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

Gene name - Cyclin E

Synonyms - DmcycE

Cytological map position - 35D5-7

Function - G1-S cyclin - Regulatory subunit of cyclin dependent kinase

Keywords - cell cycle

Symbol - CycE

FlyBase ID:FBgn0010382

Genetic map position - 2-[51]

Classification - cyclin

Cellular location - nuclear



NCBI links: Entrez Gene

Cyclin E Orthologs: Biolitmine

Recent literature
Meserve, J. H. and Duronio, R. J. (2015). Scalloped and Yorkie are required in Drosophila for cell cycle re-entry of quiescent cells after tissue damage. Development [Epub ahead of print]. PubMed ID: 26160905
Summary:
Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. This study used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. Hippo signaling was found to regulate compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage.

Parker, D., Iyer, A., Shah, S., Moran, A., Hjelmeland, A., Basu, M. K., Liu, R. and Mitra, K. (2015). A novel mitochondrial pool of Cyclin E, regulated by Drp1, is linked to cell density dependent cell proliferation. J Cell Sci [Epub ahead of print]. PubMed ID: 26446260
Summary:
The regulation and function of the crucial cell cycle regulator Cyclin E (CycE) remains elusive. Among other cyclins, CycE can be uniquely controlled by mitochondrial energetics, the exact mechanism being unclear. Using mammalian cells (in vitro) and Drosophila (in vivo) model systems in parallel this study shows that CycE can be directly regulated by mitochondria by its recruitment to the organelle. Active mitochondrial bioenergetics maintains a distinct mitochondrial pool of CycE (mtCycE) lacking a key phosphorylation required for its degradation. Loss of the mitochondrial fission protein Drp1 augments mitochondrial respiration and elevates the mtCycE-pool allowing CycE deregulation, cell cycle alterations and enrichment of stem cell markers. Such CycE deregulation after Drp1 loss attenuates cell proliferation in low cell density environments. However, in high cell density environments elevated MEK-ERK signaling in the absence of Drp1 releases mtCycE to support escape of contact inhibition and maintain aberrant cell proliferation. Such Drp1 driven regulation of CycE recruitment to mitochondria may be a mechanism to modulate CycE degradation during normal developmental processes as well as in tumorigenic events.
Shu, Z. and Deng, W.M. (2017). Differential Regulation of Cyclin E by Yorkie-Scalloped Signaling in Organ Developmen G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28143945
Summary:
Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. This study reports a role of a growth modulator Yorkie (Yki), the Drosophila homolog of Yes-associated protein (YAP), which interacts with its transcriptional partner, Scalloped (Sd), the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell-cycle regulator Cyclin E (CycE) in wing imaginal discs. Interestingly, when Yki is coexpressed with Fizzy-related (Fzr), a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells display larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression is not detected in the wing disc, coexpression of Fzr and Yki does not cause size differences between cells along the proximal-distal axis of the disc. Ectopic CycE promotes cell proliferation and apoptosis, and inhibits transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis.

