Drosophila melanogaster Cyclin G coordinates cell ...

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Cell Cycle 10:5, 1-14; March 1, 2011; © 2011 Landes Bioscience

Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation Floria Faradji, Sébastien Bloyer, Delphine Dardalhon-Cuménal, Neel B. Randsholt and Frédérique Peronnet* This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Laboratoire de Biologie du Développement UMR 7622; Université Pierre et Marie Curie-Paris 6; Centre National de la Recherche Scientifique; Paris, France

Key words: Drosophila, cell cycle, cell growth, cyclin, cyclin G Abbreviations: CCNG1/2, human cyclin G1/2 genes; CDK, cyclin-dependent kinase; CG1-1, UAS::cyclin G; CycG, Drosophila gene; Cyclin G, Drosophila protein; da, daughterless; dsCycG2, UAS::dsCycG2; dsCycG3s, UAS::dsCycG3s; EdU, 5-ethynyl-2'-deoxyuridine; en, engrailed; ey, eyeless; FACS, fluorescence activated cell sorting; FLP, flipase; GAK, Cyclin G-associated kinase; GFP, green fluorescent protein; GMR, glass multimer reporter; GOF, gain of function; LOF, loss of function; RCG23.3, UAS::mRFP-cyclin G; RCG76, UAS::mRFP-cyclin G; Rbf, retinoblastoma family; SEM,scanning electron microscopy; UAS, upstream activation sequence; WDB, Widerborst

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, we show 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.

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Introduction Cyclins are a family of proteins primarily characterized as cell cycle regulators, which all present a highly conserved domain called the cyclin box. Cyclins bind and activate Cyclin-Dependent Kinases (CDK) (reviewed in ref. 1 and 2). Cyclin/CDK complexes phosphorylate substrates that regulate the cell cycle as, for example, the retinoblastoma (Rb) family of transcriptional repressors.3 Up to now, fourteen cyclins have been characterized.4 Only a few, Cyclins A, B, D and E, are directly involved in cell cycle regulation. The role of the other cyclins in this process is less clear. Cyclins C, K, H or T are involved in transcriptional regulation.5-10 Cyclins I, S and F play roles in cell survival, shortterm memory or proteolysis.11-14 Mammalian Cyclins G1 and G2 are examples of such unconventional cyclins, whose role in regulating the cell cycle is not well understood. Their sequence is most closely related to those of I-type and A-type cyclins.15 Cyclins G1 and G2 define, together with Cyclin I, a family of cyclins expressed in terminally differentiated tissues.16 Cyclin G1 was identified in screens

for rat fibroblast cDNAs hybridizing with c-src family kinase domains and for transcriptional targets of tumor suppressor p53 in a mouse cell line.17,18 Cyclin G1 can act as either a negative or a positive cell cycle regulator. CCNG1, which encodes Cyclin G1, is induced after DNA damage.19 Cyclin G1 promotes G2 /M arrest of the cell cycle in response to DNA damage and facilitates apoptosis.19,20 On the other hand, Cyclin G1 can promote growth after cellular stress,21 and CCNG1 is overexpressed in several human cancers, suggesting a positive role in cell cycle regulation.22,23 In normal breast cells, CCNG1 expression peaks at the S and G2 /M phases of the cell cycle, whereas in cancer breast cells as well as in lymphocytes, no cell cycle-dependent expression has been observed.15,23 CCNG2 was cloned and characterized as a CCNG1 homolog and Cyclin G2 exhibits 53% amino-acid identity with Cyclin G1.15 In murine cells, CCNG2 is upregulated in response to growth inhibition.24 Furthermore, its ectopic expression induces G1/S cell cycle arrest.16,24 Hence, Cyclin G2 may be an important negative regulator of cell cycle progression.24 In normal cells, CCNG2 mRNA oscillates during the cell cycle with a peak in late S phase.15 Indeed, contrary to

*Correspondence to: Frédérique Peronnet; Email: [email protected] Submitted: 12/03/10; Revised: 01/26/11; Accepted: 01/26/11 DOI: www.landesbioscience.com

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Cyclin G1 that contains no degradation box (ubiquitin-binding site or PEST sequence), Cyclin G2 contains a PEST sequence at its carboxy-terminal extremity suggesting that it could cycle.24 Cyclin G1 interacts with two kinases: Cyclin G-associated kinase (GAK) and CDK5.25 GAK is involved in chromosome congression.26 CDK5 is a regulator of central nervous system cytoarchitecture, but no role in cell cycle regulation has been reported.27 Both G-type cyclins also interact with phosphatase PP2A, an interaction that is unique among cyclin family members.16,28,29 Cyclin G2/PP2A complexes inhibit cell cycle progression.16 On the other hand, PP2A inhibits ubiquitination of Cyclin G1 and stabilizes it in unstressed conditions as well as upon DNA damage.30 The only Drosophila Cyclin G gene (CycG) was isolated in a two-hybrid screen against the chromatin factor Corto, a member of the Enhancer of Trithorax and Polycomb group of genes.3133 CycG is expressed ubiquitously throughout development. Inactivation of CycG by RNA interference throughout development leads to high lethality, showing that this gene is essential. The cyclin domain of Drosophila Cyclin G is highly similar to those of vertebrate Cyclin G1 and G2, showing 42% and 46% identity, respectively. Moreover, genome-wide two-hybrid screens suggest that Cyclin G interacts with Widerborst (WDB), the regulatory sub-unit of PP2A, as well as with different CDKs, including CDK2 and CDK4.34,35 We have previously shown that Cyclin G is a transcriptional regulator that binds chromatin at many sites and controls expression of the homeotic gene Abdominal B.31,32 Interestingly, the few adult escapers obtained after CycG inactivation present an incomplete abdominal cuticle, which suggests that proliferation of abdominal histoblasts has been impaired. This led us to investigate the role of Cyclin G in regulating cell proliferation. Here we first examine the evolution of Cyclin G proteins. We then show, by overexpressing CycG or inactivating it by RNA interference, that Drosophila Cyclin G acts as a negative regulator of both cell growth and cell cycle progression during development.

