| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
,3,4
* Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia
Department of Genetics/Centre for the Molecular Genetics of Development and Department of Molecular Biosciences, University of Adelaide, Adelaide, 5005, South Australia, Australia
5 Corresponding author: Peter MacCallum Cancer Centre, 7 St. Andrews Pl., East Melbourne, Victoria, 3002, Australia.
E-mail: helena.richardson{at}petermac.org
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
|---|
PS3-integrin—cell adhesion), phyllopod (signaling), dEB1 (microtubule-binding protein—cytoskeletal), and moira (chromatin remodeling). These interactions of the cytoarchitectural suppressor gene, scribble, with cell adhesion, signaling, cytoskeletal, and chromatin remodeling genes, suggest that these genes may act in a common pathway to negatively regulate cyclin E or S-phase entry.
The key players in the regulation of the G1- to S-phase transition are highly conserved between mammals and Drosophila (EDGAR and LEHNER 1996). Genetic analysis in Drosophila has shown that both Cyclin E and Cyclin D act to regulate Drosophila Rb (Rbf; XIN et al. 2002). Drosophila Cyclin E is essential for the G1- to S-phase transition during embryogenesis and is downregulated in G1-arrested cells (RICHARDSON et al. 1993, 1995; KNOBLICH et al. 1994). In contrast, Drosophila Cyclin D primarily acts to regulate cell growth (increase in cell mass) and through the coupling of cell growth to G1- to S-phase progression, stimulates cell proliferation (DATAR et al. 2000; MEYER et al. 2000). As in mammalian cells, Drosophila Cyclin E/Cdk2 activity is regulated via a homolog of p21CIP1, Dacapo, which is required during exit into a terminal G1 arrest prior to differentiation (DE NOOIJ et al. 1996; LANE et al. 1996). Degradation of Cyclin E protein also plays an important role in limiting cell proliferation, and mutations in the ago gene (encoding a homolog of Cdc4, an F-box-containing component of the G1 phase ubiquitin ligase) result in increased Cyclin E protein stability and excessive cell proliferation during eye development (MOBERG et al. 2001). However, relatively little is known about the upstream signals that regulate Drosophila cyclin E transcription or the downstream targets of Drosophila Cyclin E/Cdk2 that lead to the initiation of DNA replication within a whole-animal context.
The developing Drosophila eye presents an ideal system to study the relationship between cell proliferation and differentiation. The eye develops from a single cell layer epithelium at the third larval instar stage, where a wave of morphogenesis moves from the posterior to the anterior of the eye imaginal disc (THOMAS and WASSARMAN 1999). Associated with this wave of morphogenesis is the morphogenetic furrow (MF), where the cell cycle becomes coordinated with differentiation. Within and anterior to the MF cells are arrested in G1, while posterior to the MF a subset of cells begins to differentiate into the photoreceptor cell preclusters and the surrounding cells enter a synchronous S phase, after which a subset of these cells undergoes mitosis. Hedgehog signaling has been shown to be important for Cyclin D and Cyclin E expression in this post-MF cell division (DUMAN-SCHEEL et al. 2002). Perturbations to the organized arrangement of cell division in the developing eye by, for example, ectopic expression of S-phase inducers, Cyclin E or E2F/Dp, or the negative cell cycle regulators, human p21 or Drosophila Rbf, result in defects in eye development leading to disorganized or rough adult eyes (DE NOOIJ and HARIHARAN 1995; RICHARDSON et al. 1995; ASANO et al. 1996; DU et al. 1996; XIN et al. 2002). The eye phenotypes resulting from overexpression of Cyclin E, E2F/Dp, or Rbf in the posterior differentiating cells of the eye disc have been used as the basis of genetic screens of EMS-mutagenized flies to identify dominant modifiers, revealing novel regulators of the cell cycle (STAEHLING-HAMPTON et al. 1999; BOULTON et al. 2000; LANE et al. 2000; DUMAN-SCHEEL et al. 2002).
A hypomorphic mutation in Drosophila cyclin E, DmcycEJP, which results in a rough eye phenotype, has provided an opportunity to carry out genetic screens to identify novel genes involved in the regulation of cyclin E expression and function. We have previously shown that DmcycEJP exhibits a rough eye phenotype due to a reduction in Cyclin E levels and S phases in the developing eye and that this phenotype is sensitive to the dosage of G1- to S-phase genes known to interact with Cyclin E (SECOMBE et al. 1998). This article reports the results of mutagenesis and deficiency screens to identify genes that dominantly modify the DmcycEJP rough eye phenotype and presents initial characterization of DmcycE suppressor genes, predicted to act as negative regulators of Cyclin E and/or the G1- to S-phase transition.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
|---|
100 in each) and treated with 4000 rad of X rays in a CIS Biointernational X-ray machine using a 137Cs radiation source (activity 3400 Ci). Mutagenized flies were then allowed to recover for 4 hr with food before being added to 3-day-old virgin females. The flies were turned into new bottles after 2 days and removed after 4 days. EMS mutagenesis was carried out as previously described (GRIGLIATTI 1998). For both EMS and X-ray mutageneses, DmcycEJP males isogenic on the second and third chromosomes were mutagenized and crossed en masse to b DmcycEJP females. The progeny from this cross were scored for dominant modification of the DmcycEJP rough eye phenotype. In addition, F1 progeny were scored for black-bodied flies to estimate the mutation frequency. From the number of black mutant flies obtained, we calculated that the X-ray mutagenesis frequency was 2.3 x 10–3 and the EMS mutagenesis frequency was 3 x 10–4, which are within the ranges described by previous studies (GRIGLIATTI 1998). Flies selected as having enhanced or suppressed eyes were crossed to a DmcycEJP strain to ensure that the modification of the DmcycEJP rough eye phenotype observed initially was heritable and reproducible and then crossed to second or third chromosome balancers to generate stocks. To simplify the screen and stock generation, only interactors that mapped to the second or third chromosome were kept. Once a stock was generated, flies were crossed to a DmcycEJP strain to ensure that the enhancer or suppressor mutation segregated away from the balancer chromosome. Any mutations that resulted in a dominant eye roughening in the absence of DmcycEJP were discarded.