Morozova, T. V., Hussain, Y., McCoy, L. J., Zhirnov, E. V., Davis, M. R., Pray, V. A., Lyman, R. A., Duncan, L. H., McMillen, A., Jones, A., Mackay, T. F. C. and Anholt, R. R. H. (2018). A Cyclin E centered genetic network contributes to alcohol-induced variation in Drosophila development. G3 (Bethesda). PubMed ID: 29871898
Summary:
Prenatal exposure to ethanol causes a wide range of adverse physiological, behavioral and cognitive consequences. However, identifying allelic variants and genetic networks associated with variation in susceptibility to prenatal alcohol exposure is challenging in human populations, since time and frequency of exposure and effective dose cannot be determined quantitatively and phenotypic manifestations are diverse. This study harnessed the power of natural variation in the Drosophila melanogaster Genetic Reference Panel (DGRP) to identify genes and genetic networks associated with variation in sensitivity to developmental alcohol exposure. Development time from egg to adult and viability were measured of 201 DGRP lines reared on regular or ethanol- supplemented medium, and polymorphisms were identified associated with variation in susceptibility to developmental ethanol exposure. Genotype-dependent variation in sensorimotor behavior was measured after developmental exposure to ethanol using the startle response assay in a subset of 39 DGRP lines. Genes associated with development, including development of the nervous system, featured prominently among genes that harbored variants associated with differential sensitivity to developmental ethanol exposure. Many of them have human orthologs and mutational analyses and RNAi targeting functionally validated a high percentage of candidate genes. Analysis of genetic interaction networks identified Cyclin E (CycE) as a central, highly interconnected hub gene. Cyclin E encodes a protein kinase associated with cell cycle regulation and is prominently expressed in ovaries. Thus, exposure to ethanol during development of Drosophila melanogaster might serve as a genetic model for translational studies on fetal alcohol spectrum disorder.
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
Summary:
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.
Ma, Y., McKay, D. J. and Buttitta, L. (2019). Changes in chromatin accessibility ensure robust cell cycle exit in terminally differentiated cells. PLoS Biol 17(9): e3000378. PubMed ID: 31479438
Summary:
During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. This study provides evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. During terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. These results uncover a mechanism that acts as a cell cycle-independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, a new molecular description is provided of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.
Gadre, P., Chatterjee, S., Varshney, B. and Ray, K. (2020). Cyclin E and Cdk1 regulate the termination of germline transit-amplification process in Drosophila testis. Cell Cycle 19(14): 1786-1803. PubMed ID: 32573329
Summary:
An extension of the G1 is correlated with stem cell differentiation. The role of cell cycle regulation during the subsequent transit amplification (TA) divisions is, however, unclear. This paper reports that in the Drosophila male germline lineage, the transit amplification divisions accelerate after the second TA division. The cell cycle phases, marked by Cyclin E and Cyclin B, are progressively altered during the TA. Antagonistic functions of the bag-of-marbles and the Transforming-Growth-Factor-β signaling regulate the cell division rates after the second TA division and the extent of the Cyclin E phase during the fourth TA division. Furthermore, loss of Cyclin E during the fourth TA cycle retards the cell division and induces premature meiosis in some cases. A similar reduction of Cdk1 activity during this stage arrests the penultimate division and subsequent differentiation, whereas enhancement of the Cdk1 activity prolongs the TA by one extra round. Altogether, the results suggest that modification of the cell cycle structure and the rates of cell division after the second TA division determine the extent of amplification. Also, the regulation of the Cyclin E and CDK1 functions during the penultimate TA division determines the induction of meiosis and subsequent differentiation.
Gaziova, I., Gazi, M., Mar, J. and Bhat, K. M. (2020). Restriction on self-renewing asymmetric division is coupled to terminal asymmetric division in the Drosophila CNS. PLoS Genet 16(9): e1009011. PubMed ID: 32986715
Summary:
Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. This study shows that the T-box transcription factor, Midline (Mid), couples these events. In mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. The results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.
Wang, X. F., Liu, J. X., Ma, Z. Y., Shen, Y., Zhang, H. R., Zhou, Z. Z., Suzuki, E., Liu, Q. X. and Hirose, S. (2020). Evolutionarily Conserved Roles for Apontic in Induction and Subsequent Decline of Cyclin E Expression. iScience 23(8): 101369. PubMed ID: 32736066
Summary:
Cyclin E is a key factor for S phase entry, and deregulation of Cyclin E results in developmental defects and tumors. Therefore, proper cycling of Cyclin E is crucial for normal growth. This study found that transcription factors Apontic (Apt) and E2f1 cooperate to induce cyclin E in Drosophila. Functional binding motifs of Apt and E2f1 are clustered in the first intron of Drosophila cyclin E and directly contribute to the cyclin E transcription. Knockout of apt and e2f1 together abolished Cyclin E expression. Furthermore, Apt up-regulates Retinoblastoma family protein 1 (Rbf1) for proper chromatin compaction, which is known to repress cyclin E. Notably, Apt-dependent up-regulation of Cyclin E and Rbf1 is evolutionarily conserved in mammalian cells. These findings reveal a unique mechanism underlying the induction and subsequent decline of Cyclin E expression.
BIOLOGICAL OVERVIEW

The biochemical basis of the transition to the S phase of the cell cycle (during which DNA synthesis takes place) requires complex regulatory events in organisms as diverse as yeast and man, as well as Drosophila. Proteins involved in Drosophila include at least two cyclins (DmcycD and DmcycE, herein referred to as cyclin D and cyclin E), a cyclin dependent kinase (cdc2c), and a transcription factor (E2F) that is a complex protein made up of two subunits (referred to here as E2F).

