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

Gene name - Cyclin A

Synonyms -

Cytological map position - 68E1--68E2

Function - Activates cdc2 kinase

Keywords - cell cycle

Symbol - CycA

FlyBase ID:FBgn0000404

Genetic map position - 3-[36]

Classification - G2-M cyclin

Cellular location - nuclear

NCBI links: | Entrez Gene
Recent literature
Afonso, D.J., Liu, D., Machado, D.R., Pan, H., Jepson, J.E., Rogulja, D. and Koh, K. (2015). TARANIS functions with Cyclin A and Cdk1 in a novel arousal center to control sleep in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26096977
Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. This study identifies Taranis (Tara), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, tara was isolated as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. Tara regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of Tare and CycA in sleep regulation. Finally, a novel wake-promoting role was described for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. The study proposes that Taranis controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center.
Bourouh, M., Dhaliwal, R., Rana, K., Sinha, S., Guo, Z. and Swan, A. (2016). Distinct and overlapping requirements for Cyclins A, B and B3 in Drosophila female meiosis. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27652889
Meiosis, like mitosis depends on the activity of the mitotic Cyclin dependent kinase, Cdk1 and its cyclin partners. This study examined the specific requirements for the three mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3 in meiosis of Drosophila melanogaster. All three cyclins were found to contribute redundantly to nuclear envelope breakdown, though Cyclin A appears to make the most important individual contribution. Cyclin A is also required for bi-orientation of homologues in meiosis I. Cyclin B3, as previously reported, is required for anaphase progression in meiosis I and in meiosis II. Cyclin B3 also plays a redundant role, with Cyclin A, in preventing DNA replication during meiosis. Cyclin B is required for maintenance of the metaphase I arrest in mature oocytes, for spindle organization and for timely progression through the 2nd meiotic division. It is also essential for polar body formation at the completion of meiosis. With the exception of its redundant role in meiotic maturation, Cyclin B appears to function independently of Cyclins A and B3 through most of meiosis. The study concludes that the 3 mitotic Cyclin-Cdk complexes have distinct and overlapping functions in Drosophila female meiosis.

Rotelli, M. D., Policastro, R. A., Bolling, A. M., Killion, A. W., Weinberg, A. J., Dixon, M. J., Zentner, G. E., Walczak, C. E., Lilly, M. A. and Calvi, B. R. (2019). A Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles. PLoS Genet 15(7): e1008253. PubMed ID: 31291240
Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.

Studies of the grapes (grp) gene, which encodes a homolog of the Schizosaccharomyces pombe Chk1 kinase, provide clues about the role of Cyclin A in coordinating mitotic events early in Drosophila development. Grapes is known to regulate a cell-cycle checkpoint that delays mitosis in response to inhibition of DNA replication. Grp is also required in the undisturbed early embryonic cycles: in its absence, mitotic abnormalities appear in cycle 12 and chromosomes fail to fully separate in subsequent cycles. In other systems, Chk1 kinase phosphorylates and suppresses the activity of the Cdc25 phosphatase String: the resulting failure to remove inhibitory phosphate from cyclin-dependent kinase 1 (Cdk1) prevents entry into mitosis. Because in Drosophila embryos Cdk1 lacks inhibitory phosphate during cycles 11- 13, it is not clear that known actions of Grp/Chk1 suffice in these cycles. In early cell cycles, the loss of grp compromises Cyclin A proteolysis and delays mitotic disjunction of sister chromosomes. These defects occur prior to previously reported grp phenotypes. It is concluded that Grp activates Cyclin A degradation, and functions to time the disjunction of chromosomes in the early embryo. As Cyclin A destruction is required for sister chromosome separation: a failure in Grp-promoted cyclin destruction can also explain the mitotic phenotype. The mitotic failure described previously for cycle 12 grp embryos might be a more severe form of the phenotypes that are describe here in earlier embryos and it is suggested that the underlying defect is reduced degradation of Cyclin A (Su, 1999).

When syncytial embryos are exposed to cycloheximide, a protein synthesis inhibitor, nuclear cycles arrest in interphase and cyclin A levels decline whereas Cyclin B is stable under these conditions. This suggests that a steady state of synthesis and degradation maintains the interphase levels of Cyclin A during the syncytial divisions. grp mutants were found to be deficient in Cyclin A degradation in the presence of cycloheximide. This defect is seen in embryos in cycles 4- 8, earlier than other reported grp phenotypes and is therefore unlikely to be secondary to these phenotypes. It is inferred that Grp normally destabilizes Cyclin A (Su, 1999).

In wild-type embryos, Cyclin A is unstable not only in interphase but also during mitotic arrest caused by microtubule destabilization. Thus, in embryos treated with colchicine, blocking protein synthesis with cycloheximide leads to a decline in Cyclin A levels, whereas cyclin B is stable under these conditions. grp embryos are compromised for Cyclin A proteolysis at such a colchicine-induced mitotic arrest. As for interphase destruction of Cyclin A, this defect is seen during early syncytial cycles, before the onset of other reported grp phenotypes. It is concluded that Grp promotes Cyclin A degradation in colchicine-arrested early embryos (Su, 1999).

If normal levels of Cyclin A are maintained by a steady state of synthesis and destruction, it would be expected that the levels of Cyclin A would be high in grp embryos as a result of increased stability. Western blotting shows that grp embryos have slightly higher levels of Cyclin A than wild-type embryos (1.5-3-fold). Although this relatively small increase suggests that significant degradation of Cyclin A still occurs in grp embryos, this degradation is not apparent at the arrests induced by cycloheximide or by colchicine (Su, 1999).