For complementation analysis, inter se crosses were carried out between all lethal alleles on each chromosome and allele combinations that resulted in trans-heterozygous lethality to >98% were considered to be within the same gene group.
Genetic mapping of second chromosome genes was carried out using either the b1, cn1, bw1 or al1, dpov1, b1, pr1, c1, px1, sp1 multiply marked chromosomes, while third chromosome genes were mapped using the ru1, h1, th1, st1, cu1, sr1, es, ca1 multiply marked chromosomes. The deficiency kit (Bloomington Stock Center) was used for deficiency mapping. Chromosome cytology of third chromosome suppressors was analyzed after Giemsa staining of polytene chromosomes prepared from non-Tubby larvae from a cross of the suppressor (over TM6B) to Canton-S.
DmcycE interactions:
To test X and third chromosome deficiencies for interaction with DmcycEJP, stocks were generated using balancers that contained the deficiency chromosome and DmcycEJP and the stock was crossed to homozygous DmcycEJP flies and progeny containing the deficiency and DmcycEJP were examined. To examine second chromosome candidate genes for interaction with DmcycEJP, the candidate gene mutant was recombined onto a marked DmcycEJP chromosome (using a dp, b, DmcycEJP, cn, bw chromosome) balanced over CyO and then crossed to a homozygous DmcycEJP stock and non-Curly flies were examined. X or third chromosome candidate genes were tested for interaction with DmcycEJP after generating stocks containing the candidate gene mutant and DmcycEJP, by crossing to b, DmcycEJP, bw flies and examining non-FM7 or non-TM6B flies. The eyes of at least 50 flies of the appropriate genotype were examined and compared with b, DmcycEJP, bw/DmcycEJP flies.
Phenotypic analysis of cyclin E suppressors:
To determine whether a suppressor was acting at the level of S-phase regulation, second chromosome modifiers were crossed to the Curly-Tubby (Cy-Tb) second chromosome balancer, which carries the Tubby dominant larval marker, and were crossed to homozygous DmcycEJP flies, and non-Tubby larvae were selected for examination of S phases by BrdU labeling. Third chromosome modifiers were balanced over TM6B (marked by Tubby) and crossed to homozygous DmcycEJP flies and non-Tubby larvae were picked for BrdU labeling. BrdU labeling was carried out as described previously (SECOMBE et al. 1998). Cyclin E antibody staining was carried out using a polyclonal Cyclin E antibody raised in rats, as previously described (CRACK et al. 2002). To determine whether the stage of lethality was before or after the third instar larval stage, each suppressor stock balanced over Cy-Tb or TM6B was examined for the presence of any homozygous modifier (non-Tubby) larvae that survived to or beyond the third larval instar stage. Scanning electron microscopy of adult eyes was carried out as previously described (SECOMBE et al. 1998).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
|---|
A total of 20 suppressor regions and 16 enhancer regions on the X and third chromosomes were identified by generating homozygous DmcycEJP flies that were also heterozygous for the deficiency chromosome (Table 1). Consistent with results described previously (SECOMBE et al. 1998), deficiencies removing genes already known to interact with DmcycEJP such as RBF, roughex, E2F1, and string behaved as expected (Table 1), with the exception of Df(3R)vin2 that removes cyclin A. We have previously shown that cyclin A mutants dominantly enhance DmcycEJP phenotype (SECOMBE et al. 1998), while Df(3L)vin2 and the overlapping deficiency Df(3L)vin5 suppressed the DmcycEJP rough eye phenotype. The most likely explanation for this is that these deficiencies also delete a dose-sensitive suppressor of DmcycEJP (Table 1). Suppression of DmcycEJP was also observed with a deficiency (of the region 63F4–64C15) removing the Drosophila cdc4(ago) gene, which encodes an F-box protein of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex involved in Cyclin E protein degradation and can dominantly suppress the DmcycEJP rough eye phenotype (MOBERG et al. 2001).
|
|
Tumor suppressors and oncogenes:
From the DmcycEJP deficiency screen, a number of regions that showed suppression removed Drosophila tumor suppressor genes, while many that enhanced removed potential oncogenes (Table 1). These candidate genes, as well as other potential oncogenes or tumor suppressors, were specifically tested where possible (Table 2).
|
In addition, a deficiency removing fused (17A1–18A2), an effector of the Hh pathway, showed enhancement of DmcycEJP. Although we have not specifically tested fused to determine whether it represents the interacting gene, this interaction is consistent with the recent observation that the Hedgehog (Hh) pathway acts to upregulate cyclin E transcription in the eye (DUMAN-SCHEEL et al. 2002) and that upregulation of the Hh pathway is oncogenic in mammals (WETMORE 2003). To explore this further, we analyzed the effect of halving the dose of Hh or patched (a negative regulator of the Hh receptor, Smoothened) on the DmcycEJP rough eye phenotype (Table 2; data not shown). As expected, Hh alleles dominantly enhanced while patched alleles dominantly suppressed DmcycEJP, consistent with a role for the Hh pathway in positively regulating cyclin E and inducing S-phase entry. We also examined other signaling pathways for dominant interactions with DmcycEJP (Table 2; data not shown). In mammalian cells, the EGF receptor, the Wnt/Wingless, and Notch signaling pathways have a growth and/or cell cycle stimulatory role in many cells and can be oncogenic when upregulated (ALLENSPACH et al. 2002; CHANG et al. 2003; GILES et al. 2003). Consistent with the interaction of Ras85D with DmcycEJP, loss-of-function mutations in the EGF receptor (Egfr) enhanced DmcycEJP while gain-of-function mutations (Ellipse) suppressed. Other downstream components of the Egfr-Ras pathway also interacted with DmcycEJP in a manner consistent with the Egfr having a positive role in regulating Cyclin E and entry into S phase (Table 2). Reducing the dose of Notch, however, showed no effect on the DmcycEJP phenotype. Interestingly, halving the dosage of wingless (wg), disheveled (encoding a Wg-signaling mediator), and armadillo (arm; encoding a ß-catenin homolog, the Wg signaling transcriptional effector) resulted in suppression of DmcycEJP. In contrast, halving the dosage of axin (encoding an inhibitor of Wg signaling) enhanced DmcycEJP. While contrary to the expected role of the Wg pathway, an inhibitory proliferative function for Wg has been observed in the zone of nonproliferation in the third instar wing pouch (JOHNSTON and EDGAR 1998; JOHNSTON et al. 1999; JOHNSTON and SANDERS 2003). Similarly, we have previously shown that the Dpp (TGFß homolog), although growth stimulatory earlier in development, acts to negatively regulate cell cycle progression in the third instar eye imaginal disc and mutants that disable the Dpp signaling pathway dominantly suppress DmcycEJP (HORSFIELD et al. 1998).