At least two types of regulation take place: activation of gene transcription and transcription-independent regulatory events. Adding to this already complex picture of Drosophila development are at least five types of cell cycles, each requiring different modes of regulation. In such a complicated system, it is often difficult to order the regulatory processes involved in initiation of DNA synthesis. This is a problem of central importance to the understanding of differentiation, since it deals with the question of which cells will divide and which cells will remain quiescent.

Cyclin E is supplied as a maternal transcript. Sufficient protein is made to carry the embryo through the first 14 cleavage divisions. Subsequently, sufficient zygotic transcript is made for the next three cell division cycles. These three cycles are marked by the absence of a G1 phase: cells that exit mitosis go directly into S phase. During these three cycles cyclin E shows no cell-cycle-associated variation in transcription. Nevertheless cyclin E serves a critical function, discovered through observation of the roles of cyclin E in two later events: endoreduplication (the replication of cell DNA without subsequent mitosis) and division of neuroblasts.

In endoreduplication, a process that takes place in abdominal histoblasts, transcription of cyclin E is triggered by E2F. Since supplying cyclin E exogenously during this period is sufficient to induce cell division, it is believed that cyclin E is limiting for the induction of endoreduplication. New cyclin E synthesis requires E2F, a pivotal transcription factor involved in the regulation of many genes necessary for DNA synthesis. Therefore it has been concluded that cyclin E lies downstream of E2F (Duronio, 1995b). The mammalian homolog of E2F likewise targets cyclin E. E2F binding sites are also found in the human cyclin E promoter (Ohtani, 1995).

In the CNS, cyclin E expression does not require E2F: on the contrary, expression of E2F targets requires cyclin E. Thus the hierarchy appears to be reversed -- cyclin E is required for the transcription of E2F dependent genes. The chain of events here is unknown, but in the CNS, cyclin E activates E2F that in turn activates E2F targets (Duronio, 1995b). The regulation of E2F by cyclins is not at all surprising. Human E2F1 is activated by both cyclin D plus its associated kinase and by cyclin E and its associated kinase (Johnson, 1994).

In the fly, Cyclin E is required for induction of S-phase in a process that does not require transcription. Ectopic cyclin E can bypass the S-phase requirement for E2F in epidermal cells arrested in G1 at stage 17. A similar function for cyclin E is found in Xenopus, where the p21 cyclin-dependent kinase inhibitor prevents DNA replication. This inhibition can be restored by addition of cyclin E to p21-arrested extracts (Strausfeld, 1994). This replication-independent requirement for cyclin E points to the critical function of cyclins. They act as the regulatory subunit of a protein dimer, partnering cyclin dependent kinases that transduce signals by phosphorylation, activating critical components necessary for DNA replication.

Cyclin E regulates endocycling and is required for chorion gene amplification within of follicle cells during oogenesis: endocycling and chorion gene amplification. (1) Endocycling -- first the cells become polyploid, a process in which DNA replicates but no mitosis intervenes. Endocycling is accompanied by a balanced replication of euchromatin. Three endocycles give rise to a 16C nuclear DNA content and terminate by the end of stage 10A. (2) Chorion gene amplification -- a striking exception to balanced replication of euchromatin, such amplification occurs in the follicle cells during Drosophila oogenesis through repeated initiation of replication forks from these loci. This amplification occurs during the last hours of oogenesis. The polyploid follicle cells rapidly synthesize and secrete high levels of chorion proteins that comprise the Drosophila eggshell. Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. Two clusters of chorion genes on the X and third chromosomes (hereafter X chorion and third chorion) are amplified above the copy number of the remainder of the follicle cell genome before and during a time of high-level transcription. This increase occurs through repeated initiation of replication forks from these loci. Because successively initiated replication forks continue to move outward, by the end of oogenesis each gene cluster lies at the peak of a gradient of copy number that extends ~40 kb in both directions. The final amplification level for the third chorion genes is 60- to 80-fold, and the X chorion genes 15- to 20-fold, above the remainder of the genome. Several partially redundant cis sequences that mediate high-level amplification have been identified, interspersed among the chorion transcription units, suggesting amplification is an amenable model for investigation into the little understood nature of metazoan origins of DNA replication (Calvi, 1998).

