InteractiveFly: GeneBrief

Cyclin G: Biological Overview | References

BIOLOGICAL OVERVIEW

Morphological consistency in metazoans is remarkable given the pervasive occurrence of genetic variation, environmental effects, and developmental noise. Developmental stability, the ability to reduce developmental noise, is a fundamental property of multicellular organisms, yet its genetic bases remains elusive. Imperfect bilateral symmetry, or fluctuating asymmetry, is commonly used to estimate developmental stability. It was observed that Drosophila overexpressing Cyclin G (CycG) exhibit wing asymmetry clearly detectable by sight. Quantification of wing size and shape using geometric morphometrics reveals that this asymmetry is a genuine-but extreme-fluctuating asymmetry. Overexpression of CycG indeed leads to a 40-fold increase of wing fluctuating asymmetry, which is an unprecedented effect, for any organ and in any animal model, either in wild populations or mutants. This asymmetry effect is not restricted to wings, since femur length is affected as well. Inactivating CycG by RNAi also induces fluctuating asymmetry but to a lesser extent. Investigating the cellular bases of the phenotypic effects of CycG deregulation, it was found that misregulation of cell size is predominant in asymmetric flies. In particular, the tight negative correlation between cell size and cell number observed in wild-type flies is impaired when CycG is upregulated. These results highlight the role of CycG in the control of developmental stability in D. melanogaster. Furthermore, they show that wing developmental stability is normally ensured via compensatory processes between cell growth and cell proliferation. The possible role of CycG as a hub in a genetic network that controls developmental stability is discussed (Debat, 2011).

CycG gene of Drosophila melanogaster encodes a cyclin involved in transcriptional regulation, cell growth and cell cycle (Salvaing, 2008a; Faradji, 2011). Upregulation of CycG in a context where genetic and environmental variations were minimal induces extremely high levels of fluctuating asymmetry (FA) in several traits, suggesting that Cyclin G is a major factor of developmental stability (Debat, 2011).

Cell growth is markedly downregulated by CycG as CycG inactivation increases adult wing cell size while CycG overexpression reduces it. In wing imaginal discs, however, although cell size is also reduced by CycG overexpression, inactivation of CycG only induces a slight increase in cell size (Faradji, 2011). This suggests that in flies where CycG is inactivated, extra cell growth occurs during post-larval stages. Furthermore, CycG impairs not only cell growth but also cell proliferation. Indeed, both inactivation and overexpression lead to a reduction in cell number in females. The fact that wings of flies where CycG was inactivated reach a size comparable to that of wild type flies suggests that cell growth compensates for lack of cell proliferation (Debat, 2011).

A tight negative correlation between cell size and cell number is observed in control flies suggesting that wing size stability is ensured by compensation between cell proliferation and cell growth. This has also been observed in natural populations, where cell size and cell number tend to show negative covariance. In addition, genetic manipulation of cell size using cdc2 mutants, dMyc mutants or deregulation of cell cycle regulators confirms that cell growth and proliferation can compensate each other to reach normal organ size. Hence, the final size of the wing seems to be determined by a compensatory mechanism between cell size and cell number. This mechanism is deeply impaired in CycG overexpressing flies. Remarkably, although the negative correlation is significant in LOF flies, the variance of the regression residuals presents a sharp increase relative to the controls, indicating a loosening in the relationship. Altogether these results suggest that deregulating CycG alters the link between cell growth and proliferation. This in turn suggests that compensation between cell growth and division is one key factor in maintaining wing size - and thus wing developmental stability - and that CycG is critical for ensuring this compensation (Debat, 2011 and references therein).

In natural populations, differences in wing size between sexes have been suggested to involve both cell size and cell number. In control isogenic lines though, wing sexual size dimorphism was only due to cell number, cell size being strikingly similar in both sexes. This suggests that adaptation to laboratory conditions or genetic drift might affect the cellular basis of sexual size dimorphism (Debat, 2011).

Cell size is affected similarly in both sexes when manipulating CycG expression. In contrast, the effects on cell number are different between sexes: while no effect is detectable in males, both GOF and LOF females have fewer cells than the controls. During the pupal stage, wing cells undergo two rounds of division. As G-type cyclins are known to be important in terminally differentiated cells (Bennin, 2002), it is tempting to speculate that these last divisions are differentially regulated in males and females and are controlled by Cyclin G. The last divisions in the pupal wing might be a crucial determinant of the sexual dimorphism of wing size (Debat, 2011).

The altered wing shape in both GOF and LOF flies suggests that the ubiquitous da deregulation of CycG across the wing blade induced heterogeneous effects on cell size and cell number, possibly reflecting an interference with morphogens driving wing growth. Nevertheless, the changes in mean wing shape found in GOF flies are clearly different from those in LOF flies. As the patterns of shape change remained different in LOF and GOF flies after correcting for size - only the GOF flies were smaller than the controls, the hypothesis of a simple allometric effect could be ruled out. A detailed mapping of the cellular effects on the wing would be needed to relate the shape changes to morphogenetic processes. These results are nevertheless consistent with QTL analyses showing that wing shape is regulated at least partly independently of wing size. Wing veins are important determinants of wing shape and wing shape has notably been shown to be tightly associated with the Egf receptor locus that controls the amount of vein material. Interestingly, torpedo, a mutant of the EGF Receptor, shows similar abdomen cuticle defects as those observed in CycG LOF flies (Salvaing, 2008a). Thus, CycG might also interact with the Egf receptor in the wing imaginal disc to control vein specification and wing shape (Debat, 2011).

The amplitude of the asymmetry effect observed when overexpressing CycG is particularly dramatic, and such an amplitude is usually associated with directional asymmetry or antisymmetry, the two forms of conspicuous asymmetry. Similar levels of FA have not been reported previously. Comparatively, a study using deletions covering most of the Drosophila genome detected a maximum increase of 7-fold in size FA (Breuker, personal communication to Debat, 2011; Debat, 2011).

The very low level of genetic variation and the carefully controlled environmental conditions ensured that this effect was due to developmental noise and was not confounded with genetic or environmental variation for directional asymmetry (Debat, 2011).

That wing size and shape as well as femurs of the first leg are affected demonstrates that, although the strength of the effect on FA may vary across body parts, this effect is not restricted to a single trait (i.e. the wing) or a specific segment. This result is particularly important since the only previously known cases of individual genes altering FA were trait-specific (Clarke, 2000). Although wings and legs are both thoracic appendages with partly similar developmental networks, such a common FA effect suggests that the cellular processes altered by deregulation of CycG are likely common to many traits. It also provides some support to the hypothesis of an organism-wide source of developmental noise, and indirectly it suggests the existence of organism-wide stabilizing processes, a very contentious issue (Debat, 2011).

Some preliminary tests on bristle traits nevertheless suggest that bristle number FA is not affected by CycG deregulation. This is in agreement with previous studies suggesting that meristic (i.e., discrete and countable) and metric trait variation could be controlled via different processes (Debat, 2011).