Since Grp is required for destruction of Cyclin A in an arrested mitosis, it was determined whether or not mitosis is disrupted in grp embryos. The mitosis-specific phosphorylation of histone H3 (PH3) apparently acts as an in vivo reporter for cyclin/Cdk activity and its disappearance at the end of mitosis requires cyclin destruction. During syncytial cycles of wild-type embryos, PH3 staining is continuous along the length of the chromosome arms from metaphase until late anaphase, when loss of the epitope near kinetochores leads to graded staining. In embryos from grp1 homozygous females or from grp1/Df females, the gradient of PH3 is seen on chromosomes in early anaphase. Thus, PH3 loss is advanced with respect to chromosome segregation in grp embryos. This defect is seen at the earliest cycles scored (cycle 4 in grp1/grp1 and cycle 3 in grp1/Df), well before the onset of previously reported defects in grp embryos (cycle 11) (Su, 1999).

The loss of PH3 during early anaphase in grp embryos could be due to the premature loss of PH3, a scenario opposite of that expected for a mutation that stabilizes Cyclin A during mitosis. Alternatively, timely loss of PH3 staining but delayed chromosome separation would produce the same mis-coordination. Analysis of grp embryos supports the latter hypothesis: an increase in the ratio of embryos with unsegregated chromosomes (prophase/metaphase) to those with segregated chromosomes (anaphase/telophase) has been found. It ia concluded that Grp is required for timely chromosome segregation in syncytial mitoses (Su, 1999).

The mitotic phenotype in grp embryos can be understood as follows: ordinarily, the mitotic cyclins are degraded in a sequence during exit from mitosis; Cyclin A is degraded before the metaphase-anaphase transition; cyclin B is degraded at the beginning of anaphase and cyclin B3 towards the end of anaphase. The disappearance of PH3 can be prevented by stabilization of any of these mitotic cyclins; thus, it appears that loss of PH3 marks the completion of this sequence. It is suggested that grp embryos are specifically defective in the early initiation of Cyclin A degradation but they do degrade Cyclin A, perhaps in conjunction with the B cyclins. Eventual destruction of Cyclin A would explain the ability of grp-deficient nuclei to exit mitosis and lose PH3 staining, events that can be inhibited by stable Cyclin A. Destruction of Cyclin A in conjunction with the B cyclins would explain why the length of mitosis is not increased in grp embryos. Because the expression of a stable form of Cyclin A prevents chromosome disjunction, it has been suggested that the failure to degrade Cyclin A early during mitosis in grp embryos delays chromosome disjunction. The delay in chromosome separation abbreviates anaphase and, when the abbreviation is severe, decondensation of chromosomes and entry into the next interphase occurs before the separating chromosomes reach the spindle poles (Su, 1999).

Since Grp promotes Cyclin A degradation, genetic interactions might be detected between grp and Cyclin A. A test was performed to see whether grp mutants are sensitive to levels of Cyclin A. Homozygous grp flies are viable but female sterile. When a single copy of a heat-inducible Cyclin A transgene is introduced, however, homozygous grp1 progeny are not recovered. Thus, even without induction, the presence of a heat-inducible Cyclin A transgene (which by itself is viable as a heterozygote or homozygote) causes grp1 to behave as a recessive lethal. The observed synthetic lethality suggests that the Grp deficiency sensitizes the fly to low levels of Cyclin A expression from the transgene. In contrast to this strong interaction with increased Cyclin A levels, a reduction in the dose of Cyclin A fails to suppress the grp1 allele. It is perhaps not surprising that reduction of Cyclin A by half does not suppress a null allele of grp. Reduction in the dose of Cyclin A does suppress the lethality of mei-41 mutations. The mei-41 gene is a homolog of the gene ATM, which is mutated in the genetic disorder ataxia-telangiectasia; mei-41 is thought to act upstream of grp in the checkpoint pathway, and mutations in mei-41 result in a phenotype like grp, but less severe. Importantly, the suppression of the mei-41 embryonic lethality by cyclin reduction occurs without restoring interphase length. This result shows that the mitotic defect is not an inevitable consequence of premature entry into mitosis as previously thought. It is suggested that the mitotic defect is an anaphase failure as a result of defective metaphase destruction of Cyclin A (Su, 1999).

If Grp promotes the metaphase-anaphase transition, why is it dispensable at most stages of development? Before cell cycle 12, grp embryos exhibit a defective mitosis with delayed sister chromosome separation; despite this, mitosis is successful. From this observation, two inferences can be made: (1) mitosis can tolerate a limited disruption in the timing of events and, (2) as anaphase occurs in the absence of Grp, there must be a backup Grp-independent mechanism that promotes sister separation slightly later. The mitotic mis-coordination in grp embryos gets progressively more severe, and Cyclin A levels increase progressively during the syncytial cycles. This correlation, together with the ability of reduced Cyclin A dose to suppress mei-41 lethality, leads the authors to suggest that the consequence of a defect in the grp/mei-41 pathway increases in severity as Cyclin A increases, until anaphase fails at mitosis 12 and 13 (Su, 1999).