The relationship of chorion gene amplification to the follicle cell endocycles has remained unclear. To investigate the regulation of amplification, a technique was developed to detect amplifying chorion genes in individual follicle cells. Amplification occurs in two developmental phases. One of the gene clusters, the third chorion genes, begins to amplify periodically during S phases of follicle cell endocycles. The third chorion genes are 1.8 to 2.2-fold amplified in 8C follicle cell nuclei, but no amplification is found for the X chorion gene cluster. The third chorion genes are amplified 4.1- to 4.7-fold by the completion of the 16C endorepication, while chorion genes on the X are not amplified. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis (Calvi, 1998).

In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. During the endocycles, CycE oscillates, thus controlling periodic S phases. During stage 10B, as late amplification begins, all follicle cells over the oocyte simultaneously display levels of CycE comparable with earlier S phases. CycE persists and fails to cycle, at least until stage 14; however, the level of staining slowly diminished from stage 10B onward. In older chambers, the CycE staining becomes more punctate. By stage 12, several subnuclear foci of high-level staining are clearly observed, resembling in number and intensity the sites of localized BrdU incorporation observed at this time. The possible accumulation of CycE at chorion loci, the restriction of amplification to endocycle S phases, and alteration of CycE behavior during late amplification suggests CycE may be required for chorion genes to amplify (Calvi, 1998).

Cyclin E is required positively for amplification. Ectopic expression of Dacapo, a specific inhibitor of Cyclin E inhibits the late amplification process. However, ectopic expression of Cyclin E at stage 10A does not result in premature amplification. It is concluded that Cyclin E is required for amplification but alone is insufficient to promote the process. Persistent Cyclin E inhibits replication from nonchorion origins. It is therefore suggested that Cyclin E also acts negatively within a cycle, and that specific factors at chorion origins allow these origins to escape this negative rereplication control (Calvi, 1998).

How might chorion loci escape inhibition of rereplication? One critical aspect of rereplication control is a presumed dissociation of replication engendering complexes from chromatin when origins fire. This dissociation would prevent the refiring of origins until the endocycle (in polyploid tissues) or mitosis (in diploid tissues) is complete. It may be that special amplification complexes at chorion origins are not destroyed with a single firing, or are reassembled within S phase, thus engendering local resistance to rereplication inhibition. It is proposed that amplification complexes resident at chorion origins are associated with unique amplification factor(s) that impart protection from inhibition exerted by the CycE/CDK2 pathway. These may be special members of the ORC, CDC6, and MCM families that associate with origins during acquisition of replication competence, or molecules that counteract inhibitory phosphorylation known to regulate these proteins. Signals that induce expression of an amplification factor in follicle cells during endocycles would be sufficient to explain the developmental specificity for the onset of amplification (Calvi, 1998).

A screen for modifiers of Cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2

To identify genes interacting with cyclin E, a screen was carried out for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that were identified fall into four complementation groups corresponding to the genes split ends (spen), dacapo, dE2F1, and Cdk2 (Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19) to be addressed. The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, the results presented here demonstrate that the equivalent sites in Cdk2 play no essential role. The demonstration that phosphorylation of Cdk2 on Thr 18 and Tyr 19 has no essential role during normal development does not exclude its involvement in subtle or stress regulation. Moreover, vertebrate cells, in which Cdk2 phosphorylation on Thr 18 and Tyr 19 has been demonstrated to occur, express A-type cdc25 phosphatases that have been implicated in Cdk2 dephosphorylation and that do not appear to exist in Drosophila (Lane, 2000).

To characterize the effects of Cdk2 mutations on development, embryogenesis was examined. However, a requirement for zygotic Cdk2 expression during this developmental phase could not be identified. Embryos hemizygous or transheterozygous for Cdk2 allele combinations appear to have wild-type morphology. They incorporated BrdU with normal efficiency and in normal patterns throughout embryogenesis. Hatching of larvae has been observed to occur at the same rate as in control embryos (Lane, 2000).