Whereas mean wing shape is affected differently in LOF and GOF flies, the patterns of shape variation around these different means, and specifically those of shape FA, are strikingly similar. This similarity can be interpreted in different ways. First, it might indicate that stochastic variation is constrained along a limited set of directions of shape change, consistent with the view of a wing as an integrated system. Alternatively, such similarity of patterns might reflect a similarity of processes. Although mean wing shape is affected differently when increasing or reducing CycG expression, it is conceivable that shifting CycG expression level away from its normal value might destabilize development in similar ways, generating these similar patterns of shape FA. Comparable - although not identical - patterns of wing shape FA were found in control flies, suggesting that similar processes are involved in generating FA in wild type and CycG deregulated flies. This again supports the view that CycG plays an important role in developmental stability (Debat, 2011).

CycG thus appears as a serious candidate for the genetic control of developmental stability, and further studies should examine its role in FA amplitude differences across natural populations or samples submitted to various environmental treatments. Can we reconcile the reported lack of additive genetic variation for FA with the putative existence of (a) major gene(s) altering FA? It is likely that CycG is involved in a genetic network regulating cell growth, and possibly cell proliferation (Faradji, 2011), where it might act as a hub, as the high FA induced by overexpression suggests. Such a function, likely involving various pleiotropic effects and epistatic interactions, might be under strong selection, possibly leading to the elimination of any variation (Debat, 2011).

It is also conceivable that subtle variation in CycG may occur with only small effects on FA. In particular, CycG overexpression triggered by transgenic constructs is likely of larger magnitude compared to the effects of natural variation. Investigation of natural variation in CycG sequence and expression across populations differing in their degree of FA would provide some insight on this question. Investigating genes interacting with CycG would also improve understanding of its link with organ size stochastic variation (Debat, 2011).

It was recently suggested that the ability of organs to reach a stereotypical size would depend on the competition among populations of growing cells (Neto-Silva, 2009). In given developmental conditions (e.g. during the last cell divisions in the pupal wing blade), Cyclin G intracellular concentration might somehow trigger cell division. Pushing this concentration away from its usual value might interfere with the process by which cells identify the appropriate stage of growth for division, potentially generating stochasticity in cell size and decoupling cell growth and division. This might in turn compromise the normal pattern of cellular competition, causing random variation in organ size (Debat, 2011).

The extreme FA reported in this paper was generated by deregulating expression of a single gene. Consequently, the above hypothetical scenario focuses on the role of a single protein on the generation of random variation at the cellular level, but it does not preclude the existence of diverse processes working at various biological scales (Debat, 2011).

The results do not necessarily mean that CycG is a gene for developmental stability, but they clearly show, by the strength of its effect on cell size variation, that CycG normal expression is required for the formation of symmetrical flies (Debat, 2011).

Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation

Mammalian Cyclins G1 and G2 are unconventional cyclins whose role in regulating the cell cycle is ambiguous. Cyclin G1 promotes G2/M cell cycle arrest in response to DNA damage whereas ectopic expression of CCNG2, which encodes Cyclin G2, induces G1/S cell cycle arrest. The only Drosophila Cyclin G was previously shown to be a transcriptional regulator that interacts with the chromatin factor Corto and controls expression of the homeotic gene Abdominal B. It is very close to mammalian Cyclin G1 and G2 except in its N-terminal region, which interacts with Corto and seems to have been acquired in dipterans. Ubiquitous misregulation of Cyclin G (CycG) using transgenic lines lengthens development and induces phenotypes suggesting growth or proliferation defects. Using tissue-specific misregulation of CycG and FACS, this study has shown that overproduction of Cyclin G produces small cells whereas shortage produces large cells, suggesting that Cyclin G negatively regulates cell growth. Furthermore, overexpression of CycG lengthens the cell cycle, with a prominent effect on G1 and S phases. Genetic interactions with Cyclin E suggest that Cyclin G prevents G1 to S transition and delays S-phase progression. Control of cell growth and cell cycle by Cyclin G might be achieved via interaction with a network of partners, notably the cyclin-dependent kinases CDK4 and CDK2 (Faradji, 2011).

Although G-type cyclins are expressed in many normal and cancerous tissues, their role in mammals is still poorly understood. Drosophila Cyclin G is very close to mammalian Cyclin G1 and G2 except in its long N-terminal region that seems to have been acquired in dipterans. Interestingly, this region binds the ETP Corto, a chromatin factor involved in epigenetic regulation of gene expression (Salvaing, 2008a). This suggests that the transcriptional activity of Cyclin G could be restricted to dipterans. Cyclin G2, but not Cyclin G1, presents a PEST sequence in the C-terminal part. These amino acid sequences are involved in degradation via the proteasome. Interestingly, mutation of a conserved lysine in the cyclin domain, i.e., the CDK-binding domain, of mammalian Cyclin G1, increases its stability. This suggests that a function of Cyclin G1 as a CDK regulator may be required for its rapid turnover. Both Cyclin G1 and G2 are degraded by the ubiquitin-proteasome pathway. The presence of a candidate PEST sequence in the C-terminal region of Drosophila Cyclin G could make this protein functionally closer to mammalian Cyclin G2 and suggests that Cyclin G is also degraded via the proteasome. Nevertheless, no change in the amount of CycG mRNA or Cyclin G protein occurs during the cell cycle in a cultured cell line. This could be cell line-specific, since cell cycle-regulated expression of CCNG1 has been shown to vary between cell lines (Horne, 1996). This lack of periodicity could also suggest that control of Cyclin G activity may at least partly occur at the post-translational level. Prediction of phosphorylation sites with bioinformatic tools suggests that Cyclin G may, indeed, be phosphorylated on many serine, threonine or tyrosine residues, notably in the cyclin domain. Treatment of total cell extracts with lambda phosphatase shows that Cyclin G is phosphorylated and further experiments will decipher its phosphorylation pattern (Faradji, 2011).

Ubiquitous CycG misregulation produces flies with delayed development and decreased viability, but no pattern abnormalities. Ubiquitous overexpression of CycG also reduces body weight. Mutants of many genes involved in metabolism or protein synthesis, notably those of the Insulin/PI3K/AKT pathway genes, also present such phenotypes.This suggests that one of the major roles of Cyclin G is to control growth and that it has little or no role in tissue patterning or differentiation. Inactivation of CycG by RNA interference produces large cells, in contrast to its overexpression that produces small cells. This indicates that Cyclin G is directly involved in negative cell size or growth regulation. Overexpression of CycG in mitotic clones results in a decrease in cell number showing that CycG also limits cell proliferation. Indeed, overexpression of CycG in wing discs lengthens the cell cycle and modifies phasing with a predominant effect on G1 and S phases. It is generally assumed that cells delay the G1/S transition until a critical size has been reached. For instance, shortage of nutrients causes a longer G1 phase. Furthermore, cell growth and cell cycle phasing compensate each other. For example, simultaneous overexpression of Cyclin D and cdk4, which promotes cell growth during G1 phase, does not modify cell size and cell cycle phasing but only accelerates proliferation. In addition, overexpression of Rbf, which slows all phases of the cell cycle, produces large cells. Hence, Cyclin G, which both limits cell size and decreases proliferation, has a very particular behavior. In contrast, specific cell cycle regulators, such as RBF, E2F and Cyclin E, affect growth indirectly, via their effect on the cell cycle. Growth promoters like Myc, or components of the insulin signal transduction pathway (the insulin receptor, its substrate Chico, the PI3 kinase sub-units DP110 and DP60, the kinases AKT1 and dS6K), affect primarily cell growth but do not promote cell proliferation per se. One hypothesis to explain the particular behavior of Cyclin G could be that it acts at the interface between cell cycle pathways and cell growth pathways. Misregulation of CycG could thus somehow lead to loss of compensation between cell growth and cell proliferation. Eighty-two potential Drosophila Cyclin G interactors were found in genome-wide two-hybrid screens. Among them, the transcriptional regulator Cyclin K, the mitotic kinase SAK, the CDK inhibitor Dacapo (DAP), CDK4, and CDK2, share many other partners with Cyclin G. Together, they could thus represent hubs in a common network. Analysis of this network would certainly allow a better understanding of the mechanism(s) of compensation between cell growth and cell proliferation (Faradji, 2011).