The current model for Chk1 function involves the phosphorylation and inhibition of Cdc25, in part by the binding of 14-3-3 protein to the phosphorylated Cdc25 and sequestration in the cytoplasm where it is ineffective in counteracting the nuclear kinases Wee1 and Mik1. Thus, inhibitory phosphorylation of Cdk1 prevents its activation and the cell arrests in G2. Although this action of Chk1 appears general, it is possible that Chk1 activity has other consequences. Indeed, there is no substantial accumulation of inhibitory phosphate on Cdk1, and the Cdc25Stg protein is constitutively present and nuclear during interphase of syncytial cycles 11- 13 when a grp-dependent mechanism regulates the entry into mitosis. The results presented here suggest that Grp may function to destabilize Cyclin A. When a Grp-dependent cell-cycle checkpoint is induced by blocking S phase with aphidicolin in cleavage-stage Drosophila embryos, Cdc25Stg is destabilized. Thus, whether it is direct or indirect, Grp promotes the destruction of two cell-cycle proteins, Cdc25Stg and Cyclin A. It is suggested that promotion of the metaphase- anaphase transition represents a second function of the grp/mei-41 pathway, distinct from the checkpoint arrest of entry into mitosis. Nevertheless, a common mechanism might be involved because blocking entry into mitosis and promoting exit from mitosis both involve inhibition of cyclin/Cdk1 activity (Su, 1999).

In summary, Grp is required for normal Cyclin A turnover in the early Drosophila embryo. It was also found that grp mutant embryos show a delay in the timing of the metaphase- anaphase transition. Stable versions of Cyclin A block chromosome separation at the metaphase plate, at least in cellularized Drosophila embryos, suggesting that proteolysis of Cyclin A is required for this process. Thus, the proposal that Grp activates Cyclin A proteolysis can explain the mitotic phenotype as a consequence of at least temporary persistence of Cyclin A (Su, 1999).

Control of sleep by cyclin A and its regulator

How and why the brain reversibly switches from a waking to a sleep state remain among the most intriguing questions in biology. This study shows that cyclin A (CycA) and regulator of cyclin A1, essential cell cycle factors, function in postmitotic neurons to promote sleep in Drosophila melanogaster. Reducing the abundance of CycA in neurons delayed the wake-sleep transition, caused multiple arousals from sleep, and reduced the homeostatic response to sleep deprivation. CycA is expressed in ~40 to 50 neurons in the adult brain, most of which are intermingled with circadian clock neurons, suggesting functional interactions among neurons controlling sleep and circadian behavior (Rogulija, 2012).

Prolonged intervals of rest in Drosophila share many features with mammalian sleep. Flies spend a large portion of their waking time moving, and the locomotor assay developed for circadian behavior can therefore be used to screen for sleep mutants in this animal. A bout of inactivity lasting 5 min or longer is associated with an increased arousal threshold, as well as changes in neural activity, and is used to define sleep in the fly. Since the discovery of sleep in Drosophila a decade ago, the results of several genetic screens for sleep factors have been reported, and many of the genes identified have conserved roles across species. A surprisingly large fraction of these genes are ion channels or their regulators, or are involved with broadly acting neurotransmitter pathways. These findings led to the hypothesis that there are no dedicated sleep genes but rather that sleep is regulated by genes that control normal neuronal function (Rogulja, 2012).

This study carried out a screen for sleep genes solely within the nervous system, bypassing the potential confounding effects of these genes in other tissues. ~4000 UAS-RNA interference (UAS-RNAi) lines were screened using the pan-neuronal driver elavGal4 to knock down gene expression, while simultaneously driving the expression of UAS-Dicer2 to enhance RNAi efficacy. After three retesting rounds, ~20 genes were identified whose RNAi-mediated neuronal depletion led to reproducible changes in sleep pattern, quantity, or both. One sleep-promoting factor isolated was Rca1 (regulator of cyclin A1), a conserved, essential cell cycle gene (Rogulja, 2012).

Depletion of Rca1 from neurons (elavGal4 + UAS-Rca1-RNAi, denoted 'elav>Rca1-RNAi') decreased the amount of total daily sleep, without affecting the levels of activity per waking minute. It did so by decreasing the duration of individual sleep episodes (sleep bouts). The number of sleep episodes was slightly increased in elav>Rca1-RNAi animals, likely because of the highly fragmented nature of their sleep. The net reduction in sleep duration understates the severity of the elav>Rca1-RNAi phenotype. For example, elav>Rca1-RNAi flies that showed no net change in daily sleep often failed to display behavior that is organized into prolonged intervals of rest and activity seen in the control flies (Rogulja, 2012).

Drosophila Rca1 is homologous to the mammalian early mitotic inhibitor (Emi1) and plays a critical role in preventing premature cyclin degradation. Cyclins regulate cell cycle progression through activation of specific cyclin-dependent kinases (CDKs). The main target of Drosophila Rca1 regulation is cyclin A (CycA), so tests were performed to see whether depletion of CycA might affect sleep. Indeed, expression of CycA-RNAi in neurons (elavGal4 + UAS-CycA-RNAi, denoted 'elav>CycA-RNAi') caused a decrease in the time animals spent sleeping without significantly affecting levels of activity per minute while awake. This reduction was a consequence of decreased sleep bout duration, whereas the number of sleep episodes was slightly increased (Rogulja, 2012).

It was noticed that the ectopic overexpression of Rca1 or CycA in neurons produced a small and rough eye phenotype. A similar eye phenotype is associated with a mutation in a negative regulator of CycA and was used in a modifier screen to identify new genes involved in cell cycle regulation. This morphological readout of CycA abundance was used to confirm that the UAS-Rca1-RNAi and UAS-CycA-RNAi lines were effective, by showing that they suppressed eye phenotypes associated with Rca1 or CycA overexpression, respectively. Furthermore, UAS-Rca1-RNAi also suppressed the eye phenotypes resulting from CycA overexpression, indicating that Rca1 can stabilize CycA protein in neurons (Rogulja, 2012).