Several DmcycEJP suppressor regions on the X chromosome and on the third chromosome remove known Drosophila tumor suppressor genes. Specific mutations were available for some of the candidate genes encoding tumor suppressors and were therefore tested for a genetic interaction with cyclin E. Specific mutations in Ribosomal protein S6 (RpS6 air8), the best candidate for the cyclin E suppressor in the 19A–20F region, were tested and shown to suppress the DmcycEJP rough eye phenotype (Table 2; not shown). Mutations in RpS6 were identified as loss-of-function mutations that result in overproliferation of larval hematopoietic tissues and give rise to variable melanotic tumor phenotypes (GATEFF et al. 1996). RpS6 is phosphorylated in response to mitogen stimulation and phosphorylated RpS6 is preferentially incorporated into polysomes, resulting in an increased rate of translation of a subset of transcripts (AMALDI and PIERANDREI-AMALDI 1997; MARTIN and BLENIS 2002). However, disruption of Drosophila S6 kinase leads to reduced growth and smaller flies and mutation of the upstream kinase Tor causes cell cycle arrest that can be rescued by cyclin E expression (ZHANG et al. 2000). Furthermore, conditional knockout of RpS6 in mice results in a specific block in cyclin E expression (VOLAREVIC et al. 2000). Given this role for RpS6 in mammalian cells, it is unknown how halving the dosage of RpS6 leads to the suppression of DmcycEJP; however, it is consistent with the tumor suppressor function of Drosophila RpS6.
Other Drosophila tumor suppressors were tested for interaction with DmcycEJP (Table 2), and those that showed suppression were hop-air (an activating mutation in JAK kinase), consistent with a role for Drosophila Jak in cell proliferation and that Cyclin D-Cdk4 and Cyclin E-Cdk2 bind and regulate STAT92E protein stability (CHEN et al. 2003); fat (encoding an atypical Cadherin involved in planar polarity); expanded (encoding a FERM domain protein involved in actin remodeling); and the unidentified air7, air10, and air16 (GATEFF et al. 1996; DE LORENZO et al. 1999). The Drosophila E-cadherin gene, shotgun (shg; TEPASS et al. 1996; UEMURA et al. 1996), when halved in dosage, was also shown to slightly suppress DmcycEJP. In contrast, lethal (3) malignant brain tumor [l(3)mbt] and hyperplastic discs (hyd; GATEFF et al. 1996; DE LORENZO et al. 1999), which were considered candidates for the regions 97A–98A2 and 85D8–85E13, respectively (Table 1), did not modify the DmcycEJP phenotype when specific alleles were tested (Table 2; data not shown). Taken together these data suggest that there are specific pathways that show rate-limiting effects on Cyclin E and thereby entry into S phase, in the eye imaginal disc.
Identification of cyclin E interactors using a mutagenic dominant modifier screen:
As described above, screening for dominant genetic modifiers of DmcycEJP using deficiencies and candidate gene approaches has revealed some interesting interactors. However, this approach is limited in that the deficiencies may remove more than one modifier, confounding the identification of interacting genes. For these reasons, an unbiased genetic screen for DmcycEJP modifiers using mutagenized flies was carried out, to generate specific modifier mutations that could be further characterized. To randomly generate mutations that could then be examined for their effect on the DmcycEJP phenotype, we utilized X-ray mutagenesis, which causes deletions and chromosomal rearrangements (SANKARANARAYANAN and SOBELS 1976) that are expected to aid in the identification of the modifier, and EMS mutagenesis, which causes nucleotide substitutions resulting in missense or nonsense mutations (LIFSCHYTZ and FALK 1968). For the X-ray mutagenesis, 39,234 F1 flies were screened for modification of the DmcycEJP rough eye phenotype and stocks of 104 suppressors and 59 enhancers that consistently modified the DmcycEJP phenotype on the second or third chromosomes were generated (summarized in Table 3). For the EMS mutagenesis a total of 15,049 F1 flies were screened and 29 suppressors and 54 enhancer mutations on the second or third chromosomes were isolated (Table 3).
|
|
|
Basic characterization was then carried out on second chromosome multimember complementation groups and most of the third chromosome suppressors (summarized in Tables 4 and 5). We determined whether the suppression of DmcycEJP was occurring at the level of S phases during eye development (shown for representatives of the identified suppressors in Figure 1). In all cases examined, there was a significant increase in the size of the eye disc as well as in the number of S phases in the anterior and the post-MF S-phase band. Thus all suppressors tested act to suppress DmcycEJP by increasing S phases in the normal pattern. The stage of lethality of the homozygous modifier mutation was also determined by counting the number of hatched embryos and examining whether homozygous third instar larvae were present (Tables 4 and 5). This analysis led to the observation that group 2.1 and 63S15 homozygotes died as overgrown larvae, a phenotype that occurs with Drosophila neoplastic tumor suppressors (GATEFF et al. 1996; DE LORENZO et al. 1999; see below).