For the characterization of larval development, progeny were analyzed from parents with Cdk2 alleles over a balancer chromosome carrying Tb. Scoring for the Tb phenotype, which can readily be distinguished from wild type after development beyond the second larval instar, suggests that Cdk2 mutants do not reach this stage. However, experiments involving the expression of Hs-Cdk2 in Cdk2 mutant larvae indicates that these mutants survive for longer time periods than what is normally required to reach second instar. As indicated above, periodic Hs-Cdk2 expression by heat shocks allows Cdk2 mutants to develop into morphologically normal and fertile adults. Some Cdk2 mutants are still observed to develop into adults even if the onset of the periodic Hs-Cdk2 expression is delayed until 116 hr after egg deposition. Cdk2 mutant larvae, therefore, fail to grow but some survive for several days and can be rescued by providing Cdk2 again. The dependence of larval growth on zygotic Cdk2 expression is confirmed by comparing the size of Cdk2+ and Cdk2 mutant larvae derived from parents with Cdk2 alleles over a balancer chromosome marked by an Act-GFP transgene. GFP-negative Cdk2 mutant larvae are observed in decreasing numbers for at least 4 days after egg deposition, but their size fails to increase significantly after 1.5 days (Lane, 2000).

The normal initial development that is observed in the absence of zygotic Cdk2 function could be explained by maternally derived Cdk2. The presence of a maternal Cdk2 contribution in the Drosophila egg has been demonstrated. To demonstrate the functional role of this maternal contribution, the development was analyzed of eggs derived from Cdk2 mutant females that had been rescued by periodic Hs-Cdk2 expression. These mutant females (Cdk22, Hs-Cdk2/Cdk23 or Cdk22, Hs-Cdk2/Cdk21) readily lay eggs as long as they are subjected to periodic heat shocks. However, after termination of periodic heat shocks, egg deposition decreases rapidly and stops completely within 2-3 days. This arrest of egg deposition is readily reversed within 7 days after resumption of periodic heat shocks (Lane, 2000).

The eggs from mutant females collected 1 day after the termination of periodic Hs-Cdk2 expression were fixed and stained for DNA. For comparison, the eggs from mutant females that had been maintained with periodic Hs-Cdk2 expression were also analyzed. In addition, eggs from w control females exposed to periodic heat shocks or 1 day after termination of these heat shocks were analyzed. The great majority of the eggs from these w control females reveal normal DNA staining patterns. Conversely, the majority of the eggs collected from mutant females (Cdk22, Hs-Cdk2/Cdk23 or Cdk22, Hs-Cdk2/Cdk21) display abnormal DNA staining patterns. The spatial distribution of nuclei and the appearance of chromatin is often aberrant, indicating that progression through the syncytial division cycles is severely perturbed in these embryos. This finding suggests that the maternal contribution is required during the syncytial division cycles. In addition, a significant fraction of embryos contains very few nuclei, suggesting that these embryos had failed to commence progression through the syncytial divisions (although it is not excluded that a minor fraction of these eggs was fixed while progressing normally through the first three cycles). Double labeling with an antibody recognizing a sperm tail epitope indicates that about two-thirds of these eggs with less than five nuclei are not fertilized. Compared to continuously heat-pulsed Cdk2 mutant females, those withdrawn from heat-shock treatment generate a higher fraction of eggs containing less than five nuclei at the expense of the eggs with normal appearance. Termination of periodic Hs-Cdk2 expression in Cdk2 mutant females, therefore, is accompanied by a transient production of eggs that cannot be fertilized followed by a rapid arrest of egg laying. A significant fraction (60%) of abnormal eggs is produced even when periodic Hs-Cdk2 expression is maintained. The production of abnormal eggs despite periodic Hs-Cdk2 expression is likely to reflect the fact that the germ line is refractory to induction of heat-shock genes during stages 10-12 of oogenesis. These observations demonstrate that Cdk2 expression is crucial for oogenesis and early embryogenesis (Lane, 2000).