Flies homozygous for CycA null alleles (CycA-) do not survive and could not be examined. However, CycA- heterozygotes are viable and showed a decrease in total sleep amounts, a phenotype further enhanced by expression of CycA-RNAi in this background, even in the absence of Dicer2 overexpression. An additive effect was observed between the heterozygous CycA- mutation and neuronal Rca1-RNAi as well. The reduction in total sleep seen in these experiments may not fully reflect the influence of CycA and Rca1 in sleep control, as CycA protein was still detectable in the brains of all of the experimental genotypes (Rogulja, 2012).

As CycA is thought to be the main target of Rca1 regulation during the cell cycle, and behavioral and genetic analyses indicated a similar relation with respect to the effects of these genes on sleep, focus was placed on CycA for subsequent studies (Rogulja, 2012).

It took significantly longer for elav>CycA-RNAi flies to fall asleep after lights went off than it did for the controls. This was not the case for all genes that were identified in the screen. This increased latency to sleep suggests that a mechanism regulating wake-sleep transition is disrupted in elav>CycA-RNAi flies. To determine if sleep can be homeostatically controlled in these animals, their response to sleep deprivation was examined. The flies were mechanically stimulated during the night, and their sleep pattern was examined during the subsequent morning, when 'rebound' sleep is normally seen in wild-type flies that have been sleep-deprived the night before. Although some increase was seen in rebound sleep in flies of all the genotypes tested, this response was reduced in elav>CycA-RNAi flies, suggesting a defective sleep homeostat. In accordance with the idea that sleep serves an essential function, neuronal depletion of CycA shortens life span, presumably due to chronic sleep deprivation experienced by these animals. However, it is also possible that CycA contributes to life span through other functions in postmitotic neurons (Rogulja, 2012).

Sleep is regulated in a circadian fashion, so it was asked whether some of the sleep defects observed in elav>CycA-RNAi animals resulted from a disrupted circadian clock. Most elav>CycA-RNAi flies showed robust rhythms in constant darkness, with normal periods. Circadian rhythmicity was observed even in animals with severe sleep defects. Furthermore, elav>CycA-RNAi flies eclosed with strong circadian rhythms. Finally, Period (PER), a core component of the circadian clock, accumulated rhythmically in the pacemaker neurons of elav>CycA-RNAi animals. Thus, the sleep defects observed in elav>CycA-RNAi animals appear not to reflect circadian clock anomalies (Rogulja, 2012).

Staining with two different antibodies to CycA showed a restricted expression pattern in adult brains. Both antibodies detected the same ~12 dorsal neurons, and one of the antibodies labeled cells in several other brain regions, including the pars intercerebralis, a known fly sleep center. The antibody that detected CycA in more cells was raised against the whole CycA protein, whereas the one that labeled fewer cells was raised against the CycA N terminus. Both appear to be specific to CycA, because their staining was strongly reduced in embryos in which CycA-RNAi was driven by a strong, ubiquitous Gal4 driver. The broader CycA expression pattern in the brain was supported by colocalization with CycAGal4-driven GFP, although GFP was detected in additional cells in all the clusters. Overall, CycA protein was detected in ~40 to 50 neurons in the brain (Rogulja, 2012).

In the adult fly brain, ~150 clock neurons are organized into six major clusters: three dorsal clusters (DN1, DN2, and DN3), a dorsal-lateral cluster (LNd), and two groups of ventral-lateral neurons -- small (s-LNv) and large (l-LNv). The small and large ventral-lateral neurons include neurons that produce the neuropeptide pigment dispersing factor (PDF), a major regulator of clock output. s-LNvs are the major pacemaker neurons which also regulate sleep. Costaining of brains for CycA and PDF revealed that termini of PDF-producing s-LNvs project near the dorsal CycA neurons. Indeed, as judged by PDFrGal4-driven GFP, which marks presumptive PDF receptor-producing neurons, these CycA cells appear to express the PDF receptor. Such coexpression was found in all CycA neurons and suggests a molecular and neuronal connection between circadian and sleep systems. Coexpression with PDFr also provides an opportunity to knock down CycA abundance in a more limited set of neurons than the pan-neuronal driver affords. PDFrGal4-driven CycA-RNAi results in a severe sleep reduction (Rogulja, 2012).

To determine if dorsal CycA neurons are in fact DN1 circadian neurons, it was asked whether they express per. A subset of the dorsal CycA-containing neurons expressed perGal4-driven GFP. A similar partial overlap was seen in all other CycA clusters, with the exception of the pars intercerebralis. As perGal4 was expressed in a smaller subset of CycA neurons than PDFrGal4, the former was used to knock down CycA in a more limited neuronal subpopulation. In these experiments, sleep was consistently lower than in control animals, although the extent of sleep loss was less extreme than with pan-neuronal CycA knockdown. Notably, CycA-containing dorsal neurons that expressed perGal4 were not stained by an antibody to PER, and thus do not detectably express PER protein. Therefore, dorsal CycA neurons are closely associated with the DN1 cluster, but do not appear to be clock neurons themselves. A similar relationship exists between CycA-expressing and DN3 or LNv neurons. In contrast, CycA and PER proteins were occasionally found in the same cell within a subset of dorsal-lateral neurons (LNds), a group of clock neurons that regulate the evening peak of activity. Finally, a bilaterally symmetric pair of large ventral neurons expressed both CycA and PER proteins. These ventral neurons do not belong to any known clock neuronal clusters. In summary, CycA-containing neurons are often intermingled with circadian pacemaker neurons. As CycA neurons appear to express the receptor for the circadian neuropeptide PDF, such an arrangement suggests that signals produced by neurons regulating circadian rhythmicity may be directed to neurons that control sleep (Rogulja, 2012).