Mapping and identification of DmcycEJP suppressors:
The cytological location of the lethal mutation for the complementation groups and some of the single alleles was determined by crossing suppressors to the deficiency collection (Bloomington Stock Center). In addition, a crude map position was determined for most of the third chromosome interactors and some of the second chromosome interactors by genetic mapping of the DmcycEJP suppressor mutation. In all cases tested, the map location of the suppressor by genetic mapping was consistent with the map location of the lethal by deficiency mapping. In some cases, chromosome cytology was examined to map aberrations (Tables 4 and 5). Knowledge of the location of the modifier gene then enabled likely candidate genes to be investigated by testing mutant alleles, where available, for failure to complement the modifier mutant.
This strategy enabled the identification of 5 of the 13 second chromosome (2.1, 2.2, 2.5, 2.11, and 2.12) and 6 of the 20 third chromosome suppressors (3.5, 2S1, 35S1, 43S2, 63S15, and 65S19; summarized in Table 4). Of the remaining groups, although map positions were well defined for 7 of 13 of the second (2.3, 2.4, 2.7, 2.8, 2.9, 2.10, 2.13) and 8 of 20 third chromosome suppressor groups (3.1, 3.2, 3.4, 1S3, 13S1, 59S9, 59S18, 68S10), and available candidate gene alleles were tested for each of the suppressor genes, the identity of the suppressors is not yet known (Tables 5 and 7). In these cases, it is likely that these suppressor mutations define novel genes. For two of the suppressor groups (2.6 and 3.3) and five of the single alleles (20S1, 42S12, 42S33, 43S1, 47S8) a precise location for the suppressor was not determined, since none of the available deficiencies failed to complement the suppressor. In these cases the lethal mutation must map to a region not covered by the deficiency collection. A brief description of the identification of the more precisely localized suppressors is detailed below and summarized in Tables 4 and 5.
The identified suppressors:
By complementation tests to known gene alleles the identities of five second chromosome suppressors (2.1, 2.2, 2.5, 2.11, 2.12) and six third chromosome suppressors (3.5, 2S1, 35S1, 43S2, 63S15, 65S19) were revealed. These suppressor genes fall into the functional groups of chromatin remodeling and transcription factors (four genes), signaling (two genes), cytoskeletal (one gene), cell adhesion (two genes), and neoplastic tumor suppressors (two genes). Specific details on the verification and characterization of these suppressors are discussed under these functional groupings (summarized in Table 4).
Chromatin remodeling and transcription factor genes:
3.5 . (Brahma):
3.5 was mapped to 71F1–72D1 (Table 4) and alleles of brahma, a SWI2 homolog, encoding a component of the SWI/SNF chromatin-remodeling complex (PAPOULAS et al. 1998), failed to complement both 3.5 alleles. Furthermore, Df(3R)brm11 (71F1–4; 72D1–10) was identified as a dominant suppressor of DmcycEJP in the screen of third chromosome deficiencies (Table 1). Consistent with suppressor 3.5 being brahma, we showed that previously isolated alleles of brahma also dominantly suppressed DmcycEJP (BRUMBY et al. 2002).
35S1 (Moira):
35S1 was mapped to 89A11–89B10 (Table 4). Candidate mutants in this region were tested for allelism with 35S1, and alleles in moira, a SWI3 (BAP155) homolog, failed to complement 35S1. Consistent with the suppressor 35S1 being moira, we demonstrated that previously isolated alleles of moira also dominantly suppressed DmcycEJP (BRUMBY et al. 2002).
Brahma and Moira are components of the Drosophila Brahma (SWI/SNF-related) chromatin remodeling complex (PAPOULAS et al. 1998), which has been shown to play a role in negatively regulating S phase (STAEHLING-HAMPTON et al. 1999; HARBOUR and DEAN 2000). Consistent with this notion, alleles of other Brahma complex genes, snr1 and osa, as well as a deficiency that removes the brahma-associated protein 60 (BAP60) or BAP111, dominantly suppress the DmcycEJP phenotype (BRUMBY et al. 2002; Table 1).
65S19 (Trithorax-like):
65S19 was only semilethal; however, genetic and deficiency mapping was still possible, and 65S19 was located to 70D4–71C3 (Table 4). Complementation tests of candidate genes in the region revealed that Trithorax-like (Trl) was allelic to 65S19. Consistent with this, previously characterized alleles of Trl also dominantly suppressed DmcycEJP (BRUMBY et al. 2002).
43S2[l(3)72Dk (zn72D)]:
43S2 was localized to 72D1–72D10 (Table 4) and complementation tests of mutations in the 72D1–10 region revealed that In(3)Taf4XS-2884, an inversion affecting expression of Taf4 (Taf110) and Zn72D (SAUER et al. 1996), failed to complement 43S2. A specific EMS allele of Taf4, l(3)72Dj, however, complemented 43S2, suggesting that 43S2 is most likely allelic to zn72D (CG5215). Indeed, another EMS allele in the region, l(3)72Dk, which failed to complement In(3)Taf4XS-2884, also failed to complement 43S2, suggesting that l(3)72Dk is an allele of zn72D. The zn72D gene encodes a zinc finger protein, but has not been characterized. In an attempt to verify the identity of 43S2 suppression as being due to a mutation of zn72D, l(3)72Dk was crossed into the DmcycEJP background. However, l(3)72Dk did not suppress the DmcycEJP adult eye phenotype or the S-phase defect of DmcycEJP eye discs as effectively as 43S2 did (Figure 1), which may be due to l(3)72Dk being a weaker allele than 43S2. Molecular characterization of the 43S2 and l(3)72Dk lesion will be required to confirm this. Interestingly, Zn72D was identified in a differential expression screen as a gene expressed specifically in the differentiating region of the eye disc (JASPER et al. 2002), consistent with a role for Zn72D in cell cycle arrest or differentiation.