Cyclin G functions as a positive regulator of growth and metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. This study identifies Cyclin G (CycG) as a regulator of growth and metabolism in during larval development in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, it was shown that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants are rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, these data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila (Fischer, 2015).

This study analyzed the role of Cyclin G in growth regulation and metabolism of Drosophila. Two different cycG null mutant alleles were used, thereby allowing the following of the developmental consequences resulting from the absence of cycG gene activity instead of drawing conclusions from overexpression or RNAi experiments. Misexpression studies initially raised the assumption that CycG negatively regulated cell growth and cell proliferation in Drosophila. The current results now indicate that CycG is required for normal growth, affecting both cell size and cell number. In fact, clonal analysis revealed a cell autonomous requirement of CycG not only in the wing but also the eye anlagen. In addition, the cycG null mutants show signs of metabolic disorder. Evidence is provided that CycG facilitates InR/TORC1 mediated growth regulation via PP2A, thereby helping to sustain nutrient dependent growth in Drosophila (Fischer, 2015).

Drosophila CycG appears to have extraordinarily diverse roles. It has been involved in epigenetic regulation of homeotic gene activity, in cell cycle regulation, developmental stability and in DNA repair, and now also in metabolic homeostasis. The current work confirmed molecular interactions between CycG and Wdb proteins in vivo that had been predicted from genome-wide proteome analyses in vitro. Interestingly, similar molecular interactions have been described before for mammalian CycG1 and CycG2: both proteins interact with several B' subunits, thereby mediating the recruitment of PP2A to its different substrates. In contrast to mammals, the genetic relationship between CycG and PP2A is antagonistic in Drosophila as a reduction of PP2A activity ameliorates the consequences of CycG loss. The cycG mutation could be formally explained by a gain of PP2A activity. It is tempting to speculate that the diversity of CycG functions results from a regulation of PP2A by CycG. PP2A affects a plethora of developmental and cellular processes, hence, pleiotropy is expected in case of its misregulation. Most likely, this hypothesis is too simplified. For example, loss of cycG in the female germ line results in an increase of phosphorylated H2Av (gamma-H2Av), a known target of PP2A activity. One might have expected a reduced amount of gamma-H2Av if loss of CycG equated with a gain in PP2A activity. Instead, this study has shown that CycG is found in a protein complex together with Rad9 and BRCA2 that primarily acts in the sensing of DNA double strand breaks. The importance of Drosophila CycG in DNA double strand break repair is reminiscent of functions described for mammalian CycG proteins: albeit CycG1 and CycG2 mutant mice are viable and healthy, they are both sensitive to DNA damaging reagents. Moreover, upregulation of CycG2 was involved in the activation of Chk2 and in damage induced G2/M cell cycle arrest, i.e. in DNA damage response in mammals as well. Whether the other phenotypes and interactions reported for Drosophila CycG are linked to the regulation of PP2A remains to be addressed in more detail (Fischer, 2015).

The cycG mutants display several phenotypic characteristics of a diminished TORC1 signaling activity, including weight reduction, a reduced egg laying rate, impaired endoreplication and a general increase in lipid mobilization. Moreover, CycG activity promotes phosphorylation of the primary TORC1 targets, i.e. S6K and 4E-BP. In contrast to TOR mutants, however, cycG mutants are viable, implying that CycG facilitates InR/TOR signaling rather than being an essential factor. Overall, cycG mutant flies show typical signs of nutritional starvation distress even under normal food conditions, suggesting a problem in their capacity to take up food and/or to sense and utilize the food. This defect is not due to a general inability of the animal to grasp the feed, but instead reflects a defect in coordinating the energy status with the regulation of systemic growth. As dILP accumulation in the brain is altered in cycG mutants, it is known that the signals transmitted from the nutritional sensor fat body must be disturbed. The fact, that the growth defects of cycG mutants can be strongly ameliorated by an induction of Akt1 specifically in the fat body rules out a function of CycG in the endocrine signal emanating from the fat body. Instead, all of the data indicate that CycG acts genetically at the level of Akt1, thereby controlling TOR signaling activity (Fischer, 2015).

Akt1 is negatively regulated by PP2A, supporting a model whereby CycG exerts its positive input on Akt1 via an inhibition of PP2A. In accordance, mutations in wdb efficiently rescue the growth and metabolic defects observed in cycG mutants. Likewise a downregulation of Wdb ameliorates the weight deficits resulting from a loss of Akt1 activity. In Drosophila, Wdb acts as a tissue-specific negative regulator of Akt1: it modulates lipid metabolism in the ovary as a result of a direct interaction with Akt1, whereas no such influence was seen in eye tissue. This study has shown that Wdb-Akt1 binding in the adult head is favored in the absence of CycG, i.e. CycG is able to influence the interaction between Wdb and Akt1 presumably by its direct binding to Wdb. A consequence of CycG loss may be the enhanced binding of PP2A to Akt1 and an enforced dephosphorylation of Akt1, resulting in the inhibition of downstream TOR signaling activity and affecting lipid metabolism and growth. Moreover, the second B'-subunit of Drosophila PP2A (also called Well rounded, Wrd) is involved in the negative regulation of the S6K. Assuming a molecular interaction of Wrd and CycG, a likewise regulatory input of CycG on PP2A containing the Wrd B'-subunit is conceivable. In this case, CycG might influence S6K activity as well, having a regulatory input on InR/TOR signaling also downstream of TORC1. This scenario is complicated by the negative feed back regulation of InR signaling by S6K and of Akt1 by TORC1. Circular regulation of InR/TOR signaling has been described at several levels, implementing a tight control of dietary signals and growth but complicating genetic analyses (Fischer, 2015).

In conclusion, the identification of CycG as a novel regulator of InR/TOR signaling in Drosophila highlights the importance of studying the regulatory network at the Akt1—PP2A nexus. Based on the high conservation of the InR/TOR signaling pathway and its regulation by PP2A, mammalian fat homeostasis is likely to involve similar regulatory control mechanisms to those that have been uncovered in Drosophila. This work raises the possibility of an involvement of CycG in InR/TOR-associated diseases that might be modulated by PP2A. A better understanding of the underlying mechanisms could therefore open up avenues for new strategies to fight InR/TOR-associated disorders in the future (Fischer, 2015).

p53 and cyclin G cooperate in mediating genome stability in somatic cells of Drosophila

One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. This study identified Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. This study finds it in protein complexes together with p53, and shows that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor, it was shown that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity it is proposed that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila (Bayer, 2017).