There are other known instances of the repurposing of cell cycle machinery in neurons. Examples include the recent observations that two CDK pathways are essential for proper axonal targeting of presynaptic components in Caenorhabditis elegans, and that cyclin E regulates synaptic plasticity and memory formation in mice. No obvious defects were observed in the gross morphology or number of CycA-containing neurons in elav>CycA-RNAi flies, no obvious rhythmic patterns of CycA protein expression were detected. Nevertheless, such features might become evident in studies focused on specific cells or specific subcellular regions, or in developing or aging flies. Future understanding of the role that CycA plays in determining the structure or activity of neurons specialized for the regulation of sleep seems likely to shed light on the molecular mechanisms underlying this enigmatic behavior. As the role of CycA in cell cycle regulation is conserved throughout metazoans, its role in sleep regulation might be conserved as well (Rogulja, 2012).

Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease (Rotelli, 2019).

Endoreplication is a common cell cycle variant that entails periodic genome duplication without cell division and results in large polyploid cells. Two variations on endoreplication are the endocycle, a repeated G/S cycle that completely skips mitosis, and endomitosis, wherein cells enter but do not complete mitosis and / or cytokinesis before duplicating their genome again. In a wide array of organisms, specific cell types switch from mitotic cycles to endoreplication cycles as part of normal tissue growth during development. Cells also can switch to endoreplication in response to conditional inputs, for example during wound healing, tissue regeneration, aging, and cancer. It is still not fully understood, however, how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication (Rotelli, 2019).

There are common themes across plants and animals for how cells switch to endoreplication during development. One common theme is that developmental signaling pathways induce endoreplication by inhibiting the mitotic cyclin dependent kinase 1 (CDK1). After CDK1 activity is repressed, repeated G / S cell cycle phases are controlled by alternating activity of the ubiquitin ligase APC/CCDH1 and Cyclin E / CDK2. Work in Drosophila has defined mechanisms by which APC/CCDH1 and CycE / Cdk2 regulate G / S progression, and ensure that the genome is duplicated only once per cycle. Despite these conserved themes, how endoreplication is regulated can vary among organisms, as well as tissues within an organism. These variations include the identity of the signaling pathways that induce endoreplication, the mechanism of CDK1 inhibition, and the downstream effects on cell cycle remodeling into either an endomitotic cycle (partial mitosis) or endocycle (skip mitosis). In many cases, however, the identity of the developmental signals and the molecular mechanisms of cell cycle remodeling are not known (Rotelli, 2019).

To gain insight into the regulation of variant polyploid cell cycles, two-color microarrays have been used to compare the transcriptomes of endocycling and mitotic cycling cells in Drosophila tissues (Maqbool, 2010). Endocycling cells of larval fat body and salivary gland have been shown to have dampened expression of genes that are normally induced by E2F1, a surprising result for these highly polyploid cells given that many of these genes are required for DNA synthesis. Nonetheless, subsequent studies showed that the expression of the E2F-regulated mouse orthologs of these genes is reduced in endoreplicating cells of mouse liver, megakaryocytes, and trophoblast giant cells. Drosophila endocycling cells also had dampened expression of genes regulated by the Myb transcription factor, the ortholog of the human B-Myb oncogene (MYBL2). Myb is part of a larger complex called Myb-MuvB (MMB), whose subunit composition and functions are mostly conserved from flies to humans. One conserved function of the MMB is the induction of periodic transcription of genes that are required for mitosis and cytokinesis. It was these mitotic and cytokinetic genes whose expression was dampened in Drosophila endocycles, suggesting that this repressed MMB transcriptome may promote the switch to endocycles that skip mitosis. It is not known, however, how E2F1 and Myb activity are repressed during endocycles, nor which of the downregulated genes are key for the remodeling of mitotic cycles into endocycles (Rotelli, 2019).

In addition to endoreplication during development, there are a growing number of examples of cells switching to endoreplication cycles in response to conditional stresses and environmental inputs. These cells will be called induced endoreplicating cells (iECs) to distinguish them from developmental endoreplicating cells (devECs). For example, iECs contribute to tissue regeneration after injury in flies, mice, humans, and the zebrafish heart, and evidence suggests that a transient switch to endoreplication contributes to genome instability in cancer. Cardiovascular hypertension stress also promotes an endoreplication that increases the size and ploidy of heart muscle cells, and this hypertrophy contributes to cardiac disease. It remains little understood how similar the cell cycles of iECs are to devECs (Rotelli, 2019).

Similar to the developmental repression of CDK1 activity to promote endocycles, it has been shown that experimental inhibition of CDK1 activity is sufficient to induce endoreplication in flies, mouse, and human cells. These experimental iECs in Drosophila are similar to devECs in that they skip mitosis, have oscillating CycE / Cdk2 activity, periodically duplicate their genome during G / S cycles, and repress the apoptotic response to genotoxic stress. This study uses these experimental iECs to determine how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication cycles, and how similar this remodeling is between iECs and devECs. The findings indicate that the status of a CycA-Myb-AurB network determines the choice between mitotic cycles and endoreplication cycles in both iECs and devECs (Rotelli, 2019).

