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).

A novel mitochondrial pool of Cyclin E, regulated by Drp1, is linked to cell density dependent cell proliferation

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 (Parker, 2015).

A Cyclin E centered genetic network contributes to alcohol-induced variation in Drosophila development

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 (Morozova, 2018).

Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E

In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. This study shows that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization (Mar, 2022).

What makes precursor cells divide a certain number of times and how the progeny cells become post-mitotic has remained enigmatic. These questions are among some of the most fundamental questions in neurobiology. The work described in this study provides a clue and indicates that Pros and Cyclin E may be some of the key players in these processes. The results indicate that the cytoplasmic, non-phosphorylated Pros becomes phosphorylated and nuclear and binds to chromatin in cells destined to divide once. It must directly or indirectly regulate gene expression in the nucleus. One such gene regulated by Pros appears to be Cyclin E. Ample data indicates that Cyclin E is essential but also sufficient to drive entry of precursor cells to the cell cycle, although within a temporal window of developmental time. The data presented in this study show that Pros regulates Cyclin E levels in opposing directions between precursor cells and their progeny. This is an elegant, yet simple mechanism by which Pros through Cyclin E confers the one-division potential to MP2, GMC4-2a, or GMCs from NB7-3, and then helps commit their progeny to a post-mitotic state. How many lineages in the CNS also utilize this mechanism remains unknown (Mar, 2022).

The situation is not an ON/OFF scenario. A clear ON/OFF scenario will also be evolutionarily prohibitive as it would negatively affect the neuronal number, plasticity, and diversity. Instead, Pros appears to augment the upregulation of Cyclin E level in the precursor enough to commit that cell to divide once. Once it divides to generate two daughters, Pros augments the downregulation of levels of Cyclin E such that progeny cells do not enter the cell cycle, but instead become post-mitotic. The evidence to support this model comes from the fact that in pros loss of function mutants, cells such as MP2 and GMC4-2a fail to divide. The levels of Cyclin E were also downregulated in MP2 in pros mutant embryos, indicating a positive role for Pros via augmenting Cyclin E expression in MP2 division. However, in pros loss of function mutants at a later developmental stage, the level of Cyclin E was upregulated, indicating a repression role for Pros in older stage embryos. Thus, a repression of Cyclin E by Pros could lead to the post-mitotic status of progeny neurons. A switch from an activator to a repressor can be achieved by partnering with different transcription regulators before and after cell division. These results are further supported by the finding that MP2 fails to divide in loss of function for cyclin E, and gain of function for cyclin E, or gain of function for pros leads to extra divisions. The penetrance of these defects is not very high, but it is thought that this is to be expected since players such as Cyclin E will be tightly regulated during development. There is also the issue of maternal deposition when loss of function mutations is in question (Mar, 2022).

The opposing role of Pros in cyclin E regulation, depending upon the developmental stage, is meant to switch from facilitating a single division of precursors to facilitating a post-mitotic commitment of progeny cells. De-repression of Cyclin E alone in progeny cells in late-stage embryos either in the pros mutant or by over-expression of Cyclin E in post-mitotic progeny cells does not appear to be sufficient to make them re-enter the cell cycle. It may be that the Cyclin E level was not high enough in those stages of development, or the process that makes cells post-mitotic involves additional players. Thus, elevating Cyclin E alone at the 'Cyclin E-insensitive' stage may not be enough to make them re-enter cell cycle. Additionally, differentiation genes also begin to express in progeny cells, and then there is the physical process of differentiation that gets underway with neurites sprouting and axon forming. These structural changes in post-mitotic neurons could also prevent them, in addition to new gene expression programs, from re-entering the cell cycle despite elevated levels of Cyclin E. Furthermore, in 12 hpf or older pros mutant embryos, there is a general up-regulation of Cyclin E, not only in the MP2 lineage, but also in other cells in the nerve cord. The consequence of this upregulation, such as if those lineages produce extra cells, is not known. This question is currently being examined. It is also not known if Pros plays a similar role in type II NBs in the embryonic nervous system or during neurogenesis of the adult brain (Mar, 2022).

These results also argue that there may not be a dedifferentiation of cells in pros mutants as previously thought, at least not in every lineage. Pros, with its chromatin localization in cells committed to a differentiation pathway, appears to control many genes. Cyclin E alone, at least in earlier stages of development, is sufficient to make cells divide or not divide depending on the levels, and Pros fits in this Cyclin-E-mediated model by its ability to regulate cyclin E expression. In how many lineages Pros confers the single-division potential to precursor cells is not clear. It is not known if the asymmetrically localized cytoplasmic Pros in NBs has any role in cell division or if it simply is a mechanism to segregate Pros to GMCs. In any event, Pros is unlikely to regulate Cyclin E in NBs (other than MP2/NBs that have single division potential) (Mar, 2022).

Finally, these results are consistent with previous finding that in embryos mutant for a gene called midline, MP2 undergoes multiple self-renewing asymmetric divisions. Pros was cytoplasmic in MP2 in midline mutants [24], which further indicates that the single division potential of MP2 correlates with a nuclear/chromatin-bound Pros. A recent paper indicated that Pros remodels H3K9me3+ pericentromeric heterochromatin by recruiting Heterochromatin Protein 1 during neuronal differentiation (Liu, 2020). This conclusion is consistent with the supposition that Pros augments post-mitotic commitment and neuronal differentiation of progeny cells, and regulation of Cyclin E and modulating heterochromatin are essential to these developmental events. Neuronal differentiation is a complex and evolutionarily crucial process for survival; therefore, it is not surprising that various mechanisms will augment the process as a shared phenomenon. A partial redundancy for a gene or a pathway is a common theme during neurogenesis or development (Mar, 2022).

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 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).

date revised: 22 July 2023

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.