref2P and LC3/Atg8a in flies. Nrf2 is further a central transcriptional regulator of the proteasome in both Drosophila and mammals. Whether and how Nrf2 also influences proteostatic gene expression in somatic SCs remains unclear (Rodriguez-Fernandez, 2019).

This study shows that Drosophila CncC links cell cycle control with proteostatic responses in ISCs via the accumulation of dacapo, a p21 cell cycle inhibitor homologue, as well as the transcriptional activation of genes encoding proteases and proteasome subunits. This study establish that this program constitutes a transient 'proteostatic checkpoint', which allows clearance of protein aggregates before cell cycle activity is resumed. In old flies, this checkpoint is impaired and can be reactivated with a CncC activator (Rodriguez-Fernandez, 2019).

The central role of Nrf2/CncC in the proteostatic checkpoint is consistent with its previously described and evolutionarily conserved influence on longevity and tissue homeostasis, and is likely to be conserved in mammalian SC populations, as Nrf2 has for example been shown to influence proliferative activity, self-renewal and differentiation in tracheal basal cells. It may be unique to somatic SCs, however, as CncC or Nrf2-mediated inhibition of cell proliferation is not observed during development (such as in imaginal discs) or in other dividing cells. Assessing the existence of an Nrf2-induced proteostatic checkpoint in mammalian SC populations will be an important future endeavor (Rodriguez-Fernandez, 2019).

Mechanistically, the results support a model in which the presence of protein aggregates activates CncC through Atg8a-mediated sequestration of Keap1. In mammals, Nrf2 activation can also be achieved through the interaction of Keap1 with the Atg8a homologue LC3 and p62, and ref2p/p62 contributes to the degradation of polyQ aggregates in Drosophila, suggesting that a conserved Atg8a/p62/Keap1 interaction may be involved in the activation of the proteostatic checkpoint. The activation of CncC after cytosolic proteostatic stress described in this study thus differs mechanistically and in its consequence from the regulation of CncC after other types of protein stress in ISCs: in response to unfolded protein stress in the ER, CncC is specifically inactivated by a ROS/JNK-mediated signaling pathway. This mechanism allows ISC proliferation to be increased in response to tissue damage, but can also contribute to the loss of tissue homeostasis in aging conditions. The activation of CncC after cytosolic protein stress, in turn, allows arresting ISC proliferation during protein aggregate clearance. The distinct responses of ISCs to cytosolic or ER-localized proteostatic stress has interesting implications for understanding of the maintenance of tissue homeostasis. While the XBP1-mediated UPR-ER allows the expansion of the ER and the induction of ER chaperones to deal with a high load of unfolded proteins in the ER, it also stimulates ISC proliferation through oxidative stress and the activation of PERK and JNK. It is tempting to speculate that the sequestration of unfolded proteins within the ER allows ISCs to proceed through mitosis without the possibility of major misregulation, while the presence of cytosolic protein aggregates may be a unique danger to the viability of the cell and its daughters. It seems likely that constitutive activation of autophagy and proteasome pathways during the clearance of cytosolic aggregates is incompatible with the need for intricate regulation of these same pathways during the cell cycle in proliferating ISCs. It will be of interest to explore this hypothesis further in the future (Rodriguez-Fernandez, 2019).

The data suggest that the coordination of cell cycle arrest and aggregate clearance is achieved by the simultaneous induction of the cell cycle inhibitor Dacapo and a battery of genes encoding proteins involved in proteolysis. While it was possible to detect dacapo transcript expression in ISCs by fluorescent in situ hybridization at 24h after HttQ138 expression, it remains unclear whether Dacapo is induced directly by CncC or via the action of a CncC target gene. It is surprising that transcriptional induction of autophagy genes in was not seen in a RNAseq experiment, but it is possible that this is due to the fact that only one timepoint was sampled after induction of protein aggregates. Since the transcriptional response of autophagy genes is likely very dynamic, a more time-resolved transcriptome analysis during aggregate formation and clearance may have captured such a response (Rodriguez-Fernandez, 2019).

It is further notable that dap deficient ISC clones exhibit a significantly higher aggregate load in these experiments than wild-type ISC clones. This suggests that the induction of proteolytic genes and of cell cycle regulators is not only coincidentally linked by CncC, but that aggregate clearance and the cell cycle arrest mediated by Dacapo need to be tightly coordinated for effective ISC proteostasis. It will be interesting to explore the mechanism of this requirement in the future. It is tempting to speculate that, as the elimination of protein aggregates requires an increase in proteasome activity, and proteasome activity can influence cell cycle timing, cell cycle inhibition is a critical safeguard against de-regulation of normal cell cycle progression (Rodriguez-Fernandez, 2019).

The data suggest that Atg8a induction in ISCs experiencing proteostatic stress may serve a dual purpose: sustained activation of the proteostatic checkpoint as well as increased autophagy flux. This dual role is distinct from other autophagy components like Atg1, since Atg1 over-expression, an efficient way of promoting autophagy in Drosophila cells, counteracts the checkpoint rather than promoting it. Exploring the relative kinetics of Atg8a and Atg1 induction in ISCs after proteostatic stress is likely to provide deeper mechanistic insight into the regulation of the proteostatic checkpoint (Rodriguez-Fernandez, 2019).

Critically, the proteostatic checkpoint is reversible. Based on the current data and previous studies, it is proposed that upon clearance of aggregates, the Keap1/Atg8a interaction is decreased, thus releasing Keap1 to inhibit CncC. Lineage-tracing studies show that this allows re-activation of ISC proliferation and recovery of normal regenerative responses (Rodriguez-Fernandez, 2019).

The loss of proteostatic checkpoint efficiency in ISCs of old guts is likely a consequence of the age-related inactivation of CncC in these cells (possibly caused by chronic oxidative stres. Accordingly, reactivating Nrf2/CncC in the gut by overexpressing CncC is sufficient to restore epithelial homeostasis in the intestine of old flies, and this study found that exposing animals to Otipraz intermittently late in life promotes epithelial barrier function and extends lifespan (Rodriguez-Fernandez, 2019).

Since Nrf2/CncC and other components required for the proteostatic checkpoint are conserved across species, it is anticipated that the current findings will be relevant to homeostatic preservation of adult SCs in vertebrates. Supporting this view, mammalian Cdkn1a (p21) has been described as an Nrf2 target gene. Transient activation of Nrf2 may thus be a viable intervention strategy to improve proteostasis and maintain regenerative capacity in high-turnover tissues of aging individuals (Rodriguez-Fernandez, 2019).


Amino Acids - 245

Structural Domains

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).

dacapo: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 JAN 96 

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