This study has investigated how the cell cycle is remodeled when mitotic cycling cells switch into endoreplication cycles, and how similar this remodeling is between devECs and experimental iECs. Repression of a CycA-Myb-AurB mitotic network promotes a switch to endoreplication in both devECs and iECs. Although a dampened E2F1 transcriptome of S phase genes is a common property of devECs in flies and mice, this study found that repression of the Myb transcriptome is sufficient to induce endoreplication in the absence of reduced expression of the E2F1 transcriptome. Knockdown of different components of the CycA-Myb-AurB network resulted in endoreplication cycles that repressed mitosis to different extents, which suggests that regulation of different steps of this pathway may explain the known diversity of endoreplication cycles in vivo. Overall, these findings define how cells either commit to mitosis or switch to different types of endoreplication cycles, with broader relevance to understanding the regulation of these variant cell cycles and their contribution to development, tissue regeneration, and cancer (Rotelli, 2019).

The findings indicate that the status of the CycA-Myb-AurB network determines the choice between mitotic or endoreplication cycles (The CycA-Myb-AurB network regulates the choice between cell cycle programs). These proteins are essential for the function of their respective protein complexes: CycA activates CDK1 to regulate mitotic entry, Myb is required for transcriptional activation of mitotic genes by the MMB transcription factor complex, and AurB is the kinase subunit of the four-subunit CPC. While each of these complexes were previously known to have important mitotic functions, the data indicate that they are key nodes of a network whose activity level determines whether cells switch to the alternative growth program of endoreplication. The results are consistent with previous evidence in several organisms that lower activity of the Myb transcription factor results in polyploidization, and further shows that repressing the function of the CPC and cytokinetic proteins downstream of Myb also promotes endoreplication. Importantly, genetic evidence indicates that not all types of mitotic inhibition result in a switch to endoreplication. For example, knockdown of the Spc25 and Spc105R kinetochore proteins or the Polo kinase resulted in a mitotic arrest, not a switch to repeated endoreplication cycles. These observations are consistent with CycA / CDK, MMB, and the CPC playing principal roles in the mitotic network hierarchy and the decision to either commit to mitosis or switch to endoreplication cycles (Rotelli, 2019).

While knockdown of different proteins in the CycA-Myb-AurB network were each sufficient to induce endoreplication cycles, these iEC populations had different fractions of cells with multiple nuclei diagnostic of an endomitotic cycle. Knockdown of cytokinetic genes pav and tum resulted in the highest fraction of endomitotic cells, followed by the CPC subunits, then Myb, and finally CycA, with knockdown of this cyclin resulting in the fewest endomitotic cells. These results suggest that knocking down genes higher in this branching mitotic network (e.g. CycA) inhibits more mitotic functions and preferentially promotes G / S endocycles that skip mitosis, whereas inhibition of functions further downstream in the network promote endomitosis. Moreover, different levels of CPC function also resulted in different subtypes of endoreplication. Strong knockdown of AurB inhibited chromosome segregation and cytokinesis resulting in cells with a single polyploid nucleus, whereas a mild knockdown resulted in successful chromosome segregation but failed cytokinesis, suggesting that cytokinesis requires more CPC function than chromosome segregation. It thus appears that different thresholds of mitotic function result in different types of endoreplication cycles. This idea that endomitosis and endocycles are points on an endoreplication continuum is consistent with evidence that treatment of human cells with low concentrations of CDK1 or AurB inhibitors induces endomitosis, whereas higher concentrations induce endocycles. The results raise the possibility that in tissues of flies and mammals both conditional and developmental inputs may repress different steps of the CycA-Myb-AurB network to induce slightly different types of endoreplication cycles that partially or completely skip mitosis. Together, these findings show that there are different paths to polyploidy depending on both the types and degree to which different mitotic functions are repressed (Rotelli, 2019).

The findings are relevant to the regulation of periodic MMB transcription factor activity during the canonical mitotic cycle. Knockdown of CycA compromised MMB transcriptional activation of mitotic gene expression, and their physical association suggests that the activation of the MMB by CycA may be direct. The MMB-regulated mitotic genes were expressed at lower levels in CycA iECs, even though Myb protein levels were not reduced. This result is consistent with the hypothesis that CycA / CDK phosphorylation of the MMB is required for its induction of mitotic gene expression. Moreover, misexpression of Myb in CycA knockdown follicle cells did not prevent the switch to endoreplication, further evidence that CycA / CDK is required for MMB activity and mitotic cycles. While the dependency of the MMB on CycA was not previously known in Drosophila, it was previously reported that in human cells CycA / CDK2 phosphorylates and activates human B-Myb in late S phase, and also triggers its degradation. While further experiments are needed to prove that CycA / CDK regulation of the MMB is direct, interrogation of the results of multiple phosphoproteome studies using iProteinDB indicated that Drosophila Myb protein is phosphorylated at three CDK consensus sites including one, S381 that is of a similar sequence and position to a CDK phosphorylated site on human B-Myb (T447). The hypothesis is favored that it is CycA complexed to CDK1 that regulates the MMB because, unlike human cells, in Drosophila CycA / CDK2 is not required for S phase, and Myb is degraded later in the cell cycle during mitosis. Moreover, it is known that mutations in CDK1, but not CDK2, induce endocycles in Drosophila, mouse, and other organisms. A cogent hypothesis is that CycA / CDK1 phosphorylates Myb, and perhaps other MMB subunits, to stimulate MMB activity as a transcriptional activator of mitotic genes, explaining how pulses of mitotic gene expression are integrated with the master cell cycle control machinery. It remains formally possible, however, that both CycA / CDK2 and CycA / CDK1 activate the MMB in Drosophila. The early reports that CycA / CDK2 activates B-Myb in human cells were before the discovery that it functions as part of the MMB and the identification of many MMB target genes, and further experiments are needed to fully define how MMB activity is coordinated with the central cell cycle oscillator in fly and human cells (Rotelli, 2019).