Earlier work has shown that Drosophila CycG is important for the meiotic recombination checkpoint in the female germline. In cycG mutant germaria, DSB repair is delayed, and CycG protein is found in conjunction with the 9-1-1 complex suggesting that it may be involved in DSB sensing. This study extends this analysis to somatic tissue, where again problems were noted in DNA damage repair as detected by persistent γ-H2Av signals in irradiated cycG mutants. This indicates that in the absence of CycG, repair of double-strand breaks in the DNA is compromised. Accordingly, cycG mutants fail to repair DSBs with the fidelity of wild type, display more chromosomal aberrations upon irradiation, and are hypersensitive to genotoxic stress. No evidence was found for an involvement of CycG in DSB sensing in somatic cells, however. Instead, CycG appears to perform its role through modulating the activity of p53. Since a retardation and/or erroneous DNA repair was notice in the absence of CycG, it is proposed that CycG is required to resolve IR-induced DNA damage presumably as co-factor of p53. From genetic data it is concluded that CycG is a positive mediator of p53 activity, and indeed mutants in either gene resemble each other not only in life span but also in radiation sensitivity. The physical interaction of CycG and p53, however, strongly suggests that CycG directly promotes p53 activity, regardless of whether it may also regulate downstream or upstream components of the DNA damage repair machinery (Bayer, 2017).

Unlike in vertebrates, Drosophila cycG is not under the transcriptional control of p53. Instead a robust protein-protein interaction is seen in a yeast two-hybrid assay between p53 and CycG proteins, involving the cyclin repeats of CycG and the tetramerization domain of p53. Direct binding in vivo, however, required genotoxic stress. It is proposed that complex formation, rather than being permanent, occurs only in response to DNA damage and perhaps requires additional factors and/or protein modifications. With the help of a p53 biosensor this study showed that CycG is crucial for p53 mediated transcriptional response to genotoxic stress in the germline as well as in somatic tissue, suggesting that CycG may be involved in the activation or stabilization of p53 itself, or in the assembly of active transcriptional complexes (Bayer, 2017).

The CycG-p53 axis might have been expected given their close interrelationship in the mammalian system. Here, the two cyclin G homologues Ccng1 and Ccng2 have been involved in growth control to genotoxic stress. Ccng1 but not Ccng2 is a direct transcriptional target of p53. Both are found in complexes with protein phosphatase 2A, and together with Mdm2 Ccng1 is involved in Mdm2 mediated degradation of p53. As the two mammalian cyclin G proteins appear to act differently on cell proliferation, a lot of work has been invested to understand their respective roles. More recently it was proposed that observed discrepancies may arise from dose dependency of Ccng1. In fact, also Drosophila tissues and cells appear to respond differentially to the dose of CycG, as for example overexpression may impact the cell cycle in a dominant negative manner, and RNAi downregulation causes effects different from the gene deletion phenotypes. The role of Ccng1 in response to genotoxic stress has been analysed in quite some detail. Here, Ccng1 not only forms a complex with Mdm2, resulting in destabilizing p53. Moreover, it also interacts with ARF, thereby stabilizing and activating p53. It hence has been proposed that Ccng1 is required for a timely and proper response to genotoxic stress, first for the activation of p53 to allow for DNA damage repair, and then for p53 degradation to protect cells from apoptosis that have recovered from the initiating stress (Bayer, 2017).

Intensive searches in the Drosophila genome failed to uncover Mdm2 or ARF homologues to date. Recently, however, a Mdm2 analogue called Corp has been identified that shares several Mdm2 properties: Corp is a transcriptional target of p53 in response to genotoxic stress, it binds to p53 protein and results in reduced p53 protein levels presumably by proteolytic degradation. Hence, like Mdm2 Corp acts in a negative feed back loop on p53 activity. Whether Corp is likewise inactivated by phosphorylation and/or an ARF-like molecule remains to be shown. Moreover, it will be interesting to see, whether Corp can recruit CycG, and whether PP2A plays any role in its regulation. It is known already that Drosophila CycG also binds to the PP2A-B' subunit, similar to the two mammalian CycG proteins. Unlike in vertebrates, however, it acts negatively on PP2A activity by genetic means. Despite the similarity of the respective components and the processes they are involved in, there is not a 1:1 conformity when comparing flies and mammals. Perhaps, the manifold feed back loops weaved into the system, elude genetic analyses. Perhaps, as in mammals, Drosophila CycG forms protein complexes with disparate activities (i.e., repair or apoptosis) depending on tissue, cell cycle phase, or phase of response to DNA damage (Bayer, 2017).

Cyclin G is involved in meiotic recombination repair in Drosophila melanogaster

Cyclin G (CycG) belongs to the atypical cyclins, which have diverse cellular functions. The two mammalian CycG genes, CycG1 and CycG2, regulate the cell cycle in response to cell stress. Detailed analyses of the role of the single Drosophila cycG gene have been hampered by the lack of a mutant. A null mutant was generated in the Drosophila cycG gene that is female sterile and produces ventralised eggs. This phenotype is typical of the downregulation of epidermal growth factor receptor (EGFR) signalling during oogenesis. Ventralised eggs are also observed in mutants (for example, mutants of the spindle class) that are defective in meiotic DNA double-strand break repair. Double-strand breaks (DSBs) induce a meiotic checkpoint by activating Mei-41 kinase (the Drosophila ATR homologue), thereby indirectly causing dorsoventral patterning defects. Evidence is provided for the role of CycG in meiotic checkpoint control. The increased incidence of DSBs in cycG mutant germaria may reflect inefficient DSB repair. Therefore, the downregulation of Mei-W68 (an endonuclease that induces meiotic DSBs), Mei-41, or Drosophila melanogaster Chk2 (a downstream kinase that initiates the meiotic checkpoint) rescues the cycG mutant eggshell phenotype. In vivo, CycG associates with Rad9 and BRCA2. These two proteins are components of the 9-1-1 complex, which is involved in sensing DSBs and in activating meiotic checkpoint control. Therefore, it is proposed that CycG has a role in an early step of meiotic recombination repair, thereby affecting EGFR-mediated patterning processes during oogenesis (Nagel, 2012a).

Dorso-ventral axis formation of the Drosophila oocyte requires Cyclin G

In general, cyclins control the cell cycle. Not so the atypical cyclins, which are required for diverse cellular functions such as for genome stability or for the regulation of transcription and translation. The atypical Cyclin G (CycG) gene of Drosophila has been involved in the epigenetic regulation of abdominal segmentation, cell proliferation and growth, based on overexpression and RNAi studies, but detailed analyses were hampered by the lack of a cycG mutant. For further investigations, the cycG locus was subjected to a detailed molecular analysis. Moreover, a cycG null mutant was studied that was recently established. The mutant flies are homozygous viable, however, the mutant females are sterile and produce ventralized eggs. This study shows that this egg phenotype is primarily a consequence of a defective Epidermal Growth Factor Receptor (EGFR) signalling pathway. By using different read outs, it was demonstrated that cycG loss is tantamount to lowered EGFR signalling. Inferred from epistasis experiments, it is concluded that CycG promotes the Grk signal in the oocyte. Abnormal accumulation but regular secretion of the Grk protein suggests defects of Grk translation in cycG mutants rather than transcriptional regulation. Accordingly, protein accumulation of Vasa, which acts as an oocyte specific translational regulator of Grk in the oocyte is abnormal. A role is proposed of cycG in processes that regulate translation of Grk and hence, influence EGFR-mediated patterning processes during oogenesis (Nagel, 2012b).