The largest complementation group identified in this screen has not previously been implicated in Cyclin E function. Genetic and molecular analysis of this complementation group corresponds to the spen gene. Independent work has proven this suggestion (Wiellette, 1999). spen encodes a 600-kD ubiquituously expressed nuclear protein containing three RNP-type RNA binding domains and a novel characteristic C-terminal domain defining a family of homologous metazoan genes. Mutations in spen result in peripheral nervous system defects and interact with raf kinase signaling and E2F/DP transcription factors and with Cyclin E. It is attractive to speculate that spen is particularly important for the transition from cell proliferation to terminal differentiation. spen mutant ommatidia in the eye display the same defects as those resulting from the Sev-CycE transgene. The affected ommatidia are of variable composition, often lacking either R7 or one or more other photoreceptors, but also occasionally containing extra photoreceptors. Sev-CycE transgene expression forces differentiating cells through an extra cell cycle, presumably explaining the presence of extra cells. In addition, extra divisions of differentiating cells are likely to disturb the regular arrangement of the ommatidial cluster and consequently might cause apoptosis, potentially explaining the observed loss of cells as well. Similarly, coexpression of GMR-E2F1 and GMR-DP transgenes in all eye imaginal disc cells posterior to the morphogenetic furrow has been shown to result in ectopic BrdU incorporation and apoptosis. spen mutations dominantly enhance both the Sev-CycE and GMR-E2F1/DP rough eye phenotype. Conversely, spen mutations suppress the eye phenotypes resulting from GMR-dap expression in a CycE heterozygous background. While spen function opposes the mitogenic activity of CycE and dE2F1, it remains to be analyzed whether the phenotypic interactions observed between spen and Hox and Raf involve deregulated cell proliferation as well (Lane, 2000 and references therein).

The identification of mutations in Drosophila dE2F1 in this screen was expected on the basis of the large body of evidence demonstrating the tight functional relationship between Cyclin E and E2F/DP transcription factors. However, the fact that dE2F1 mutations result in enhancement rather than suppression of the Sev-CycE phenotype would not necessarily have been predicted since the results of genetic analysis in Drosophila so far have suggested that E2F/DP activity has a positive role in stimulating the transcription of S phase genes (Cyclin E, RNR2, DNA polalpha, PCNA, and Orc1) and cell proliferation. In contrast, the enhancement of the Sev-CycE phenotype observed with dE2F1 alleles points to a growth-suppressive role of dE2F1. Similarly, the E2F1 knock-out phenotype observed in mice has clearly demonstrated a tumor-suppressing function. Moreover, while vertebrate E2F/DP functions as a transcriptional activator in some promoters, it acts as a corepressor in conjunction with pRB in many other promoters. A decrease in E2F/DP levels, therefore, might also result in derepression of unknown proliferation-stimulating genes and synergy with ectopic Cyclin E expression (Lane, 2000 and references therein).

The chromatin-remodeling Protein Osa interacts with CyclinE in Drosophila eye imaginal discs

Coordinating cell proliferation and differentiation is essential during organogenesis. In Drosophila, the photoreceptor, pigment, and support cells of the eye are specified in an orchestrated wave as the morphogenetic furrow passes across the eye imaginal disc. Cells anterior of the furrow are not yet differentiated and remain mitotically active, while most cells in the furrow arrest at G1 and adopt specific ommatidial fates. Microarray expression analysis was used to monitor changes in transcription at the furrow, and genes were identified whose expression correlates with either proliferation or fate specification. Some of these are members of the Polycomb and Trithorax families that encode epigenetic regulators. Osa is one; it associates with components of the Drosophila SWI/SNF chromatin-remodeling complex. Studies of this Trithorax factor in eye development implicate Osa as a regulator of the cell cycle: Osa overexpression caused a small-eye phenotype, a reduced number of M- and S-phase cells in eye imaginal discs, and a delay in morphogenetic furrow progression. In addition, evidence is provided that Osa interacts genetically and biochemically with CyclinE. The results suggest a dual mechanism of Osa function in transcriptional regulation and cell cycle control (Baig, 2010).

This work applied DNA microarray hybridization to investigate the differences between mitotically active anterior and differentiating posterior eye-disc cells. Advantage was taken of the program of ommatidial differentiation to identify genes with essential roles at the stage of eye development when logarithmic growth transitions to mitotic arrest and adoption of specific cell types. Several recent studies have cataloged transcripts in whole eye discs with SAGE or DNA microarray hybridization, but this is the first genomic analysis that combines an analysis of purified posterior eye-disc fragments with mutant conditions that alter the program of photoreceptor differentiation. 866 transcripts were identified with differential anterior or posterior expression. Supporting the validity of this approach, functions that correlate with the mitotic activity of committed, but still undifferentiated, anterior cells segregate to the 'anterior' group, while neuronal functions are overrepresented in the 'posterior' group. This analysis and a recent SAGE-based investigation of regional differences in expression levels in eye imaginal discs identified several chromatin factors including PcG and TrxG members and proteins involved in heterochromatization, suggesting that chromatin-based transcriptional regulation plays a role in regional specific cell functions in eye development (Baig, 2010).