the longest (549–587 residues), whereas mosquito Cyclin Gs are shorter (477-476 residues for Aedes aegypti or Culex quinquefasciatus). Lepidopteran (Bombyx mori), hymenopteran (Nasonia vitripennis, Apis mellifera) and coleopteran (Tribolium castaneum) Cyclin Gs are comparable in size to mammalian Cyclin Gs (307– 363 residues). ClustalW2 alignments showed that C-ter regions are highly conserved among insects (Sup. Fig. 1A and data not shown), length differences mainly affecting N-ter regions. Drosophila and Glossinia Cyclin G N-ter regions are the longest and contain three conserved blocks separated by less conserved stretches rich in homopolymers (Sup. Fig. 1B). N-ter regions are shorter in other Diptera (Fig. 1B and data not shown) and contain about 40 residues in non-dipteran insects, as in mammals. Together these data suggest that long N-ter regions could be specific to Diptera, in particular true flies, and could thus mediate fly or even Drosophila-specific functions. Cyclin G misregulation causes global growth or proliferation defects. Consequences of ubiquitous CycG misregulation were analyzed by crossing da::Gal4 virgin females with UAS::dsCycG (lines UAS:dsCycG2 or UAS::dsCycG3s) or UAS::CycG (lines RCG76 or RCG23.3) males to either inactivate or overexpress CycG in their progeny, respectively. Both inactivation (LOF) or overexpression (GOF) of CycG using a da::Gal4 driver led to high lethality: total lethality for da>dsCycG3s individuals, up to 31% lethality for females and 56% for males da>dsCycG2,31 and up to 45% for females and 72% for males da>RCG76. LOF and GOF also increased total development time by 24 h at 25°C. While the average weight of LOF adult escapers was similar to that of their control siblings, GOF escapers showed a severe reduction in weight (54% for females and 63% for males) (Fig. 2A), suggesting that overexpression of CycG impeded growth. Interestingly, many male escapers presented rotated genitalia (44% for LOF and 26% for GOF escapers) (Fig. 2B). During metamorphosis, the male genital plate undergoes a 360° dextral rotation that positions it along the dorso-ventral axis, thus causing a loop of the spermiduct around the gut.36 After dissection, we observed that LOF and GOF CycG male escapers never presented inversion of looping (not shown). Therefore, mispositioning of the genital plate probably resulted either from under- or over-rotation of the genital plate. Interestingly, LOF male genitalia were over-rotated and GOF male genitalia were under-rotated. These phenotypes could be due to defects in male genitalia primordium growth or to apoptosis.37-40 We also specifically deregulated CycG in the eye-antenna imaginal discs using two different Gal4 drivers: ey::Gal4, which drives Gal4 from early embryogenesis throughout development, and GMR::Gal4, which is restricted to cells undergoing differentiation, i.e., posterior to the morphogenetic furrow that moves across the eye disc during the third larval instar. Using GMR::Gal4, we did not observed any defect in the eyes, suggesting that late induced GOF or LOF of CycG has no effect on the second mitotic wave that occurs just after morphogenetic


Results Sequence conservation of Cyclin G. H. sapiens Cyclins G1 and G2 contain respectively 295 and 344 amino acids, vs. 566 for the longest D. melanogaster Cyclin G isoform. A ClustalW2 multiple alignment reveals that Cyclin G presents overall similarity with both Cyclin G1 and G2 (Fig. 1A). Cyclin G2 contains a PEST motif in C-ter,15 and the epestfind algorithm predicted PEST motifs in C-ter of both Cyclin G2 and Cyclin G (Fig. 1A), suggesting functional conservation between these regions. A major difference concerns the Cyclin G N-ter region, most of which has no counterpart in H. sapiens Cyclin G1 and G2 (Fig. 1A and data not shown). To gain insight into Cyclin G evolutionary history, we compared insect Cyclin Gs. Among Diptera, Drosophila or tsetse fly (Glossina morsitans morsitans) Cyclin G sequences are

Figure 1 (See opposite page). Cyclin G conservation. (A) ClustalW2 alignment of D. melanogaster Cyclin G with H. sapiens Cyclin G1 and G2. The cyclin box is underlined in red and PEST motifs are underlined in black. (B) Multiple ClustalW2 alignment of Cyclin G N-ter regions from 8 insect species: Drosophila melanogaster, Drosophila virilis, Glossina morsitans morsitans, Nasonia vitripennis, Apis mellifera, Aedes aegypti, Bombyx mori and Tribolium castaneum. Note the striking length differences of N-ter regions. Black lines indicate three sequence blocks conserved among true flies. 2

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Figure 1. For figure legend, see page 2.