This study has shown the ventralized phenotype of cycG mutant eggs results from a downregulation of the EGFR signalling pathway. CycG is required for the translational rather than the transcriptional regulation of Grk within the oocyte. One may think of several mechanisms through which CycG might influence grk mRNA translation. DroID, a comprehensive resource for gene interactions in Drosophila, identified several protein interaction partners of CycG that may relate to its role as translational regulator of grk mRNA. Notably, CycG was identified in vivo in protein complexes together with RNA binding proteins, several potential splice factors and translational regulators, for example Eukaryotic translation initiation factor 4AIII (eIF4AIII), Bicoid stability factor (Bsf), Barentz (Btz), Cap binding protein 80 (Cbp80), and SC35. Moreover, a large scale yeast-two hybrid screen picked Gustavus (Gus) as a partner of CycG. This interaction gives a direct link to dorso-ventral axis formation, since Gus is required for the correct localization of Vasa in the Drosophila egg. Most interestingly, Vasa protein levels appear reduced in cycGHR7 mutant ovaries. This may suggest that CycG is a cofactor of Gus, which acts on Vasa stability in the oocyte. In the absence of CycG, Vasa may be degraded more rapidly. Since Vasa is required for efficient translation of Grk, downregulation of Vasa could affect Grk accumulation and result in ventralized eggs. Alternatively, CycG may affect Grk translation indirectly. It was shown that a meiotic checkpoint induced by unrepaired double-strand breaks affects efficient translation of Grk, thereby causing a ventralized eggshell phenotype. A typical example are mutants in the spindle-A (spn-A) gene, which encodes a homologue of the Rad51 recombinase and which is required for double-strand break repair. Spn-A and CycG were found as molecular partners in yeast-two hybrid screens, and hence, CycG may in fact be involved in meiotic recombination repair. Finally, mutants affecting the rasi-RNA pathway cause similar ventralized eggs: in these mutants DNA breaks accumulate due to defects in transposon silencing, effecting the meiotic checkpoint as well. Because CycG has been involved in radiation sensitivity in both Drosophila and mammals, it is tempting to speculate that it may be involved in double-strand break repair during meiosis, as well. Hence, in the absence of CycG, meiotic double-strand breaks would accumulate, thereby activating the meiotic checkpoint and indirectly affecting grk mRNA translation and axis formation of the oocyte (Nagel, 2012b).

The enhancer of trithorax and polycomb corto interacts with cyclin G in Drosophila

Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called 'Enhancers of Trithorax and Polycomb' (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown. In a two-hybrid screen, Cyclin G was identified as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene. These results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG (Salvaing, 2008a).

In Drosophila, the Bithorax-complex (BX-C) contains the three Hox genes, Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B), that specify the identities of the third thoracic segment (T3) and the eight abdominal segments (A1 to A8). These genes are expressed in spatially regulated patterns during embryonic development thanks to maternal, gap and pair-rule proteins. Their large cis-regulatory sequences are modular and allow parasegmental regulation. These sequences contain different classes of elements such as initiation elements that respond to early segmentation gene products, insulators and promoter targeting sequences (Salvaing, 2008a).

Hox expression is maintained in the original pattern during later stages of development by the Polycomb-group (PcG) and trithorax-group (trxG) genes. In mutants of PcG or trxG genes, Hox patterns are established correctly but are not maintained. PcG proteins keep Hox genes silenced whereas TrxG proteins keep Hox genes activated thus counteracting PcG action. PcG and TrxG proteins are required for the maintenance of many gene expression patterns. These maintenance proteins form heteromultimeric complexes that bind to chromatin and alter its structure. Current models propose that PcG complexes lead to compact, transcriptionally inactive chromatin, whereas TrxG complexes maintain chromatin in an open conformation that facilitates transcription. In Drosophila, several PcG and TrxG complexes have been purified so far: the Polycomb Repressive Complex 1 (PRC1), the Polycomb Repressive Complex 2 (PRC2), the PhoRC complex, the Pcl-PRC2 complex, the Trithorax Activating Complex 1 (TAC1) and the Brahma Complex (BRM) also called SWI/SNF complex. They are extremely large complexes that contain several proteins including chromatin modifying enzymes such as histone methyl-transferases, acetyl-transferases or deacetylases (Salvaing, 2008a).

Although most PcG mutations suppress trxG mutations and vice versa, a large screen to identify modifiers of the trxG gene ash1 allowed isolation of enhancers that were previously identified as PcG [E(z), E(Pc), Asx, Scm, Psc and Su(z)2]. These genes were then called Enhancers of Trithorax and Polycomb (ETPs). Further molecular data showed that some ETPs encode members of PRC complexes, such as E(Z), PSC or SCM, while some do not. Recently, reclassification of these maintenance proteins has been proposed, the label PcG being kept for members of PRC silencing complexes and the label TrxG for members of complexes that counteract PcG-mediated silencing. A third class of proteins would be represented by PcG/TrxG DNA-binding recruiters or specific co-factors. This study keeps the term ETP for those maintenance proteins that play a dual role in PcG and TrxG functions without belonging to any PcG or TrxG complexes identified so far. The GAGA factor, Gaf, encoded by Trithorax-like (Trl), falls into this category. Indeed, it was first described as an activator of Hox genes, and later shown to play a role in the recruitment of PcG complexes without co-purifying with any PRC silencing complexes. The HMG protein DSP1 also meets the criteria to be an ETP: dsp1 mutants exhibit Hox gene loss-of-function phenotypes but DSP1 is also important for PcG recruitment to chromatin. corto behaves genetically as an ETP. corto mutants present PcG as well as trxG phenotypes and enhance the phenotypes of some PcG, trxG and ETP mutants (Lopez, 2001). Corto directly interacts with Gaf and DSP1 suggesting that ETPs are involved in collaborative processes (Salvaing, 2003; Salvaing, 2006; Salvaing, 2008a).

PcG, TrxG and ETP proteins bind DNA sequences called PRE/TRE that carry the information for the active or silent state of the gene they control. Some PRE/TRE have been shown to maintain this transcriptional state throughout cellular divisions in absence of the initial activator or repressor. Despite massive efforts towards identification of PcG complex targets at genome scale, the mechanism by which the active or inactive state of PRE/TRE is conserved throughout several cell cycles remains still largely unknown. Many PcG and ETP mutants [Asx, corto, E(z), Pc, ph, Psc, Su(z)2, Trl] exhibit proliferation defects as well as chromosome condensation and segregation defects. This suggests that maintenance proteins play a general role in cell cycle control. An attractive hypothesis is that ETPs are critical to maintain the correct association of PcG or TrxG complexes with chromatin during the cell cycle (Salvaing, 2008a).

In a two-hybrid screen using Corto as bait, Cyclin G (CycG), the Drosophila homologue of the mammalian Cyclin G1 and G2 (CycG1, CycG2), was isolated. Vertebrate CycG1 is a transcriptional target of the tumor suppressor p53 (Tamura, 1993; Okamoto, 1994). It is possibly involved in cell proliferation as it is overexpressed in certain cancer cells (Reimer, 1999; Baek, 2003). However, CycG1 induces G2/M arrest and cell death in response to DNA damage (Okamoto, 1999; Kimura, 2001; Seo, 2006). Vertebrate CycG2 acts as a negative regulator of cell cycle, as shown by its high level (Bates, 1996; Bennin, 2002) in cells in which G1/S arrest has been induced by growth inhibitory signals (Salvaing, 2008a).