This study investigated the role of the BAP chromatin-remodeling complex subunit Osa at the MF. Several observations link the TrxG factor Osa to cell cycle control. First, the BAP components osa and moira have been implicated in a regulatory network of cell proliferation and cell cycle progression by evidence that they are transcriptional targets of the DNA replication-related element-binding factor (Nakamura, 2008). Second, phenotypes of osa mutant cells suggest that Osa is required for both differentiation and proliferation. Finally, by analyzing the osa overexpression phenotype, evidence was found for genetic and biochemical interaction of Osa with DmCycE. Interestingly, whereas expression of cell cycle regulators such as string/cdc25 is dependent on Osa's chromatin-remodeling function, the reduction in cell cycle progression that results from overexpression of Osa appears to be independent of string/cdc25 and CycE transcription rates. These results support a dual mechanism to link chromatin remodeling with cell cycle control (Baig, 2010).

CycE function appears to be modulated by BAP, the Osa-containing form of the SWI/SNF complex. Genetic interactions of CycE with several core components of both the BAP and PBAP forms of the SWI/SNF complex have been described previously. Consistent with the current observations, these studies also detected a genetic interaction between osa and CycE. Furthermore, a direct or indirect physical association between CycE and SWI/SNF components was detected by co-immunoprecipitation with Brm or Snr1. These results now show that CycE also immunoprecipitates with the BAP signature protein Osa. Although the PBAP signature proteins Polybromo or BAP170 were not tested in this study, the Osa overexpression small-eye phenotype and lack of cell cycle defects in single and double mutants for Polybromo and BAP170 suggest that the cell cycle function is specific to the BAP version of the Drosophila SWI/SNF complexes (Baig, 2010).

CycE-SWI/SNF complex interactions appear to be evolutionarily conserved since BRG1 (Brahma Related Gene 1, one of two mammalian orthologs of Drosophila Brm) and BAF155 (orthologous to Moira) copurify with CycE from human cells. In addition, expression of the SWI/SNF complex components BRG1 or INI/hSNF5 (orthologous to Snf1) causes G1 cell cycle arrest in human tissue culture cells. Interestingly, the cell cycle arrest can be rescued by co-expression of hCycE or hCycD1, respectively. These data are therefore consistent with a function of the Drosophila BAP and human SWI/SNF-like complexes as cell cycle regulators. Furthermore, the genetic and biochemical interaction data suggest that this function requires Cyclin activity (Baig, 2010).

Chromatin-remodeling activity and the function of SWI/SNF in cell cycle regulation must be tightly controlled to assure proper development and to prevent the transition of normal cells into cancer cells. The current findings are consistent with a function of Osa in negatively controlling cell cycle progression. A fine-tuned balance of repressive and activating signals seems to coordinate cell cycle progression by controlling Osa protein levels and downstream events such as CycE interaction or string/cdc25 expression. The elevated Osa protein levels anterior to the MF that are observed in normal development might reflect the contribution of Osa in the transition of these cells into a G1-arrested state. The G1 arrest of these cells requires the function of several signaling pathways: Hh, Dpp, Wg, Egfr, and Notch. By downregulating CycE activity, the increased Osa protein levels in these cells might contribute to counteracting the mitogenic activity of these signaling pathways that is observed in other developmental contexts (Baig, 2010).