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furrow passage, or on retina cell differentiation. On the opposite, inactivation of CycG with ey::Gal4 (ey>dsCycG2) induced a weak increase in the number of ommatidia (2% for both sexes) that was significant in females only (Fig. 2C). However, overexpression of CycG using the same driver (ey>RCG76 ) led to highly significant decreases in the number of ommatidia (32% for females and 38% for males) (Fig. 2C and D). Hence, inactivation of CycG with the ey::Gal4 driver slightly increased proliferation of eye-antenna imaginal disc cells in females, whereas overexpression of CycG from the embryonic stage onwards limited their proliferation in both sexes. To estimate cell size, we measured the mean size of ommatidia by counting their number in a 5500 μm 2 square located in the center of the eye and dividing this area by the number of ommatidia.41,42 Ommatidia of females ey>dsCycG2 and their ey::Gal4/+ sibling sisters had the same size whereas ommatidia of ey>dsCycG2 males were significantly larger than controls (mean size 265.3 μm 2 for ey>dsCycG2 males vs. 246.0 μm2 for ey::Gal4/+ males). Furthermore, ommatidia of ey>RCG76 flies were significantly smaller than those of their ey::Gal4/+ siblings (mean size 216.4 μm2 for ey>RCG76 females vs. 242.0 μm2 for ey::Gal4/+ females; 198.2 μm2 for ey>RCG76 males vs. 247.2 μm 2 for ey::Gal4/+ males) (Fig. 2E). As each ommatidium corresponds to the corneal lens secreted by a group of four cone cells,43 these data suggested that cone cells tended to be larger when CycG was RNAi inactivated and smaller when CycG was overexpressed. Hence, inactivation of CycG with the ey::Gal4 driver could slightly increase growth of retina cell in males, whereas overexpression of CycG could limit their growth in both sexes. As proliferation might also be affected, we looked for effects of a CycG misregulation on cell numbers in imaginal wing discs where normal cell proliferation has been extensively described.44 Next, we deregulated CycG in the posterior compartment of wing imaginal discs, using the en::Gal4 driver associated with a UAS::GFP transgene (Fig. 3). While inactivation of CycG in posterior cells (en>GFP, dsCycG3s) did not change posterior compartment area (Fig. 3B and B’), CycG overexpression (en>GFP, CG1-1) led to a marked decrease of this area (Fig. 3C and C’). To determine whether this reflected a negative effect of CycG on mitosis, we stained these wing discs with an anti-H3pS10 antibody to highlight mitoses. This revealed no detectable differences between the anterior and posterior compartments for any of the three genotypes (en>GFP; en>GFP, dsCycG3s; en>GFP,CG1-1) (Fig. 3A”–C”). To establish whether cell numbers were affected, wing imaginal discs were then dissociated with trypsin, and their cell composition was analyzed by FACS. In control wing discs (en>GFP), 42.6% of the cells belonged to the posterior compartment (i.e., GFP positive cells, Fig. 3D). This percentage was similar in wing imaginal discs where CycG was inactivated in the posterior compartment (en>GFP, dsCycG3s; 43.0%) and significantly decreased in those where CycG was overexpressed (30.8% for en>GFP, RCG76 and 34.9% for en>GFP, RCG23.3) (Fig. 3D). This reduced cell number could reflect either induction of apoptosis or a negative effect on cell proliferation.

To test the first hypothesis, we simultaneously overexpressed CycG and the baculovirus caspase inhibitor p35 in the posterior compartment to impair apoptosis (en>GFP, p35, RCG76 ). Cell number in the posterior compartment was not restored by p35 expression (Fig. 3D). Furthermore, apoptotic cells were not detected in wing imaginal discs where CycG had been inactivated (en>GFP, dsCycG3s) or overexpressed (en>GFP, CG1-1), neither with acridine orange staining (not shown) nor with activated Caspase 3 antibodies (Fig. 3A’”–C’”). We concluded from these data that misregulation of CycG did not induce apoptosis in posterior compartments. Taken together, the data presented above suggest that Cyclin G might negatively control cell proliferation and cell growth in many tissues during Drosophila development. CycG misregulation affects cell growth. Cell size in wings of da>dsCycG2 and da>RCG76 adult escapers was measured by automatically counting the total number of bristles in three squares located between vein 4 and vein 5, using Image J software (Fig. 4A). Cell size was estimated by dividing the square surface by the mean number of bristles in a square, as each cell carries one bristle.45 Interestingly, wing cells of da>dsCycG2 flies were significantly larger than those of their da::Gal4/+ siblings (mean size 219.7 μm2 for da>dsCycG2 females vs. 156.9 μm2 for da::Gal4/+ females; 177.6 μm2 for da>dsCycG2 males vs. 161.1 μm2 for da::Gal4/+ males), whereas wing cells of da>RCG76 flies were significantly smaller (mean size 138.6 μm2 for females, 126.9 μm2 for males) (Fig. 4B). We next used FACS to compare cell size of third instar larval wing imaginal discs from control discs (en>GFP) and from discs where CycG was inactivated (en>GFP, dsCycG3s) or overexpressed (en>GFP, RCG76 ). In control wing discs, posterior cells were slightly smaller than anterior cells (Fig. 4C), as previously reported in reference 44. In en>GFP, dsCycG3s wing discs, posterior cells had the same size as anterior cells (Fig. 4D). In en>GFP, RCG76 wing discs, posterior cells were very small as compared to anterior cells (Fig. 4E). To compare the three genotypes, we first eliminated small variations of cell size due to genetic background by normalizing posterior compartment median size to anterior compartment median size (Fig. 4F). Cells where CycG had been RNAi inactivated tended to be larger than control en>GFP cells. Strikingly, their size was more variable than that of control cells, as shown by the enlarged box on the box plot (Fig. 4F). On the other hand, cells where CycG had been overexpressed were significantly smaller than control en>GFP cells. Taken together, these data suggest that Drosophila CycG negatively controls cell growth. Cyclin G is involved in G1 and S-phase regulation. To determine whether CycG mRNA or protein varied during the cell cycle, S2R+ cells were sorted according to cell cycle phases as described in Materials and Methods. Quantitative RT-PCR and western blot analyses showed that CycG mRNA and protein were present all along the cell cycle (Sup. Fig. 2). Nevertheless, since ubiquitous CycG misregulation increased total development time, we hypothesized that it could have an effect on the cell cycle. To test this, we generated mitotic clones with overexpressed or RNAi inactivated CycG using the flip-out system. FLP was induced by


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Figure 2. Phenotypes induced by Cyclin G (CycG) misregulation. (A) Effect on adult weight of ubiquitous overexpression or inactivation of Cyclin G (CycG) using the da::Gal4 driver and the UAS::CycG RCG76 or the UAS::dsCycG2 line. Error bars represent standard deviation from the mean. Individuals overexpressing CycG (da>RCG76) were significantly lighter than their sibling controls (da::Gal4/+) (*t-test, p < 0.001). (B) Effect on male genitalia of ubiquitous inactivation or overexpression of CycG using the da::Gal4 driver and the UAS::dsCycG2 line (LOF, da>dsCycG2) or the RCG76 line (GOF, da>RCG76). 44% of males with inactivated CycG presented over-rotated genitalia whereas 26% of those with CycG overexpression presented under-rotated genitalia. These phenotypes were never observed in control siblings. (C) Effect on ommatidium number of inactivation or overexpression of CycG in the eye-antenna imaginal disc throughout development using the ey::Gal4 driver. Females with inactivated CycG (ey>dsCycG2) presented a weak, but significant increase in ommatidium number (*Mann-Whitney test, p = 0.05), as compared to control siblings (ey::Gal4/+). Females and males overexpressing CycG presented a significant reduction in ommatidium number as compared to control siblings (*Mann-Whitney test, p = 0.001). Error bars represent standard deviation from the mean. (D) SEM pictures of eyes from an ey::Gal4/+ control female (left) and an ey>RCG76 representative female (right). The white bar corresponds to 100 μm. (E) Effect on ommatidium size of inactivation or overexpression of CycG in the eye-antenna imaginal disc throughout development using the ey::Gal4 driver. Males with inactivated CycG (ey>dsCycG2) presented a significant increase in ommatidium size (*Mann-Whitney test, p = 0.015), as compared to control siblings (ey::Gal4/+). Females and males overexpressing CycG presented a significant reduction in ommatidia number as compared to control siblings (**Mann-Whitney test, p = 0.0001). Error bars represent standard deviation from the mean.