This study addresses the interactions between Corto and CycG both in vitro and in vivo. CycG is shown to play an essential role during development. Moreover, CycG is targeted to many sites on polytene chromosomes where it co-localizes partially with Corto and with the PcG factor Ph. As an ETP, corto maintains Abd-B repression in embryos. This could be achieved by association between Corto and CycG since both proteins bind to Abd-B regulatory elements, including the iab-7 PRE and the promoter (Salvaing, 2008a).

This study has identified Cyclin G as a new binding partner of the ETP. CycG inactivation leads to lethality showing that this gene is essential in flies. Mammalian genomes encode two G-type cyclins, CycG1 and CycG2, the first one being mainly nuclear whereas the second is mainly cytoplasmic (Horne, 1996). Drosophila has a single homologue, however, it produces at least two different protein isoforms, only the larger being associated with chromatin. These isoforms could combine CycG1 and CycG2 functions. In Drosophila, large scale two-hybrid screens suggested binding of CycG to various Cyclin-Dependent Kinases (CDK) (Cdc2 and Cdk4). Corto and CycG interact in vitro as well as in vivo and form a complex in embryos and presumably also on chromatin. Moreover, Corto interacts with the amino-terminal domain of CycG, which is compatible with the simultaneous binding of CDK and cell-cycle control function of CycG (Salvaing, 2008a).

Requirement of PcG, trxG and ETP genes in cell-cycle control has already been shown in Drosophila. Interestingly, PcG and trxG genes are also involved in self-renewal and proliferation of hematopoietic stem cells in vertebrates. One way they might control cell proliferation is by an epigenetic regulation of genes involved in cell cycle and cell proliferation. Indeed, homologues of Drosophila E(z) and Brm participate in the transcriptional regulation of Cyclin A and E in vertebrates, and in Drosophila, Cyclin A is a PcG target. Alternatively, PcG, TrxG or ETP proteins may interact directly with cell cycle regulatory proteins. Indeed, it has been shown that Brm interacts with Cyclin E, that Mel-18, a human homologue of Posterior Sex Combs, interacts with Cyclin D2 possibly blocking its interaction with Cdks, and this study shows here that the ETP Corto interacts with CycG. These interactions reveal a potential role for these maintenance proteins in regulating the cell cycle independently of transcriptional regulation. This could be a widespread mechanism by which PcG, TrxG and ETP coordinate the chromatin activity status (Salvaing, 2008a).

CycG and Corto co-localize on many sites on polytene chromosomes suggesting that they may have regulated associations. The data show that Corto represses Abd-B in embryos and although it was not possible to test the role of CycG in regulating Abd-B expression in embryos, it was observed that both Corto and CycG bind the iab-7 PRE and the promoter of Abd-B suggesting that they could cooperate in this function. Nevertheless, neither Corto nor CycG were detected on the BX-C locus in salivary glands suggesting that they regulate Abd-B in a tissue-specific manner. The role of the CycG-Corto interaction needs to be further investigated. CycG could regulate Corto activity directly on chromatin by recruiting other factors like kinases or phosphatases thus modifying the phosphorylation status of Corto itself, of histones or other proteins at PRE/TRE and promoters. It has been shown that binding of the PcG protein Bmi1 to chromatin correlates with its phosphorylation status. It will be interesting to investigate whether Corto and CycG bind the iab-7 PRE and promoter of Abd-B simultaneously, to examine their phosphorylation status when bound to chromatin, and to determine if their presence correlates with Abd-B transcriptional activity. One interesting possibility would be that CycG is involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG (Salvaing, 2008a).

Regulation of Abd-B expression by Cyclin G and Corto in the abdominal epithelium of Drosophila

Polycomb-group (PcG) and trithorax-group (trxG) genes encode important regulators of homeotic genes, repressors and activators, respectively. They act through epigenetic mechanisms that maintain chromatin structure. The corto gene of Drosophila encodes a co-factor of these regulators belonging to the Enhancer of Trithorax and Polycomb class. Corto maintains the silencing of the homeotic gene Abdominal-B in the embryo and it interacts with a cyclin, Cyclin G, suggesting that it could be a major actor in the connection between Polycomb/Trithorax function and the cell cycle. This study shows that inactivation of Cyclin G by RNA interference leads to rotated genitalia and cuticle defects in the posterior abdomen of pupae and that corto genetically interacts with Cyclin G for generating these phenotypes. Examination of these pupae shows that development of the dorsal histoblast nests that will give rise to the adult epithelium is impaired in the posterior segments which identity is specified by Abdominal-B. Using a line that expresses LacZ in the Abdominal-B domain, it was shown that corto maintains Abdominal-B repression in the pupal epithelium whereas Cyclin G maintains its activation. These results prompt a proposal that the interaction between the Enhancer of Trithorax and Polycomb Corto and Cyclin G is involved in regulating the balance between cell proliferation and cell differentiation during abdominal epithelium development (Salvaing, 2008b).

Ubiquitous downregulation of CycG by RNA interference (using da::Gal4 or Act::Gal4 drivers) led to a high percentage of lethality in late third instar larvae or pharates depending on the CycG line and on the sex (Salvaing, 2008a). Lethality was complete in Act::Gal4/+; UAS::dsCycG2/+ males which intriguingly never underwent pupariation and stopped their development as third instar larvae, dying after a few days. In contrast, most females died as late pharates. UAS::dsCycG2/+; da::Gal4/+ as well as Act::Gal4/+; UAS::dsCycG2/+ emerging animals presented defects in the abdominal cuticle restricted to the posterior tergites A4 to A6. Apart from disorientation of abdominal bristles, the tergites of these segments exhibit unsclerotized patches of variable size. Males were more strongly affected than females and also frequently exhibited rotated genitalia (Salvaing, 2008b).

Genetic interactions between CycG and the loss-of-function alleles corto⁴²⁰ and corto⁰⁷¹²⁸ were examined. Their combination with ubiquitous RNAi inactivation of CycG increased lethality, cuticle defects and rotated genitalia. These data suggest that CycG and corto interact genetically and corroborate the existence of a functional relationship between CycG and corto (Salvaing, 2008b).

To understand the underlying defects of the cuticular phenotypes observed in RNAi-inactivated CycG flies, the development of the abdominal epithelium in pupae was addressed. In Drosophila, the abdominal epithelium of adults is derived from a fixed number of diploid histoblast cells, nested within the polyploid larval epithelium. Each abdominal hemisegment contains four histoblast nests, anterior and posterior dorsal, ventral and spiracle nests, that contribute to tergite and sternite of each abdominal segment. Histoblasts start to proliferate at the beginning of metamorphosis, replacing the larval cells, to eventually build up the adult abdominal integument. In wild-type pupae, the anterior and posterior dorsal histoblast nests of each hemisegment begin to fuse between 15 and 18 h APF. Fusion is completed at 24 h APF and the histoblasts have replaced all the polyploid larval cells at 48 h APF. In Act::Gal4/+; UAS::dsCycG2/+ 48 h APF pupae, whereas the dorsal histoblast nests of segment A3 fuse, the histoblast nests of segments A4 to A6 still remained small and unfused. Nevertheless, the development of these flies was not notably delayed, with regard either to puparium formation or to emergence of adult escapers. Therefore, it is concluded that RNAi inactivation of CycG especially impedes abdominal epithelium development of segments A4 to A6 where histoblast proliferation seemed to have stopped completely (Salvaing, 2008b).