Genetic interactions between osa and components of the Wg signal transduction pathway were also detected. These interactions could be a consequence of the small size of the eye field in Osa-overexpressing discs, since the signaling molecule Wg is normally expressed in lateral positions and has a locally restricted negative effect on Dpp-mediated MF progression. If relative Wg signaling increases in the abnormally small eye, repression of Dpp function in medial cells should increase. This model is supported by the weak dpp expression in the small discs and by the half-moon shape of the MF in Osa discs. The MF bends posteriorly in the Osa-overexpressing discs (indicating that the retarding effect is strongest in lateral positions), whereas the MF points anteriorly in wg mutant discs (presumably due to the missing repressive Wg effects in lateral positions). Partial rescue of Osa overexpression by impaired Wg signaling is consistent with this model. On the basis of these findings it is speculated that the posterior position of the MF that is caused by Osa overexpression is a manifestation of a developmental delay in eye development due to inhibition of cell proliferation and the resulting relative increase of the repressive Wg signal on dpp expression (Baig, 2010).

However, there are alternative regulatory possibilities in which the interplay of Osa and Wg signaling involves mutual transcriptional regulation and/or coregulation of common target genes at the transcriptional level. In Drosophila, expression of an activated form of the Wg signaling component Armadillo causes a small-eye phenotype that is suppressed by lowering the dosage of functional brm. Furthermore, Osa has been characterized as an antagonist of Wg signaling in wing development by inhibiting the expression of Wg target genes. A suppression of the Osa small-eye phenotype by Wg pathway mutants was detected, suggesting that Wg signaling acts synergistically with Osa in this system. These findings point at context-dependent features that appear to differ between wing and eye development. Such context-dependent functions have been reported earlier even between different cell populations of wing imaginal discs. For example, Wg signaling represses Drosophila Myc (DMyc) expression in the presumptive wing margin. In this area of the disc, repression of DMyc promotes G1 arrest via the regulation of the Drosophila retinoblastoma family (Rbf) protein, while forced expression of DMyc promotes cell cycle progression by inducing CycE expression. In contrast, Wg signaling in the hinge region of the wing imaginal disc has the opposite effect on cell proliferation. As these examples illustrate, it is difficult to generalize the relation between Osa, Wg signaling, and Myc function. However, a possible contribution of DMyc regulation to the Osa overexpression small-eye phenotype provides an interesting possibility. Observations in other systems support a role of SWI/SNF function in transcriptional regulation of cell cycle genes. In vertebrates, direct transcriptional regulation of Cyclins by SWI/SNF complex components has been implicated, and mammalian BRG1 and β-catenin (the vertebrate ortholog of Armadillo) interact with each other to activate Wnt target genes. In Drosophila, only a single osa gene exists, and it is involved in both activation and repression of target genes. In mammals, the two Osa orthologs BAF250a/b seem to have antagonistic functions in activating or repressing cell-cycle-specific genes such as cdc2, cyclin E, and c-Myc, and this regulation involves binding to the promoter sequences (Baig, 2010).

No significant changes were detected in DmCycE transcript or protein levels in osa and other BAP component mutants; instead, biochemical interaction was detected between Osa and DmCycE. To date, the functional consequence surrounding the association of Cyclin/Cdk complexes with chromatin-remodeling complexes remains unclear. Although different Cyclins possess distinct functions and tissue specificities, several reports describe roles for different CDK/cyclin complexes in transcription and RNA splicing. In many cases, CDK/cyclin complexes regulate the activity of components of the transcription machinery or other factors in a cell-cycle-dependent manner. Along these lines, CycE/CDK2 phosphorylates NPAT (nuclear protein mapped to the AT locus), which in turn activates replication-dependent transcription of histones. This function is stimulated by CycE binding to the histone genes in human tissue culture cells. It is conceivable that the kinase activity of CycE/Cdk2 modulates the activity of the BAP chromatin-remodeling complexes in a cell-cycle-dependent manner as it has been demonstrated for human Brm, BRG1, or BAF155 (Baig, 2010).


GENE STRUCTURE

The Drosophila cyclin E gene, DmcycE, encodes two proteins with a common C-terminal region and unique N-terminal regions. (Richardson, 1993).

Bases 5' UTR -744 for type 1 (zygotic) and 392 for type II (maternal)

Bases 3' UTR - 1342


PROTEIN STRUCTURE

Amino Acids - 601 for type I (zygotic) and 708 for type II (maternal)
Structural Domains

Drosophila and human cyclin E are 43% identical overall and 62% identical in the region of the cyclin box. The novel N-terminal regions of DmcycI type I and type II proteins each have a potential nuclear localization sequence (Richardson, 1993).


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

date revised: 28 December 97

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