Endocycles were experimentally induced by knockdown of CycA to mimic the repression of CDK1 that occurs in devECs. The data revealed both similarities and differences between these experimental iECs and devECs. Both iECs and SG devECs had a repressed CycA-Myb-AurB network of mitotic genes. In contrast, only devECs had reduced expression of large numbers of E2F1-dependent S phase genes, a conserved property of devECs in fly and mouse. In CycA iECs, many of these key S phase genes were not downregulated, including Cyclin E, PCNA, and subunits of the pre-Replicative complex, among others. This difference between CycA dsRNA iECs and SG devECs indicates that repression of these S phase genes is not essential for endoreplication. In fact, none of the E2F1 -dependent S phase genes were downregulated in Myb dsRNA iEC. Instead, the 12 E2F1-dependent genes that were commonly downregulated in Myb dsRNA iEC, CycA dsRNA iEC, and SG devEC all have functions in mitosis. These 12 mitotic genes are, therefore, dependent on both Myb and E2F1 for their expression, including the cytokinetic gene tum whose knockdown induced endomitotic cycles. This observation leads to the hypothesis that downregulation of the E2F transcriptome in fly and mouse devECs may serve to repress the expression of these mitotic genes, and that the repression of S phase genes is a secondary consequence of this regulation. These genomic data, together with the genetic evidence in S2 cells and tissues, indicates that in Drosophila the repression of the Myb transcriptome is sufficient to induce endoreplication without repression of the E2F1 transcriptome. The observation that both CycAdsRNA iECs and devECs both have lower CycA / CDK activity, but differ in expression of E2F1 regulated S phase genes, also implies that there are CDK-independent mechanisms by which developmental signals repress the E2F1 transcriptome in devECs (Rotelli, 2019).

The results have broader relevance to the growing number of biological contexts that induce endoreplication. Endoreplicating cells are induced and contribute to wound healing and regeneration in a number of tissues in fly and mouse, and, depending on cell type, can either inhibit or promote regeneration of the zebrafish heart. An important remaining question is whether these iECs, like experimental iECs and devECs, have a repressed CycA-Myb-AurB network. If so, manipulation of this network may improve regenerative therapies. In the cancer cell, evidence suggests that DNA damage and mitotic stress, including that induced by cancer therapies, can switch cells into an endoreplication cycle. These therapies include CDK and AurB inhibitors, which induce human cells to polyploidize, consistent with the fly data that CycA / CDK and the CPC are key network nodes whose repression promotes the switch to endoreplication. Upon withdrawal of these inhibitors, transient cancer iECs return to an error-prone mitosis that generates aneuploid cells, which have the potential to contribute to therapy resistance and more aggressive cancer progression. The finding that the Myb transcriptome is repressed in iECs opens the possibility that these mitotic errors may be due in part to a failure to properly orchestrate a return of mitotic gene expression. Understanding how this and other networks are remodeled in polyploid cancer cells will empower development of new approaches to prevent cancer progression (Rotelli, 2019).

Asymmetric assembly of centromeres epigenetically regulates stem cell fate

Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

A key player in centromere assembly in vertebrates is HJURP (holliday junction recognition protein), which localizes at centromeres during the cell cycle window of CENP-A deposition. Furthermore, centromere assembly is regulated by the cell cycle machinery. In flies, deposition of CID (the homologue of CENP-A) requires activation of the anaphase promoting complex/cyclosome (APC/C) and degradation of CYCLIN A (CYCA). In humans, centromere assembly is antagonized by Cdk1 activity, while the kinase Plk1 promotes assembly. Additionally, the CYCLIN B (CYCB)/Cdk1 complex inhibits the binding of CENP-A to HJURP, preventing CENP-A loading at centromeres. To date, little is known about centromere assembly dynamics and functions in stem cell asymmetric divisions. Drosophila melanogaster ovaries provide an excellent model to study stem cells in their native niche. In this tissue, germline stem cells (GSCs) are easily accessible and can be manipulated genetically. Moreover, centromere assembly mechanisms in GSCs and their differentiated cells, cystoblasts (CBs), could be used to epigenetically discriminate between these two cell types. In Drosophila, CID binds to CAL1 (fly functional homologue of HJURP) in a prenucleosomal complex, and its localization to centromeres requires CAL1 and CENP-C (Dattoli, 2020).