It has been shown that corto is involved in the regulation of Abd-B and that Corto and CycG bind to the iab-7 PRE and to the promoter of Abd-B in embryos (Salvaing, 2008a). Since the epithelium defects of RNAi-inactivated CycG individuals affect abdominal segments A4 to A6, and are enhanced in corto mutants, it was hypothesized that they might be associated with misregulation of Abd-B, which specifies posterior abdominal identity. To address the role of corto and CycG in Abd-B regulation in the abdominal epithelium, genetic interactions between Abd-B and corto or CycG mutants was studied. The Fab-7¹ allele was used, in which both the Fab-7 boundary and the iab-7 PRE of the Abd-B cis-regulatory sequences have been deleted. This mutation induces a higher level of Abd-B expression in A6 which leads to a shift of A6 cell identity toward A7. As there is no normal sclerotized A7 segment in wild-type males, Fab7 homeotic A6 to A7 transformation results in loss of cells. As a result, Fab-7¹/+ males thus present a half-reduced A6 segment. It was observed that corto alleles enhance the expressivity of this phenotype leading to complete disappearance of the A6 segment in 100% of the males. Next, the effect of inactivation of CycG was examined in a Fab-7¹/+ genetic context. The expressivity of the Fab-7¹ phenotype was slightly enhanced in most (86%) of the UAS::dsCycG2/+; da::Gal4/Fab-7¹ males but at least a thin A6 segment always persisted. Curiously, no cuticular defects were observed neither in A5 nor in the remaining A6 tergites of these males suggesting that they might partly result from altered Abd-B expression. Overexpression of CycG also led to enhancement of the Fab-7¹ phenotype expressivity but in this case complete disappearance of A6 was observed in 56% of UAS::CycG/+; da::Gal4/Fab-7¹ males and in 100% of Act::Gal4/+; UAS::CycG/+; Fab-7¹/+ males. Lastly, the Fab-7¹ phenotype was investigated in corto, RNAi-inactivated CycG males. Crosses of Act::Gal4; Fab-7¹ females with UAS::dsCycG2/CyO; corto⁰⁷¹²⁸/TM6b males gave only few Act::Gal4/+; UAS::dsCycG2/+; Fab-7¹/corto⁰⁷¹²⁸ male escapers that all exhibited complete disappearance of A6 segment. These results suggest that both corto and CycG participate in maintenance of A6 cell identity by regulating Abd-B expression. corto clearly acted as a repressor of Abd-B since it enhanced the gain-of-function phenotype of Fab-7¹. However, it is not possible to conclude about the precise role of CycG on Abd-B expression since overexpression as well as inactivation led to enhancement of the Fab-7¹ phenotype, although to a lesser extent in case of inactivation. To understand this issue, the expression of Abd-B was addressed in corto mutants and in RNAi-inactivated CycG or overexpressing CycG individuals (Salvaing, 2008b).

Thus, Abd-B expression was examined in the abdominal epithelium of pupae. Since monoclonal anti-Abd-B antibodies show unspecific ubiquitous staining in the pupal epithelium, the HCJ199 strain was used where a P{LacZ} element is inserted in the cis-regulatory sequences of Abd-B. In agreement with published reports, it was observed that LacZ expression mimics Abd-B expression forming a decreasing gradient from A7 (in females) to the posterior part of A4, the expression in this segment being very faint and only detectable at high magnification. This pattern was also observed in Act::Gal4/+; HCJ199/+ control female pupae showing that the Act::Gal4 driver has no effect on Abd-B expression per se. At 24 h APF, LacZ was expressed in polyploid larval cells as well as in proliferating diploid histoblasts. Later on (48 h APF), LacZ was still expressed in the proliferating diploid histoblasts of A7, A6 and A5, and a barely discernible staining could be seen in posterior . In HCJ199/corto⁴²⁰ and HCJ199/corto⁰⁷¹²⁸ 48 h APF pupae, LacZ was also expressed from A7 to the posterior part of A4 but expression in posterior A4 was much stronger than in control pupae. Moreover, some cells in the posterior region of A3 also expressed LacZ. This suggests that, in the abdominal epithelium of pupae as in embryos, corto maintains repression of Abd-B expression. In RNAi-inactivated CycG female pupae (Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+) at 48 h APF, almost complete loss of LacZ expression was observed in A5 and A6, whereas it was still expressed in A7. In contrast, 48 h APF Act::Gal4/+; UAS::CycG/+; HCJ199/+ female pupae that overexpressed ubiquitously CycG showed ectopic expression of LacZ in the whole abdomen. Taken together, these results suggest that CycG has the ability to activate Abd-B expression in the abdominal epithelium and contributes to Abd-B expression maintenance in A6 and A5. Thus, corto and CycG play opposite roles on the control of Abd-B expression in the abdominal epithelium, corto being a repressor and CycG an activator. The expression of Abd-B was addressed in pupae where corto and CycG expressions were simultaneously reduced. In Act::Gal4/+; UAS::dsCycG2/+; HCJ199/corto⁴²⁰ 48 h APF female pupae, although it was not possible to precisely determine segment borders due to impaired histoblast nest development, rescue of LacZ pattern was obseved that extended more anteriorly than in Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+ pupae. Then, Abd-B loss of expression in A5 and A6 induced by CycG inactivation was abrogated when the amount of Corto was simultaneously reduced (Salvaing, 2008b).