Signaling pathway genes:
2.2 . (phyllopod):
2.2 was localized to 51A1–51A5 (Table 4). Consistent with this, Df(2R)trix (51A1–2; 51B6) dominantly suppressed the DmcycEJP rough eye phenotype (data not shown). Mutations and P alleles within the 51A region were tested for allelism with 2.2 alleles, revealing that a null allele of phyllopod, phyl2245, failed to complement all three S(DmcycEJP) 2.2 alleles. To verify that 2.2 was indeed phyl, previously identified phyl alleles (2245 and 2366) were tested and shown to dominantly suppress the rough eye phenotype and the S-phase defects of DmcycEJP (Figure 1; M. COOMBE, L. QUINN, R. DICKINS, J. SECOMBE and H. RICHARDSON, unpublished results). These data are consistent with the mutation of phyl being responsible for the observed suppression of DmcycEJP by the 2.2 alleles.
Phyl expression is induced by the Sevenless receptor tyrosine kinase signaling pathway and is a rate-limiting component in R7 photoreceptor cell differentiation in the eye imaginal disc, but also has other roles in neural differentiation during development (DICKSON 1998). Phyl is a pioneer protein (containing no homology to other known proteins) that functions with the Ring finger protein Seven in absentia (Sina) and the F-box protein Ebi, to bind to and target the two isoforms of the neural differentiation inhibitor, Tramtrack (Ttk69 and Ttk88) and probably other proteins for destruction by the ubiquitin/proteosome pathway, allowing neural cell differentiation (LI et al. 1997; TANG et al. 1997; BOULTON et al. 2000). Consistent with this, homozygous viable mutants in sina (sina1) strongly suppressed the DmcycEJP adult rough eye and S-phase defects, while a stronger sina allele (sina2) showed weak dominant suppression (M. COOMBE, L. QUINN, R. DICKINS, J. SECOMBE and H. RICHARDSON, unpublished results). However, a deficiency removing sina showed strong dominant suppression of DmcycEJP (Table 1). This deficiency removes a sina-related gene (sina-h), located adjacent to sina, as well as Abl, which has been shown to dominantly suppress DmcycEJP (see below). Consistent with the involvement of the Sina complex in negative regulation of G1-S, ebi alleles have been shown to dominantly suppress DmcycEJP (BOULTON et al. 2000). The mechanism by which the Sina complex acts to regulate G1-S does not involve targeting Cyclin E or E2F for ubiquitin-dependent degradation (BOULTON et al. 2000) and remains to be determined.
2S1 (trio):
2S1 was mapped to 61E–62A8 (Table 4) and by crosses to mutations within the region it was revealed that trio [encoding a Rac guanine nucleotide exchange factor (Rac-GEF; BATEMAN et al. 2000)] failed to complement 2S1. To confirm this interaction, a previously isolated allele of trio (trioM89) was crossed into the DmcycEJP background. trioM89 was shown to dominantly suppress the DmcycEJP rough eye phenotype and S-phase defect (Figure 1). Rac-GEFs are involved in the activation of Rac family GTPases, which have roles in actin cytoskeletal remodeling (BLANCHARD 2000). In mammalian cells, Rac can lead to repression of Rho activity (SANDER et al. 1999), and therefore mutation of trio may lead to higher levels of Rho activity. Rho activation in mammalian cells has been shown to promote cell cycle progression by leading to downregulation of the Cyclin/Cdk inhibitors p21 and p27 (AZNAR and LACAL 2001; PRUITT and DER 2001; SAHAI and MARSHALL 2002). trio has been shown to genetically interact with Abl, encoding a nonreceptor tyrosine kinase also involved in actin cytoskeleton remodeling (LUO 2000). Consistent with this, the deficiency removing Abl (73A3; 74F) dominantly suppressed the DmcycEJP rough eye phenotype; however, this deficiency also removes sina, sina-h (see above), and the Abl pathway gene, Disabled (Dab). The Abl alleles Abl04674 and Abl1 were then tested and shown to also suppress the DmcycEJP rough eye phenotype (not shown). The precise mechanism by which reducing the dosage of trio and Abl leads DmcycEJP suppression remains to be determined.
Cytoskeletal genes:
2.5 . (dEB1):
2.5 was localized to 42B3–42C7 (Table 4). 2.558S12 was also lethal over the adjacent deficiency, Df(2R)nap1 (41D2–E1; 42B1–3), indicating that this allele is a deficiency or rearrangement that affects a larger region than 2.542S11. S(DmcycEJP)2.542S11 was crossed to P-element alleles available in the region and l(2)04524, was found to be semilethal in combination with 2.542S11. The few escaper flies, trans-heterozygous for 2.542S11 and l(2)04524, did not have any gross abnormalities, but generally died within a few days of eclosing, and the females were sterile. l(2)04524 is inserted within the 5'-UTR of the Drosophila homolog of the EB1 gene (BDGP). dEB1 encodes a cytoskeleta1 protein that binds to microtubules and plays an important role in adherens junction integrity and cell polarity (LU et al. 2001; ROGERS et al. 2002). EB1 was identified in mammalian cells as a binding partner of the adenomatous polyposis coli (APC) colon cancer tumor suppressor (SU et al. 1995); however, Drosophila APC1 and APC2 both lack the EB1-binding domain. Consistent with the identity of 2.5 being dEB1, l(2)04524 and the EMS dEB1 alleles, dEB15 (1DL) and dEB16 (GJ63/9) (obtained from J. Roote), dominantly suppressed DmcycEJP rough eye and S-phase defects (Figure 1 and data not shown). Moreover 2.542S11 and l(2)04524 disrupt dEB1 transcription (D. COATES, L. QUINN, R. DICKINS, J. SECOMBE, A. BRUMBY and H. RICHARDSON, unpublished results). How the EB1 microtubule protein is involved in G1-S regulation remains to be determined.