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Figure 3. Misregulation of CycG in wing imaginal disc posterior compartments. (A–C) Wing imaginal discs of third instar larvae stained with DAPI and expressing GFP in posterior compartments (anterior left; dorsal up). White lines represent the antero-posterior border. (A) en>GFP controls; (B) CycG inactivation (en>GFP, dsCycG3s); (C) CycG overexpression (en>GFP, CG1-1). (A’–C’) Immunostaining of same discs with guineapig anti-Cyclin G showing ubiquitous expression of CycG in en>GFP (A’), RNAi inactivation of CycG in en>GFP, dsCycG3s posterior compartment (B’), and overexpression of CycG in en>Gal4, CG1-1 posterior compartment (C’). (A”–C”) Immunostaining of wing discs of the same genotypes with rabbit anti-H3pS10 antibody showing mitosis in en>GFP discs (A”), in en>GFP, dsCycG3s discs with posterior compartment RNAi inactivated CycG (B”), or in en>Gal4, CG1-1 discs with posterior compartment overexpression of CycG (C”). (A’”–C’”) Immunostaining of wing discs of the same three genotypes with rabbit anti-cleaved Caspase 3 antibody. (D) Wing disc cells from late third instar larvae were dissociated with trypsin and analyzed by FACS. Mean percentages of GFP positive cells (posterior compartments) for at least 3 independent experiments are represented for control wing discs (en>GFP), for wing discs with RNAi inactivated CycG (en>GFP, dsCycG3s), and for wing discs overexpressing CycG (en>GFP, RCG76 or en>GFP, RCG23.3). Error bars represent standard deviation from the mean. Data were compared by Mann-Whitney tests. Percentages of posterior cells in wing imaginal discs with RNAi inactivated CycG were slightly but not significantly higher than those of control wing imaginal discs. Percentages of posterior cells in wing imaginal discs overexpressing CycG were significantly lower than those of control wing imaginal discs (*p = 0.001). This phenotype was not rescued by overexpression of the caspase inhibitor p35 (en>GFP, p35, RCG76).


heat-shock 72 h after egg laying (AEL), and wing imaginal discs were dissected at 120 h AEL. The number of cells in each clone was determined and cell doubling time was calculated as (log2/ logN)(h), where N is the median cell number per clone and h the time in hours between clone induction and disc dissection (48 h),46 (Fig. 5A for GFP controls and Sup. Fig. 3 for other genotypes). Inactivation of CycG slightly increased the global length of the cell cycle (11.3 h) as compared to GFP control clones (10.4 h). Overexpression of CycG, on the other hand, considerably slowed down the cell cycle (15.1 h for Act>RCG23.3 clones and 17.1 h for Act>RCG76 ones). To determine which phase(s) of the cell cycle were disturbed, we used again the en>GFP system to induce GFP together with CycG inactivation or overexpression in the posterior wing disc compartment. Wing discs were dissected at 120 h AEL and incubated for 20 minutes with EdU in order to label S phases. After dissociation and sorting, cells of anterior (GFP -) and posterior (GFP+) compartments were treated separately and analyzed by FACS (Fig. 5B). Distribution of cells within the cycle was slightly different in between compartments of en>GFP controls, with 19.9% G1, 35.5% S and 40.5% G2/M in the anterior compartment and 23.9% G1, 36.7% S and 34.6% G2/M in the posterior compartment (Fig. 5C). When CycG was inactivated (en>GFP, dsCycG3s), phase distribution of posterior compartment cells was slightly modified (23.9% G1, 40.4% S, 32.2% G2/M) (Sup. Fig. 3A). Interestingly, posterior compartment inactivation gave rise to a slight non-autonomous effect on the anterior compartment (20.8% G1, 40.4% S, 36.4% G2/M). When CycG was overexpressed in the posterior compartment (en>GFP, RCG76 or en>GFP, RCG23.3), distribution of cells within the cycle was also modified (20.7% G1, 49.0% S, 25.9% G2/M and 22.9% G1, 43.9% S, 30.4% G2/M, respectively)

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Figure 4. For figure legend, see page 8.