This study has show that ubiquitous downregulation of CycG in pupae results in failure of epithelium formation in the posterior abdomen. Abdominal epithelium of adults derives from imaginal histoblasts that are recruited during embryogenic stages and form small group of diploid cells nested in the polyploid larval epithelium. The anterior dorsal nest is composed of about 15 to 18 cells whereas the posterior dorsal nest is composed of about 5 to 6 cells. These cells stay quiescent being arrested in G2 during the larval stages. At the onset of metamorphosis, they first undergo a phase of rapid proliferation triggered by ecdysone signalling and consisting of three synchronous and fast divisions. Also set off by ecdysone signalling are the second phase of histoblast proliferation which is slow and asynchronous and the simultaneous death of the polyploid larval cells. At 24 h APF, the anterior and posterior histoblast nests have fused. Inactivation of CycG impedes the proliferation of histoblasts in the posterior part of the abdomen, the dorsal anterior and posterior nests being still individualized at 48 h APF. This probably results in cuticle defects in the less severely affected individuals that will become adult. Similar cuticle defects have been described in some mutants (Arrowhead, escargot, cdc2, myb, torpedo, EcR) where they extend more often over the entire abdomen. Arrowhead has been shown to be involved in the establishment of abdominal histoblasts during embryogenesis. In RNAi-inactivated CycG larvae, the number of cells in the dorsal anterior and posterior histoblast nests is identical to that of wild-type larvae, suggesting that CycG inactivation does not affect histoblast recruitment during embryogenesis. esgargot and cdc2 are required to maintain diploidy of histoblast cells. In RNAi-inactivated CycG larvae, the size of histoblast nuclei in dorsal nests appears similar to the size of the corresponding wild-type nuclei thus suggesting that CycG inactivation does not affect ploidy. Lastly, the epithelium defects could be related to defects in cell proliferation. This is the case for the myb mutant, which proliferating histoblasts exhibit mitosis defects, or the torpedo mutant, which shows loss of mitotic figures in the histoblast nests at 25 h APF. In RNAi-inactivated CycG pupae at 48 h APF, approximately 100 and 40 cells were observed in the anterior and posterior nests of the A6 segment, respectively, suggesting that they might have undergone the 3 first rounds of division. Then, it could be that cells slow down during the second phase of proliferation. Intriguingly, like in RNAi-inactivated CycG pupae, slowdown of histoblast proliferation in segments A5 and A6 has been observed in the torpedo mutant; torpedo encodes the EGF receptor. This suggests that the role of CycG in the proliferation of the abdominal epithelium could be related to MAP kinase signalling. Furthermore, the use of a dominant-negative form of the Ecdysone-receptor that blocks death of the larval epidermal polyploid cells also induces cuticle defects. In this case, cell-autonomous inhibition of EcR activity leads to abortive delamination and persistence of larval polyploid cells in the pupal epithelium. A similar phenomenon, linked to disruption of ecdysone signal reception, could arise when CycG is inactivated. Interestingly, that Act::Gal4>UAS::dsCycG males never go through pupariation; this could reflect a defect in EcR signalling reception. Although the abdominal cuticle of corto mutants seems to be unaffected, the cuticle defects were enhanced by combining them with CycG inactivation. It suggests that corto and CycG together regulate the formation of the abdominal epithelium during metamorphosis (Salvaing, 2008b).

These data also show that corto and CycG oppositely regulate the expression of the Hox gene Abd-B in the growing pupal epithelium, corto behaving as a repressor whereas CycG behaves as an activator. Since Corto also represses Abd-B in embryos, it can be considered as a global repressor of Abd-B. Nevertheless, neither Corto nor CycG were detected on the BX-C locus in salivary glands suggesting that they regulate Abd-B in a tissue-specific manner (Salvaing, 2008a). In accordance with expression data, reduction of corto or overexpression of CycG leads to enhancement of the gain-of-function phenotype of Fab-7¹ heterozygotes. Surprisingly, whereas loss of Abd-B expression was observed upon inactivation of CycG, a mild enhancement of the gain-of-function phenotype of Fab-7¹ was seen in the same genetic background. This enhancement may result from perturbation of proliferation in the remaining tergite rather than from homeotic transformation of A6 cells to A7 cells. However, it may also reflect the intrinsic mechanism of action of CycG. Indeed, it has been shown that CycG binds both the iab-7 PRE and the promoter of Abd-B. It is well known that PREs have a stronger silencing activity when present in two copies in the genome, a phenomenon called pairing-sensitive repression. Then, if CycG activates Abd-B partly by working at the promoter and partly by limiting pairing-sensitive repression, loss of Abd-B activation at promoter could be overwhelmed by loss of pairing-sensitive repression when a single copy of the iab-7 PRE is present which is the case in the Fab-7¹/+ flies (Salvaing, 2008b).

Finally, in pupae combining RNAi-inactivated CycG and corto mutation, histoblast proliferation is still impeded whereas Abd-B expression seems to be restored. It suggests that the ratio between Corto and CycG activities must be preserved to insure appropriate regulation of Abd-B in the posterior abdomen. Altogether, these results suggest that a tripartite interaction between corto, CycG and Abd-B together regulates the balance between proliferation and differentiation during the formation of the abdominal epithelium at metamorphosis. Further experiments are now required to better understand how these processes are coordinated (Salvaing, 2008b).

Search PubMed for articles about Drosophila Cyclin G

Baek, W. K., et al. (2003). Increased expression of cyclin G1 in leiomyoma compared with normal myometrium. Am. J. Obstet. Gynecol. 188: 634-639. PubMed ID: 12634633

Bates, S., Rowan, S. and Vousden, K. H. (1996). Characterisation of human cyclin G1 and G2: DNA damage inducible genes. Oncogene 13: 1103-1109. PubMed ID: 8806701

Bayer, F. E., Zimmermann, M., Fischer, P., Gromoll, C., Preiss, A. and Nagel, A. C. (2017). p53 and cyclin G cooperate in mediating genome stability in somatic cells of Drosophila. Sci Rep 7(1): 17890. PubMed ID: 29263364

Bennin, D. A., et al. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest. J. Biol. Chem. 277: 27449-27467. PubMed ID: 11956189

Clarke, G. M., Yen, J. L. and McKenzie, J. A. (2000). Wings and bristles: character specificity of the asymmetry phenotype in insecticide-resistant strains of Lucilia cuprina. Proc. Biol. Sci. 267: 1815-1818. PubMed ID: 11052530

Debat, V., et al. (2011). Developmental stability: a major role for cyclin G in Drosophila melanogaster. PLoS Genet. 7(10): e1002314. PubMed ID: 21998598

Faradji F, Bloyer S, Dardalhon-Cuménal D, Randsholt NB, Peronnet F (2011) Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation. Cell Cycle 10: 1-14. PubMed ID: 21311225

Fischer, P., La Rosa, M.K., Schulz, A., Preiss, A. and Nagel, A.C. (2015). Cyclin G functions as a positive regulator of growth and metabolism in Drosophila. PLoS Genet 11: e1005440. PubMed ID: 26274446

Horne, M. C., et al. (1996). Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue-specific and cell cycle-regulated expression. J. Biol. Chem. 271: 6050-6061. PubMed ID: 8626390

Kimura, S. H., Ikawa, M., Ito, A., Okabe, M. and Nojima, H. (2001). Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery. Oncogene 20: 3290-3300. PubMed ID: 11423978

Lopez, A., Higuet, D., Rosset, R., Deutsch, J. and Peronnet, F. (2001). corto genetically interacts with Pc-G and trx-G genes and maintains the anterior boundary of Ultrabithorax expression in Drosophila larvae. Mol. Genet. Genomics 266: 572-583. PubMed ID: 11810228

Nagel, A. C., Fischer, P., Szawinski, J., La Rosa, M. K. and Preiss, A. (2012a). Cyclin G is involved in meiotic recombination repair in Drosophila melanogaster. J Cell Sci 125: 5555-5563. PubMed ID: 22976300

Nagel, A. C., Szawinski, J., Fischer, P., Maier, D., Wech, I. and Preiss, A. (2012b). Dorso-ventral axis formation of the Drosophila oocyte requires Cyclin G. Hereditas 149: 186-196. PubMed ID: 23121330

Neto-Silva, R. M., Wells, B. S. and Johnston, L. A. (2009). Mechanisms of growth and homeostasis in the Drosophila wing. Annu. Rev. Cell Dev. Biol. 25: 197-220. PubMed ID: 19575645

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Okamoto, K. and Prives, C. (1999). A role of cyclin G in the process of apoptosis. Oncogene 18: 4606-4615. PubMed ID: 10467405

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date revised: 25 April 2018

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