Cell adhesion genes:
2.11 . (scab) (-Integrin):
Group 2.11 was defined by S(DmcycEJP)62S9 from this screen and E(sev-cycE)e93 was from the LANE et al. (2000) genetic screen (see above). 2.11 was mapped to the region 51D3–51F13 (Table 4), and by testing mutations within this region, it was revealed that the P allele, l(2)01288, failed to complement both 2.11 alleles. The insertion point of l(2)01288 has been defined (BDGP) and disrupts the scab gene, encoding an -integrin, PS3, thought to play a role in tissue morphogenesis (STARK et al. 1997). To further confirm that 2.11 is allelic to scab, previously identified EMS-derived alleles of scab (scb1 and scb2) were tested and shown to also fail to complement 2.11 alleles. Consistent with the suppressing gene being scab, l(2)01288, scb1, and scb2 were recombined onto the DmcycEJP and were shown to also suppress the rough eye phenotype and the S-phase defect of DmcycEJP (Figure 1 and data not shown). In mammalian cells, integrins in association with the extracellular matrix have a well-established role in promoting anchorage-dependent cell proliferation (DANEN and YAMADA 2001). However, recent studies have shown that integrins can also inhibit G1-S progression (HAZLEHURST et al. 2000; METTOUCHI et al. 2001). Our identification of scab in the DmcycEJP genetic screen suggests that in Drosophila integrins also act as negative regulators of G1-S.
2.12 . (CadN):
2.12 alleles 42S13 (also an allele of group 2-6) and 22S9 (Figure 1 and data not shown) were mapped to 36D1–36E4 (Table 4). Mutations and P alleles in the region were tested by complementation analysis, revealing that an allele of CadN (CadNM12) failed to complement both 2.12 alleles. CadN encodes a cadherin-like transmembrane protein (LEE et al. 2001; IWAI et al. 2002) that can bind to -catenin and ß-catenin (Armadillo), components of the adherens junction (PEREZ-MORENO et al. 2003). In mammalian cells, downregulation of N-Cadherin leads to upregulation of G1 Cyclin activity (CHARRASSE et al. 2002). Due to the close location of CadN and DmcycE, it was not possible to obtain a recombinant of the CadN allele with DmcycEJP to confirm that CadN exhibits the same modifier effect as S(DmcycEJP)2.12.
Cytoarchitectural tumor suppressor genes:
2.1 . [lethal-(2)-giant larvae]:
2.1 was localized to 21A1–21B7–8 by deficiency mapping (Table 4). The mapping of 2.1 was initially confounded by the fact that two deficiencies in the deficiency kit, Df(2L)Prl (32F1–3; 33F1–2) and Df(2L)J39 (31D1–11; 32D1–E5), also contained lesions in the 21A region and therefore failed to complement 2.1. The localization of 2.1 was confirmed by genetic mapping of 2.1 alleles, which indicated that the lethal mapped to the left of UbcD1 (32A4–5) and close to al (21C2–4). Since 2.1 homozygous mutants die as giant larvae, an allele of the lethal-(2)-giant-larvae (lgl) gene, which also gives giant larvae and is localized at 21A, was tested for complementation of 2.1 alleles and failed to complement, whereas mutations in other genes in this region that have been identified as negative cell cycle regulators in previous screens, spen (poc; STAEHLING-HAMPTON et al. 1999; LANE et al. 2000) and net (I. HARRIHARAN, personal communication), both complemented 2.1 alleles. Taken together these data suggest that lgl corresponds to 2.1. To confirm that a lesion in lgl suppresses the DmcycEJP phenotype, a null allele of lgl (lgl4) was tested for suppression of DmcycEJP. However, lgl4 did not suppress the S-phase defect or the rough eye phenotype of DmcycEJP to the same extent as 2.1 alleles did (Figure 2; and data not shown). However, halving the dosage of 2.1 alleles resulted in a greater increase in Cyclin E protein levels in DmcycEJP eye discs than halving the dosage of lgl4/+ (Figure 3). It is possible that additional mutations in the lgl4 background may account for its poorer ability to dominantly suppress DmcycEJP compared with 2.1 alleles. Consistent with lgl mutations being responsible for the suppression of DmcycEJP, lgl-2.1 and other lgl mutant clones in the eye imaginal disc showed ectopic expression of Cyclin E, which could be suppressed by expression of lgl using a UAS-lgl transgene (N. AMIN, A. BRUMBY, J. SECOMBE and H. RICHARDSON, unpublished results).
|
|
Scribble is a four-PDZ95-Dlg-ZO1 and multi-leucine-rich repeat containing protein localized to septate junctions and required for apical-basal polarity (BILDER and PERRIMON 2000; HUMBERT et al. 2003). When homozygous, 63S15, like scribble null alleles, arrest as giant overgrown larvae due to amorphous overgrowth of imaginal discs and brain lobes, which is characteristic of neoplastic tumor suppressor mutants (GATEFF et al. 1996; DE LORENZO et al. 1999; BILDER 2001). To confirm that lesions in scribble suppress DmcycEJP, the l(3)j7b3 allele and stronger EMS alleles of scrib, scrib1 and scrib2 (BILDER and PERRIMON 2000), were crossed into a DmcycEJP background. The weak P allele, l(3)j7b3, did not suppress DmcycEJP, although mild suppression was observed with scrib1 and scrib2 alleles, but not as well as with 63S15 (Figure 2 and data not shown). This suggests that 63S15 may be a stronger scribble allele than scrib1 or scrib2. In confirmation that scribble alleles suppress the DmcycEJP phenotype, halving the dosage of scribble in DmcycEJP eye discs leads to higher levels of Cyclin E (Figure 3 and data not shown) and scrib1 and scrib2 eye imaginal disc clones show ectopic expression of Cyclin E (BRUMBY and RICHARDSON 2003).
lgl and scribble are neoplastic tumor suppressor genes that together with discs-large (dlg) act in the same pathway to regulate apical-basal cell polarity (BILDER et al. 2000; HUMBERT et al. 2003). Because of this function, we have termed these proteins cytoarchitectural tumor suppressors to highlight their role in cell structure. Consistent with this pathway being important in regulation of G1- to S-phase progression, a deficiency removing dlg, Df(1)vN48, as well as a specific dlg allele (dlg6) showed suppression of DmcycEJP (Tables 1 and 2; Figure 2). Scribble, Dlg, and Lgl have been recently shown to act antagonistically to the Crumbs cell polarity complex (BILDER et al. 2003; TANENTZAPF and TEPASS 2003), and consistent with this, a deficiency removing crumbs and a crumbs allele (crb2) dominantly enhanced DmcycEJP (Table 1).