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Figure 4 (See previous page). Consequences of CycG misregulation on cell size. (A) Fly wing. Cells were automatically counted in three 256 μm2 squares located between vein 4 and vein 5 just distal to the posterior cross-vein. (B) Mean cell size in female and male adult wings where CycG was ubiquitously deregulated using the da::Gal4 driver. Cells were counted in 20 wings for each genotype and data were compared by Mann-Whitney tests. Cells of flies with RNAi inactivated CycG (da>dsCycG2) were significantly larger that those of control flies (da::Gal4/+) (*p = 0.001) whereas cells of flies overexpressing CycG (da>RCG76) were significantly smaller (*p = 0.001). (C–E) Wing imaginal disc cells of late third instar larvae were dissociated and analyzed by FACS. Size of wing disc cells was determined by Forward Scatter (FSC) measure. Plot of a representative experiment is shown for each genotype (C: en>GFP; D: en>GFP, dsCycG3s, E: en>GFP, RCG76). Black curve: GFP negative cells (anterior compartment); grey curve: GFP positive cells (posterior compartment). (F) Box plot representing the ratio of posterior compartment cell median size to anterior compartment cell median size for each genotype. At least 4 experiments were averaged. Dark lozenges represents the mean, dark grey is for the 25th percentile, light grey is for the 75th percentile. In control wing discs (en>GFP), posterior cells were slightly smaller than anterior cells (ratio GFP, dsCycG3s), posterior cells were of the same size as anterior cells (ratio ≈ 1). The increased spread of this box plot suggested that size of en>GFP, dsCycG3s cells was more variable than the one of control en>GFP cells. In wing discs overexpressing CycG (en>GFP, RCG76), posterior cells were smaller than anterior cells (ratio GFP wing disc posterior compartment cells completed G1 phase in 2.3 h, S phase in 4.1 h and G2/M phases in 4.0 h, which is very similar to published results.44 In en>GFP, dsCycG3s posterior compartments, duration of G1 and S phases increased slightly as compared to the control (2.8 h and 4.5 h, respectively) whereas G2/M length was not modified. In en>GFP, RCG76 and en>GFP, RCG23.3 posterior compartments, duration of both G1 and S phases was clearly increased (G1 3.7 h for RCG76 and 3.6 h for RCG23.3; S 8.8 h for RCG76 and 6.8 h for RCG23.3). The weak effect of CycG misregulation on phase G2/M could explain the unmodified mitosis patterns observed in such discs stained with anti-H3pS10 antibody (Fig. 3A”–C”). We concluded that Cyclin G mainly affected G1 and S phases, either blocking G1/S transition and/or slowing down S-phase progression. To clarify this point, we analyzed genetic interactions between CycG and CycE. CycE promotes entry into S phase.44 As a result, overexpression of CycE in posterior compartments (en>GFP, CycE) modified distribution of cells in the cycle by drastically lowering the number of cells in G1 phase and increasing the number of cells in S phase (Sup. Fig. 3D). By counting cells in mitotic clones overexpressing CycE (Sup. Fig. 3D), we determined the length of each phase of the cell cycle (Fig. 5D). G1 phase was very short (0.8 h), in agreement with previous reports.44 Nevertheless, total length of the cell cycle was similar to control (10.5 h), and the cells compensated mainly by extending S phase (6.2 h). We then used the CycE overexpression context to check the effect of CycG on G1 phase, by overexpressing both CycE and CycG in posterior compartment and in mitotic clones (Figs. S3E and 5D). We reasoned that if CycG was blocking G1/S transition, the extended-G1 phenotype due to overexpression of CycG would be rescued by simultaneous overexpression of CycE. Total cycle length of cells overexpressing CycG and CycE was 12.6 h, i.e., in between those of CycG or CycE single overexpression, suggesting that these two proteins could have antagonistic effects on the cell cycle. Furthermore, G1 phase was reduced (1.3 h) as compared to single CycG overexpression, suggesting that Cyclin G and Cyclin E play opposite roles in G1/S transition. The S phase in these

double-overexpressing cells was still longer than in en>GFP control cells (6.6 h vs. 4.1 h). We also jointly overexpressed CycE and inactivated CycG in the posterior compartment (Sup. Fig. 3F and Fig. 5D). Total cycle length of cells overexpressing CycE and inactivating CycG was similar to the one of en>GFP control cells (10.6 h vs. 10.4 h). The shortened-G1 phenotype due to CycE overexpression was maintained (0.8 h), and cells also presented a reduction of S-phase length as compared to cells overexpressing CycE alone (5.1 h vs. 6.2 h). These effects were compensated by an increase of G2 /M length. The last results suggested that inactivation of CycG tended to accelerate S-phase progression. All together, our data support the idea that Cyclin G slows the cell cycle by preventing G1 to S transition and by retarding S-phase progression. Cyclin G interacts with CDK2 and CDK4. We next tried to predict potential protein partners of Cyclin G in control of cell growth and cell cycle. Large-scale two-hybrid screens predicted 82 interactors for Drosophila Cyclin G, among them many transcription factors and cell cycle regulators (Fig. 6A).34,35 We built a Cyclin G interactor network based on these data, which indicated that top interactors could include the cyclin-dependent kinases CDK4 and CDK2, the cyclin-dependent kinase inhibitor Dacapo (DAP), the kinase SAK and Cyclin K (Fig. 6B). The CDK4/Cyclin D complex regulates cell growth during G1 phase47 and the CDK2/Cyclin E complex regulates entry into S phase.44,47,48 Since our data suggested that Cyclin G inhibits cell growth, prevents G1/S transition and slows down S phase, a possible explanation might be that Cyclin G could accomplish these functions by interacting with both CDK4 and CDK2. To test this, we performed co-immunoprecipitations of tagged Cyclin G, CDK4 and/or CDK2 proteins co-expressed in S2 cells (Fig. 6C and D). We observed that FLAG-Cyclin G co-immunoprecipitated with CDK4-EGFP and CDK2-EGFP, corroborating the large-scale screen predictions.


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Discussion Although G-type cyclins are expressed in many normal and cancerous tissues, their role in mammals is still poorly understood. Drosophila has a single Cyclin G coding gene (CycG), simplifying genetic analysis. Here, we investigated its role and present evidence that growth and cell cycle of non-differentiated, proliferating wing imaginal disc cells, are highly dependent upon CycG expression.

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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 5. Consequences of CycG misregulation on the cell cycle. (A) Diagram showing number of cells in Act>GFP control clones. (B) FACS analysis of wing disc cells. Dot plot displaying EdU content (ordinate) vs. DNA content (abscissa) and gates used in all experiments. (C) Diagram showing percentages of cells in G1, S and G2/M phases for anterior and posterior compartments of control wing discs (en>GFP). Number of experiments (n) is indicated below. (D) Diagrams showing cell cycle duration for each genotype. G1, S and G2/M-phase lengths were determined using, on the one hand, the total duration of the cell cycle as measured in mitotic clones and, on the other hand, the percentages of posterior compartment cells in each phase of the cell cycle (see A and C for GFP controls, and Sup. Fig. 3 for other genotypes). The long cell cycle of CycG overexpressing cells (RCG76, RCG23.3) was due to longer G1 and S phases. The short G1 phase and the long S phase of cells overexpressing CycE were partly rescued by overexpression and inactivation of CycG, respectively.