This study investigated the dynamics of CENP-A deposition in Drosophila GSCs. GSC centromeres are assembled after replication, but before chromosome segregation, with neural stem cells following the same trend. Centromere assembly in GSCs is tightly linked to the G2/M transition. Indeed, CYCA localizes at centromeres, and its knockdown is responsible for a marked reduction of centromeric CID and CENP-C, but not CAL1. Surprisingly, excessive CID deposition is prevented by CYCB, through the kinase HASPIN. Superresolution microscopy analysis of GSCs at prometaphase and metaphase shows that CID incorporation on sister chromatids occurs asymmetrically, and chromosomes that will be inherited by the stem cell are loaded with more CID. Moreover, GSC chromosomes make stronger kinetochores, which anchor more spindle fibers. This asymmetric distribution of CID between GSC and CB is maintained also at later stages of the cell cycle, while it is not observed in differentiated cells outside of the niche. This study also found that the depletion of CAL1 at centromeres blocks GSC proliferation and differentiation. Notably, overexpression of both CID and CAL1, as well as HASPIN knockdown, promotes stem cell self-renewal and disrupts the asymmetric inheritance of CID. Conversely, overexpression of CAL1 causes GSC-like tumors. Finally, CAL1 and CID knockdown at later stages of egg development have no obvious effect on cell division, suggesting that these cells inherit CID from GSCs. Taken together, these findings establish centromere assembly as a new epigenetic pathway that regulates stem cell fate (Dattoli, 2020).

In this study a detailed characterization of centromere dynamics was performed throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, this study shows that overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (a) a rapid pulse during G1 in mitotically dividing cells; and (b) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results also support this model (Dattoli, 2020).

Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with the current functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals (Stankovic, 2017). Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase (Ranjan, 2019), further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

According to the current results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (a) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (b) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids. The current data confirm this model, symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, it was observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported. This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In a favorite scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be rule out that the effects on cell fate observed with the functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

Ultimately, the results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, these data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

Earlier Treatment of Cyclin A in The Interactive Fly

In Drosophila, Cyclin A and Cyclin B appear to be functionally indistinguishable. However, differences between these cyclins exist, with respect to distribution and degradation patterns. The functional overlap between Cyclin A and B, both of which regulate the cyclin dependent kinase cdc2, is discussed in the Cyclin B site.

Following cellularization, Cyclin B, like Cyclin A accumulates during G2. Cyclin A distribution peaks in prophase, and then declines. In contrast, Cyclin B is maintained at its maximum from prophase until metaphase. Prophase cells often show a more uniform distribution of Cyclin B throughout the cell, and degradation of the protein does not occur until the metaphase-anaphase transition, well after the disappearance of Cyclin A. The accumulation and degradation of Cyclin A is similar in both colchicine treated cells and untreated control cells. However, Cyclin B accumulation seems to be independent of spindle function, and its degradation is inhibited by colchicine treatment (Whitfield, 1990). Expression of a modified form of Cyclin A, which cannot be degraded during mitosis, reveals that persistence of Cyclin A delays metaphase, suggesting a requirement for the degregadation of Cyclin A before entry into mitosis. Persistence of Cyclin B, whose degradation is required for completion of mitosis, does not effect entry into mitosis. Persistence of Cyclin A also results in abnormal spindle structures (Sigrist, 1995).

As with Cyclin B, a review of vertebrate studies highlights the functional differences between Cyclin A and B. From such perspectives, the two cyclins appear to be functionally worlds apart. Cyclin A plays a dual role in vertebrates, involved in both S phase and in the G2-M transition. In association with E2F it helps to terminate activation of the transcription of genes involved in DNA replication. Later in the cycle, depletion of Cyclin A arrests cells in G2. Thus Cyclin A is required for progression from G2 to M (Pagano, 1992). In Drosophila, no S phase role has been detected for Cyclin A. The requirement for Cyclin A in the G2-M phase transition is not well characterized: the targets are not yet known. Cyclin A peaks in prophase and then declines. These kinetics suggests little if any involvement of Cyclin A in the metaphase/anaphase checkpoint (see Cyclin B), but is consistent with an earlier requirement for the onset of mitosis.

In fly embryos, Cyclin E is the normal inducer of S phase in G1 cells. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. High-level expression of cyclin A triggers the G1/S transition in wild-type embryos and in mutant embryos lacking cyclin E. Three types of control downregulates this activity of cyclin A: (1) cyclin destruction limits the accumulation of cyclin A protein in G1; (2) inhibitory phosphorylation of cdc2, the kinase partner of cyclin A, reduces the S-phase promoting activity of cyclin A in G1 and (3) Roughex (Rux), a protein with unknown biochemical function, limits cyclin A function in G1. Overexpression of rux blocks S phase induction by coexpressed cyclin A and promotes the degradation of cyclin A. Rux also prevents a stable cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction. This inhibition also drives the nuclear localization of stable cyclin A. It is therefore considered likely that Roughex effects on the nuclear translocation of cyclin A would target cyclin A for degradation. It is concluded that cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by Rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of cyclin E. The action of Rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists (Sprenger, 1997).


The sequences of cDNAs clones reveals differences in the 5' and 3' untranslated regions, arguing for alternative mRNA processing events (Lehner, 1989)
cDNA clone length - 2385

Bases 5' UTR - 298+

Bases 3' UTR - 670


Amino Acids - 491

Structural Domains

In aligned fly and clam Cyclin A sequences within a 60 amino acid region, residues at 82% of the positions are similar and 72% are identical. Within this same region, 47% of the positions are identical among all the cyclins, whether A or B type, or whether of yeast, mollusc, echinoderm or insect origin. Conservation among this group of proteins extends throughout the carboxy-terminal two-thirds of these proteins (Lehner, 1989).

Cyclin A: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 January 2020

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