Scribble-interacting genes:
To determine whether a common pathway is involved in the mechanism by which the DmcycEJP suppressors lead to deregulation of cell proliferation, we analyzed weak scribble mutant combinations for a dominant genetic interaction with other genes identified in the DmcycE genetic screen (Table 6). The trans-heterozygous combination of scrib5/scribl(3)jB709 or scrib1/scrib5 results in adults with eye, bristle, and thorax-closure defects (not shown). Reducing the dose of the lgl (27S3, E2S31, and lgl4) showed a strong genetic interaction with the weak scrib allele phenotype, resulting in no scrib mutant progeny heterozygous for lgl. This is consistent with the previous observations that scribble mutations exhibit strong genetic interactions with lgl and dlg in the embryo (BILDER et al. 2000). Strikingly, halving the dosage of several other suppressor genes identified in the screen also resulted in very low numbers of scrib mutant progeny, most notably with dEB1 (2.5), phyl (2.2), the PS3 integrin gene scab (2.11), the Brahma complex gene moira, and to a lesser extent brahma, as well as the unidentified 2.3, 2.4, and 2.9 genes. The mechanism of these interactions requires further analysis and relies on identifying the 2.3, 2.4, and 2.9 genes.
|
|
5% escapers over l(2)37Ac. 2.365S12 is therefore likely to be a smaller lesion affecting both of these uncharacterized genes. A recently characterized gene in the 36F region, hamlet, which is a transcription factor involved in dendrite morphogenesis (MOORE et al. 2002), was also tested for allelism with 2.3 and failed to complement 2.359S16 and 2.365S12 but not l(2)36Fd. Further analysis is required to determine whether hamlet or l(2)36Fd corresponds to the 2.3 suppressor.
3.1 . (19S5, 24SX, 58S5, 62S2) location [73D–74F (74B1–74C1)]:
Consistent with the map position defined by deficiency mapping, chromosome cytology revealed that 58S5 contained a deletion in the 74A–F region, and it failed to complement several lethal alleles in the region. The cadherin-like gene, CG6445 (Cad74A), was considered a candidate, since the cadherin-like protein, Fat, is a tumor suppressor in Drosophila (GATEFF et al. 1996; DE LORENZO et al. 1999). Southern analysis failed to reveal any alterations in this gene in 3.1 alleles (data not shown). The method of male recombination (SVOBODA et al. 1995) was then used to further define the map position of the 3.1 alleles, 19S5 and 24S10 relative to several P alleles, revealing that the lethal associated with 3.1 mapped to the right of blot (74B1–2) and to the left of l(3)S070006 (allelic to l(3)L6750 = frc at 74B4), l(3)00073 (74C1–2), and EIP74EF (74D2–5). Taken together these data suggest that 3.1 maps between 74B1 and 74B4. A candidate gene within this region, CG3885, encodes a Sec3-like protein, a component of the exocyst complex involved in docking at the plasma membrane, which is a function that Lgl has also been implicated in (LEHMAN et al. 1999; MUSCH et al. 2002).
1S3 location [98A–100B (98A5–98E3)]:
Chromosome cytology showed that 1S3 contained a translocation breakpoint at 98C (data not shown). Since there is a hole in the deficiency collection between 98A5 and 98E3, it is likely that 1S3 maps within this region. A candidate in the 98A5–98E3 region was APC1 (encoding the Adenomatous polyposis tumor suppressor; AHMED et al. 1998); however, mutations in APC1 (APCQ8 and APCX1) complemented 1S3. Another candidate is raps (pins), which encodes a protein involved in asymmetric division of neuroblasts and directly interacts with Dlg (PARMENTIER et al. 2000; BELLAICHE et al. 2001). Further analysis is required to test whether raps mutations are allelic to 1S3.
59S9 location (62D2–62F5):
Consistent with this location for 59S9, cytological analysis revealed a breakpoint at 62B. A possible candidate in this region is spinophilin (neurabin), encoding an actin-binding scaffold protein, which in mammalian cells is involved in binding to and upregulating Rac and p70-S6K activity (BUCHSBAUM et al. 2003). Since another gene involved in Rac activation, trio, was identified as a suppressor of DmcycEJP it is possible that spinophilin is also a suppressor. Furthermore, Drosophila mutations in spinophilin are semilethal (KEEGAN et al. 2001), as is 59S9.
Further analysis is needed to investigate whether the potential candidates for these suppressors, listed above and in Table 7, are disrupted by the suppressor mutations and for the identification of the suppressors.
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
|---|
-integrin), cadN (N-Cadherin), shg (E-Cadherin), and fat (encoding an atypical-cadherin and hyperplastic tumor suppressor); and (5) cytoarchitectural tumor suppressor genes scribble, lgl, and dlg, required for apical-basal cell polarity and cell proliferation inhibition. While some of these genes (brm, mor, expanded, fat, scribble, and lgl) have been previously shown or implicated to play a role in negatively regulating G1-S (GATEFF et al. 1996; DE LORENZO et al. 1999; STAEHLING-HAMPTON et al. 1999; BILDER et al. 2000), a potential role for Trl, Znf72D, phyl, sina, trio, Abl, RpS6, wg, dsh, arm, dEB1, scab, cadN, and shg in inhibiting G1-S progression in Drosophila is novel. Further studies are required to determine whether Abl, RpS6, wg, dsh, arm, and shg do indeed suppress DmcycEJP by acting at the S-phase level and to understand the mechanism by which these genes act in G1-S regulation. The identification of novel classes of presumptive negative regulators of cyclin E or G1-S progression highlights the power of Drosophila whole-animal genetics as a tool for revealing new cell proliferation pathways. It is unclear at present how many of the DmcycEJP modifiers identified in our screen bear upon the role of Cyclin E in DNA replication or centrosome duplication (see Introduction). Brahma and Moira are likely to be downstream targets of Cyclin E/cdk2 that may impact upon transcriptional regulation or DNA replication (BRUMBY et al. 2002), but whether other interactors act upstream or downstream of Cyclin E remains to be determined. The only cyclin E interactor we identified that has been shown to be associated with the centrosome is EB1 (REHBERG and GRAF 2002); however, whether this reflects upon the role for Cyclin E in centrosome duplication in Drosophila is unclear. A recent study has shown that the Drosophila SkpA, a component of SCF ubiquitin ligases, regulates centrosome duplication independently of Cyclin E accumulation (MURPHY 2003).