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.31 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.49 Both Cyclin G1 and G2 are degraded by the ubiquitin-proteasome pathway.30,50 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.15 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 (not shown) and further experiments will decipher its phosphorylation pattern. 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.51-54 This suggests that one of

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Figure 6. Cyclin G interactors. (A) Large-scale two hybrid screens34,35 using Cyclin G as bait detected 82 potential interactors (http://thebiogrid.org). These included a large proportion of transcription factors and cell cycle regulators (16% and 12%, respectively). (B) Network formed by Cyclin G and its potential binding partners as predicted by the original two-hybrid screens. 34,35 Interaction between the chromatin factor Corto and Cyclin G was not from these screens but from our previous work. 31 Top-partners shared more than 16 other partners with Cyclin G, and are represented by pink circles. Their connections with Cyclin G are represented by red lines. These top-partners are the cyclin-dependent kinases CDK4,60 and CDK2,61 the cyclindependent kinase inhibitor Dacapo (DAP),58,59 the kinase SAK,57 and Cyclin K.7 Cyclin G partners that shared between 16 and 3 other partners with Cyclin G are represented with filled blue circles. Cyclin G partners that shared less than 3 partners with Cyclin G are represented by unfilled blue circles. Green circles represent proteins that are not Cyclin G direct partners but that interact with at least 16 partners of Cyclin G. These secondary partners are the kinase AKT1,68 the cyclin-dependent kinase CDK5,27 Cyclin D,69 the kinase activator PLU,70 and the transcriptional repressor E2F2.71 (C and D) Co-immunoprecipitation experiments. Plasmids allowing expression of CDK2-EGFP or CDK4-EGFP and FLAG-Cyclin G were co-transfected into S2 cells. After 48 h, extracts were prepared. Immunoprecipitations with anti-EGFP antibody, anti-FLAG antibody and anti-Myc antibody (negative control) were performed simultaneously. 5% of input were loaded onto the gel. IP: immunoprecipitated material (50%); Spnt: supernatant. Top: anti-EGFP western blot; bottom: anti-FLAG western blot. Asterisks represent bands nonspecifically recognized by the anti-FLAG antibody. (C) Co-immunoprecipitations between CDK2 and Cyclin G. (D) Co-immunoprecipitations between CDK4 and Cyclin G.

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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 conversely 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.55 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.47 In addition, overexpression of Rbf, which slows all phases of the cell cycle, produces large cells.44 Hence, Cyclin G, which both limits cell size and decreases proliferation, has a very particular behavior. On the one hand, specific cell cycle regulators, such as RBF, E2F and Cyclin E, affect growth indirectly, via their effect on the cell cycle.47 On the other hand, growth promoters like Myc,46 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.51-56 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. 82 potential Drosophila Cyclin G interactors were found in genome-wide two-hybrid screens. Among them, the transcriptional regulator Cyclin K,7 the mitotic kinase SAK,57 the CDK inhibitor Dacapo (DAP),58,59 CDK4,60 and CDK2,61 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.

aegypti, XP_001649965.1 (476 AA); Culex quinquefasciatus, XP_001844277.1 (477 AA); Hymenoptera: Nasonia vitripennis, XP_001600259.1 (363 AA); Apis mellifera, XP_392746.3 (346 AA); Lepidoptera: Bombyx mori, BGIBMGA001790-PA (361 AA); Coleoptera: Tribolium castaneum, XP_968026.1 (308 AA). Only sequences described as complete were used. Multiple sequence alignments were performed using ClustalW2 at http://www.ebi. ac.uk with standard comparison parameters. PEST motifs were identified by the epestfind algorithm at http://emboss.bioinformatics.nl. For Cyclin G sequences with short N-ter regions, which were not deduced from cDNA but from genomic data, we checked that 6 kb of genomic DNA upstream from the reported transcription start could not encode peptides similar to dipteran N-ter regions. Plasmid constructs. The full-length CycG cDNA was amplified from pJG-CycG31 using primers CycGF: 5'-cac cat gtc tgt ccc agt acg cta ctc ctc-3' and CycGR: 5'-cct aga acg cag gcc atc gtc-3'. The resulting amplicon was introduced into pENTR/D-TOPO® (Invitrogen), then transferred through LR-recombination into T. Murphy’s vectors pPRW or pPW (https://dgrc.cgb.indiana.edu/vectors). Full-length cdk2 and cdk4 cDNA were amplified by PCR from S2 cell cDNAs. For cdk2, we used primers 5'-CAC CAT GAC CAC CAT TCT AGA TAA CTT TC-3' and 5'-CGA AGC GCA GAT TGC GCC GAG CAG and for cdk4, primers 5'-CAC CAT GTC ATA TGT ACG CCA GCT GAA G-3' and 5'-GTG CTA ATG AAA GTC GTC CTT GGG GAT-3'. PCR products were cloned into pENTR/TOPO® vector (Invitrogen) and transferred by LR-recombination into T. Murphy’s vectors pAWG (cdk2 and cdk4) and pAFW (CycG). Fly stocks. Flies were raised on standard yeast-cornmeal medium at 25°C. Lines allowing CycG overexpression were generated by standard P-element mediated germline transformation.62 Lines RCG23.3 and RCG76, which express mRFP-tagged Cyclin G, resulted from an insertion on the second or third chromosome, respectively. Line CG1-1, which expresses an untagged Cyclin G, resulted from an insertion on the second chromosome. This line was specially used for immunostainings. The transgenic lines UAS::dsCycG2 and UAS::dsCycG3s were used to inactivate CycG by RNAi.31 Lines UAS::CycE,63 UAS::p35 (BL7298), da::Gal4,64 GMR::Gal4 (BL-9146) and ey::Gal4 (BL5535) were from the Bloomington Stock Center. The [en::Gal4, UAS::mitoGFP/CyO] line65 was used for FACS analyses and the [hs::FLP; Act>CD2>Gal4, UAS::GFP/CyO]44 was used to generate mitotic clones. Phenotype ratings and statistics. For weight measurements, ten da::Gal4/+ virgin females were crossed with ten males, either RCG76/+ or UAS::dsCycG2/+, at 25°C. Flies were transferred each day into new tubes. The progeny was collected at eclosion, aged 3 days in new tubes, sorted by eye color and weighed one by one after freezing. One hundred flies were weighed for each genotype. Genitalia of da>RCG76 and da>dsCycG2 males were dissected as described in reference 66. For ommatidia counting, ten ey::Gal4/+ or GMR::Gal4 virgin females were crossed with ten males, either RCG76/+ or UAS::dsCycG2/+, at 25°C. Flies were transferred every second


