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: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene

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.
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).


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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