Similar genetic screens carried out using phenotypes generated by overexpression of cyclin E (LANE et al. 2000) or the G1-S regulators E2F1/Dp (STAEHLING-HAMPTON et al. 1999), Rbf (DUMAN-SCHEEL et al. 2002), and human p21 (Cdk2 inhibitor; I. HARIHARAN, personal communication) have revealed a more restricted set of interacting genes than that obtained in our cyclin E hypomorphic allele genetic screen. The GMR-E2F1/Dp screen (STAEHLING-HAMPTON et al. 1999) revealed alleles of the chromatin remodeling genes brm, mor, and osa and of the transcription factor pointed, an effector of the Egfr-Ras signaling pathway, as enhancers. This is consistent with our identification of brm and mor as suppressors of the hypomorphic cyclin E phenotype in our mutagenesis screen. In addition, we tested alleles of osa and showed that they suppressed the hypomorphic cyclin E phenotype (BRUMBY et al. 2002). The sevenless-cyclin E screen (LANE et al. 2000) revealed alleles in identified cell cycle genes cdk2 (as a suppressor), dacapo (as an enhancer), and E2F1 (a suspected gain-of-function allele as an enhancer) and identified as an enhancer the novel gene spen (poc), also identified in the GMR-E2F1/Dp screen (STAEHLING-HAMPTON et al. 1999). Spen (Poc) is a RNP-type RNA-binding protein that has recently been shown to be required for Wg signaling in imaginal discs (LIN et al. 2003). We have not identified spen (poc) as a suppressor in our genetic screen, but alleles of spen (poc) were tested and shown to suppress DmcycEJP (Table 2), consistent with the Wg signaling pathway acting to negatively regulate G1-S progression in the eye disc. As detailed above, we have shown that one of the single alleles identified as an enhancer in the sevenless-cyclin E screen is allelic to our DmcycEJP suppressor 2.11, which we have identified as scab. In the GMR-Rbf screen, alleles of patched, encoding an inhibitor of Hedgehog (Hh) signaling, were identified as dominant suppressors (DUMAN-SCHEEL et al. 2002). Although our mutagenesis screen did not reveal alleles of patched, patched alleles strongly suppressed DmcycEJP (Table 2), consistent with the notion that Hh signaling leads to increased transcription of cyclin E (DUMAN-SCHEEL et al. 2002). The greater number of interactors that we obtained in our screen may be due to the fact that our screen was of a cyclin E hypomorphic phenotype that affected cell proliferation in early eye development as well as the post-MF S phases and may therefore have been more sensitive to gene dosage than the overexpression screens. Furthermore, unlike the overexpression screens, the cyclin E hypomorphic screen is more likely to reveal genes that are upstream of cyclin E expression.
The DmcycEJP suppressor genes we have identified from our mutagenic screen are mostly distinct from Drosophila tumor suppressors previously described (TOROK et al. 1993; GATEFF et al. 1996; DE LORENZO et al. 1999). Recently, clonal screens have revealed a novel pathway involved in inhibiting G1-S progression and cell death in the Drosophila eye (HAY and GUO 2003). This pathway includes lats (warts), salvador, and hippo, and although this pathway has been recently shown to regulate cyclin E at possibly both a transcriptional and protein stability level, we did not identify alleles of these genes in our genetic screen. Alleles of hippo, at least, have been shown to suppress the DmcycEJP phenotype (WU et al. 2003). The fact that we did not identify hippo in our mutagenesis screen may have been because the screen was not saturating. However, lats (warts) alleles did not show appreciable suppression of DmcycEJP (Table 2); therefore it is possible that only certain mutations of this pathway are capable of dominant suppression.
Also pertinent to our study is the recent Drosophila protein interaction map determined by yeast two-hybrid analyses (GIOT et al. 2003). None of our identified cyclin E genetic interactors were identical to the 15 interactors identified by the protein interaction study (GIOT et al. 2003), but many proteins identified in our screen were not analyzed in their screen (e.g., Brahma, Moira, Scab, CadN, Dsh, Scribble, Crumbs, Expanded, and Abl). Most of the 15 yeast two-hybrid interactors with Cyclin E are uncharacterized, but of the characterized proteins, Combgap, a transcription factor, has been implicated in cell proliferation via its effect on Ci expression (CAMPBELL and TOMLINSON 2000). Of the other characterized interactors, Gliolectin is involved in cell adhesion in axon pathfinding (SHARROW and TIEMEYER 2001) and Traf2 is involved in Dorsal activation (SHEN et al. 2001), but no cell proliferation role has been described for these proteins. Some of the Cyclin E yeast two-hybrid interacting genes map to regions where cyclin E genetic interactors have been mapped (not shown) and are candidates for future analysis.
Whether the genetic suppressors of cyclin E identified in our screen can all be connected in a common pathway or represent several converging pathways acting upon G1-S progression in the eye imaginal disc remains to be determined. As a first step to explore this we examined interactions between a weak scrib mutant and S(DmcycEJP) alleles, which revealed genetic interactions with lgl, phyl, dEB1 scab, mor, the unidentified suppressors 2.3, 2.4, and 2.9, and to a lesser extent brm. This analysis provides a connection between chromatin remodeling, signaling, cytoskeletal, cell-cell adhesion, and cytoarchitectural suppressor genes. How exactly these pathways may be connected and whether other genes identified in the DmcycEJP