Materials and Methods Bioinformatics. Insect Cyclin G-like sequences were identified by BLASTP using matrix BLOSUM62 at http://blast/ncbi/ nlm/nih/gov. Species, accession number and amino acid number (AA) of sequences compared were: Homo sapiens Cyclin G1 AAC41977.1, (295 AA); Homo sapiens Cyclin G2 AAC41978.1, (344 AA); Diptera, Drosophila: D. melanogaster, NP_524609.2, isoform A (566 AA); D. sechellia, XP_002043719.1 (569 AA); D. erecta, XP_001981070.1 (574 AA); D. yakuba, XP_002099524.1 (570 AA); D. ananassae, XP_001358263.2 (581 AA); D. pseudoobscura, XP_001964584.1 (566 AA); D. mojavensis, XP_001998859.1 (578 AA); D. virilis XP_002053891.1 (578 AA); D. willistoni, XP_002069809.1 (564 AA); D. grimshawi, XP_001989712.1 (549 AA); Diptera, Glossinidae: G. morsitans morsitans, ADD20326 (587 AA); Diptera, Culicidae: Aedes


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day into new tubes. Individuals were collected and fixed in paraformaldehyde 3.7% overnight at 4°C. They were deshydrated in ethanol and prepared for scanning electron microscopy using standard methods. For each genotype, ommatidia of ten eyes were counted from SEM images. Analysis of ommatidum size was performed by counting, for at least ten eyes, the number of ommatidia in a 5,500 μm2 square localized in the center of the eye and dividing this area by the number of ommatidia, as already described in references 41 and 42. Measure of cell size in adult wings was performed in 256 x 256 pixel squares displayed in the L4-L5 intervein region just behind the posterior cross-vein. Thresholding was performed and epidermal hairs were counted automatically using ImageJ software. The area was shifted twice along the proximo-distal axis and counting repeated. Numbers of cells in the three regions were averaged. Statistical analyses were performed on the web sites www.anastats.fr and www.u707.jussieu.fr/biostatgv/. Student t-tests were performed for samples of more than 30 individuals. Otherwise, Mann-Whitney U-tests were used. Immunostaining of wing discs. Third instar larvae wing discs were dissected and fixed in 250 μl of 3.7% paraformaldehyde for 20 min at room temperature, then washed three times for 5 min each in 1x PBS. They were pre-incubated for 30 min in incubation buffer (1% BSA, 0.1% Triton, PBS 1x) and incubated overnight at 4°C with primary antibody diluted at 1:400 in incubation buffer [Guinea Pig Anti-Cyclin G,31 rabbit antiH3pS10 (Upstate 06-570), rabbit anti-cleaved Caspase 3 (Cell Signalling Technology, 9661)]. Samples were washed five times for 30 minutes each in 2 ml of PBT (Triton 0.1%, PBS 1x) and blocked for 30 minutes with incubation buffer. Discs were then incubated with secondary antibody [anti-mouse Alexa 568 or anti-Guinea pig Alexa 568 (Molecular Probes)] diluted 1:1,000 in incubation buffer, for 3 hours at room temperature. Samples were washed for 30 minutes in 2 ml of PBT and four times for 15 minutes each in 2 ml of PTW (Tween 0.1%, PBS 1x). They were mounted in Mowiol with DABCO as anti-fading reagent. Discs were observed with a Nikon Eclipse 80i epifluorescence microscope equipped with a Nikon digital camera (DXMxm1200C). Proliferation rate measurements. Gal4 expressing clones were induced by the FRT flip-out method using the [hs::FLP; Act>CD2>Gal4, UAS::GFP/CyO] line.67 Larvae were heatshocked for 1 h at 37°C at 72 h ± 2 h After Egg Laying (AEL). Imaginal wing discs were dissected at 120 h ± 2 h AEL and fixed with 3.7% paraformaldehyde in 0.1% Triton-PBS for 20 minutes at room temperature. DNA was stained with DAPI (1 μg/mL). Wing discs were mounted in Mowiol with DABCO as anti-fading reagent. Clones were observed as described above. About 100 clones were counted for each genotype. Cell doubling time was obtained with the formula (log2/logN)(h) where h is the age of the clone and N the median cell number per clone.46 Flow cytometry of wing imaginal disc cells. Flies were raised on yeast-cornmeal medium containing 0.25% bromophenol blue at 25°C. Wandering third-instar larvae with almost no food in the gut and no everted spiracles were dissected. A single wing

imaginal disc was dissected per larva. Wing imaginal disc cells were dissociated as described in reference 44. Discs (20 for cell size and 75 to 100 for cell cycle analyses) were transferred to a 6 ml polystyrene tube containing 450 μL of trypsin-EDTA (Sigma, T-4174) and 50 μL of PBS. Cells were dissociated for 2 hours at 25°C with gentle shaking. Dissociation was stopped with 10% fetal calf serum. Cells were passed through a 50 μm CellTrics® filter and sorted for the presence of GFP on a BD BioSciences InfluxTM 500 cell sorter at 4°C. For cell cycle analysis, wing discs were incubated in Schneider medium containing 10% fetal calf serum and 10 μM EdU for 20 minutes at 25°C. Discs were washed three times with cold PBS before cell dissociation and sorting. GFP positive or negative cells were fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. EdU detection was performed using the Click-iT TM EdU Alexa Fluor® 647 Flow Cytometry assay kit as indicated by the supplier (Invitrogen). Cells were centrifuged at 1,700 g during 10 min at 4°C. They were rinsed twice with permeabilization solution supplemented with 1% BSA and kept overnight at 4°C. After centrifugation, cells were resuspended in 500 μl of permeabilization solution supplemented with 400 μg/ml of RNAse and 50 μg/ml of propidium iodide. DNA amount was analyzed with a Beckman Coulter CyAn ADP LX flow cytometer. Cell cycle was analyzed with the Summit V5 software. Transfections and co-immunoprecipitations. 4 x 106 S2 cells were transfected in 25 cm2 flasks using the Effecten® Transfection reagent kit as indicated by the supplier (Qiagen). Co-transfections were performed using 1 μg of each plasmid at a 1 to 10 DNA/Effecten ratio. Proteins were extracted 48 h after transfection. Cells were collected and washed twice with PBS. They were fixed in 1% paraformaldehyde on ice for 10 min. Fixation was stopped with 140 mM glycine. Cells were washed twice and resuspended in 450 μl of ELB Buffer [150 mM NaCl, 0.1% Igepal, 5 mM EDTA, 50 mM Hepes pH 7.5, 0.5 mM DTT, 10 mM NaF, 1 mM NaV, 1 mM PMSF, one tablet of complete, EDTA free, protease inhibitors (Roche Diagnostics)]. Cells were sonicated for 12 cycles (5 s ON, 5 s OFF) on medium intensity in a Bioruptor® sonicator (Diagenode). Protein concentrations were measured with Bradford assay. For co-immunoprecipitations, 300 μg of protein extract were incubated with either 3 μg of either anti-FLAG (SigmaAldrich, F-3165), anti-GFP (Roche, 11814460001) or anti-Myc (Genscript, A00704) overnight at 4°C. Immune complexes were then incubated with 30 μL of protein G magnetic beads (Ademtech) for 3 hours at 4°C. Supernatants were kept and beads were washed five times with ELB buffer containing 0.5% Igepal. Beads were resuspended in 40 μL of Laemmli buffer. 15 μg of proteins (Input), 20 μL of supernatant and 20 μL of magnetic beads were boiled and loaded on a 12% polyacrylamide gel. After transfer, filters were incubated with the same antibodies.


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Acknowledgements

The authors wish to thank Dr. I. Guénal, Dr. A. Nagel, Pr. A. Preiss, Pr. J. Silber, Dr. T. Murphy and the Bloomington Stock Center for reagents and flies, Dr C. Antoniewski, Dr. A. Audibert, Dr. V. Debat, Dr. J. Montagne, Dr. M. Gho and members of the

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Chromatin and Development team for fruitful discussions, Dr S. Ronsseray for statistics, V. Steeghs for help in cdk2 and cdk4 cDNA sub-clonings, A. Coléno-Costes for RT-qPCR, V. Ribeiro for excellent technical assistance, the Flow Cytometry facilities of IJM and IFR83 (Paris, France) for access and technical support in cytometry analyses. This work was supported by grants References 1. Errico A, Deshmukh K, Tanaka Y, Pozniakovsky A, Hunt T. Identification of substrates for cyclin dependent kinases. Adv Enzyme Regul 2010; 50:375-99. 2. Minshull J, Pines J, Golsteyn R, Standart N, Mackie S, Colman A, et al. The role of cyclin synthesis, modification and destruction in the control of cell division. J Cell Sci Suppl 1989; 12:77-97. 3. Adams PD. Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochim Biophys Acta 2001; 1471:123-33. 4. Satyanarayana A, Kaldis P. Mammalian cell cycle regulation: Several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 2009; 28:2925-39. 5. Bondos SE, Tan XX, Matthews KS. Physical and genetic interactions link hox function with diverse transcription factors and cell signaling proteins. Mol Cell Proteomics 2006; 5:824-34. 6. Coqueret O. Linking cyclins to transcriptional control. Gene 2002; 299:35-55. 7. Edwards MC, Wong C, Elledge SJ. Human cyclin K, a novel RNA polymerase II-associated cyclin possessing both carboxy-terminal domain kinase and Cdk-activating kinase activity. Mol Cell Biol 1998; 18:4291-300. 8. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loopspecific binding to TAR RNA. Cell 1998; 92:451-62. 9. Leclerc V, Tassan JP, O’Farrell PH, Nigg EA, Léopold P. Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II. Mol Biol Cell 1996; 7:505-13. 10. Fisher RP, Morgan DO. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell 1994; 78:713-24. 11. Brinkkoetter PT, Pippin JW, Shankland SJ. Cyclin I-Cdk5 governs survival in post-mitotic cells. Cell Cycle 2010; 9:1729-31. 12. Edelheit S, Meiri N. Cyclin S: A new member of the cyclin family plays a role in long-term memory. Eur J Neurosci 2004; 19:365-75. 13. Tetzlaff MT, Bai C, Finegold M, Wilson J, Harper JW, Mahon KA, et al. Cyclin F disruption compromises placental development and affects normal cell cycle execution. Mol Cell Biol 2004; 24:2487-98. 14. Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 1996; 86:263-74. 15. Horne MC, Goolsby GL, Donaldson KL, Tran D, Neubauer M, Wahl AF. Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue-specific and cell cycle-regulated expression. J Biol Chem 1996; 271:6050-61. 16. Bennin DA, Don AS, Brake T, McKenzie JL, Rosenbaum H, Ortiz L, et al. 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 2002; 277:27449-67. 17. Okamoto K, Beach D. Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J 1994; 13:4816-22. 18. Tamura K, Kanaoka Y, Jinno S, Nagata A, Ogiso Y, Shimizu K, et al. Cyclin G: a new mammalian cyclin with homology to fission yeast Cig1. Oncogene 1993; 8:2113-8.

from CNRS and UPMC. F. Faradji was supported by a fellowship from MESR. Note

Supplemental materials can be found at: www.landesbioscience.com/journals/cc/article/14959

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Cell Cycle

Volume 10 Issue 5