Mosaic Genetic Screen for Suppressors of the de2f1 Mutant ... · Duronio 2001; Dimova and Dyson...

Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.109.104661

Mosaic Genetic Screen for Suppressors of the de2f1 MutantPhenotype in Drosophila

Aaron M. Ambrus,1 Vanya I. Rasheva,1 Brandon N. Nicolay and Maxim V. Frolov2

Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607

Manuscript received May 2, 2009Accepted for publication June 6, 2009

ABSTRACT

The growth suppressive function of the retinoblastoma (pRB) tumor suppressor family is largelyattributed to its ability to negatively regulate the family of E2F transcriptional factors and, as a result, torepress E2F-dependent transcription. Deregulation of the pRB pathway is thought to be an obligatory eventin most types of cancers. The large number of mammalian E2F proteins is one of the major obstacles thatcomplicate their genetic analysis. In Drosophila, the E2F family consists of only two members. They areclassified as an activator (dE2F1) and a repressor (dE2F2). It has been previously shown that proliferation ofde2f1 mutant cells is severely reduced due to unchecked activity of the repressor dE2F2 in these cells. Wereport here a mosaic screen utilizing the de2f1 mutant phenotype to identify suppressors that overcome thedE2F2/RBF-dependent proliferation block. We have isolated l(3)mbt and B52, which are known to berequired for dE2F2 function, as well as genes that were not previously linked to the E2F/pRB pathway suchas Doa, gfzf, and CG31133. Inactivation of gfzf, Doa, or CG31133 does not relieve repression by dE2F2. Wehave shown that gfzf and CG31133 potentiate E2F-dependent activation and synergize with inactivation ofRBF, suggesting that they may act in parallel to dE2F. Thus, our results demonstrate the efficacy of thedescribed screening strategy for studying regulation of the dE2F/RBF pathway in vivo.

THE family of E2F transcription factors and retino-blastoma (pRB) family tumor suppressor proteins

playapivotal role incontrolofcellproliferation(reviewedin Cayirlioglu and Duronio 2001; Trimarchi andLees 2002; Blais and Dynlacht 2004; Dimova andDyson 2005; Degregori and Johnson 2006). AlthoughE2F is involved in a variety of cellular activities, the bestunderstood function of E2F is to regulate transcriptionof genes at the G1/S transition. Among E2F targets aregenes that encode regulators of S-phase entry andcomponents of the DNA replication machinery. Inmammals, there are eight E2F genes. E2F-1 throughE2F-6 function as heterodimers with a DP subunit, whileE2F-7 and E2F-8 do not require DP to bind to DNA. Inspite of structural similarities among E2F proteins, E2F-1, E2F-2, and E2F-3a are predominately involved inactivation of gene expression while the group E2F-3b,E2F-4, E2F-5, E2F-6, E2F-7, and E2F-8 behave asrepressors. In quiescent cells, when activity of cyclin-dependent kinases (cdks) is low, repressor E2Fs arecomplexed with the pRB family members (also calledpocket proteins) and repress expression of E2F regu-lated genes. The prevailing mechanism of the repressionis thought to be through the direct recruitment of

histone deacetylases, histone methylases, and othercorepressor complexes by pocket proteins to E2Fregulated promoters. Upon entry into the cell cycle,mitogenic stimulation leads to an increase in the activityof G1 cdks, which phosphorylate pRB family membersand disrupt their interaction with E2Fs. This coincideswith displacement of the repressor E2Fs, appearance offree activator E2Fs on the promoters of E2F responsivegenes, and induction of the E2F transcriptional pro-gram. The critical role of the E2F/pRB network in cellproliferation is underscored by obligatory inactivationof pRB control in almost all cancers (Hanahan andWeinberg 2000). The prevailing view is that the tumorsuppressor property of pRB is to constrain E2F activity.However, our knowledge of the various tiers of regula-tion by which pRB has the capacity to block cell pro-liferation in thecontext ofamulticellularorganismis stillvery limited. Thus, understanding how the growthsuppressive function of pRB/E2F can be overridden isimportant to fully understand the importance of the pRBpathway in cancer and during normal development.

Drosophila represents an ideal model system toaddress this question. This is primarily due to the abilityto carry out high-throughput genetic screens to unveilnovel functional interactors of the E2F/pRB pathway inan unbiased way. Importantly, the pRB/E2F network ishighly conserved in Drosophila, yet the families aremuch smaller than in mice and humans. The Drosophilagenome encodes an activator, dE2F1, and a repressor,

1These authors contributed equally to this work.2Corresponding author: Department of Biochemistry and Molecular

Genetics, University of Illinois, MBRB 2352, MC 669, 900 S. AshlandAve., Chicago, IL 60607. E-mail: [email protected]

Genetics 183: 79–92 (September 2009)

dE2F2, their heterodimeric partner, dDP, and two pocketproteins, RBF1 and RBF2 (reviewed in Cayirlioglu andDuronio 2001; Dimova and Dyson 2005; van den

Heuvel and Dyson 2008). Studies of the phenotypes ofde2f and rbf mutant and transgenic animals have pro-vided clear insights into the critical roles of thesegenes in cell proliferation (Duronio et al. 1995, 1998;Royzman et al. 1997; Neufeld et al. 1998; Du and Dyson

1999; Cayirlioglu et al. 2001; Frolov et al. 2001). As inmammals, dE2F1 is a potent inducer of S-phase entry.Overexpression of de2f1 drives postmitotic cells of theeye imaginal disc into the cell cycle (Asano et al. 1996;Brook et al. 1996). Consistently, inactivation of rbf1,which negatively regulates de2f1, leads to ectopic cellcycles (Du and Dyson 1999; Firth and Baker 2005).Analysis of single and double de2f mutant animalsrevealed that de2f1 and de2f2 act antagonistically duringlarval development. This conclusion is based on theobservation that the phenotype of de2f1 de2f2 doublemutant animals is less severe than the phenotype ofde2f1 mutants (Frolov et al. 2001). Cell proliferationand E2F-dependent transcription are severely reducedin de2f1 mutant animals. These defects are suppressedby a concomitant mutation of de2f2. The pattern of cellproliferation is largely normal and repression of severalexamined E2F targets is relieved in de2f1 de2f2 doublemutants. These results indicate that unchecked de2f2provides a significant contribution to the de2f1 mutantphenotype and therefore, the lack of cell proliferationin de2f1 mutants can be potentially viewed as a readoutof the de2f2 activity.

Here, we present the results of a mosaic geneticscreen aimed at identifying suppressors that rescueproliferation in de2f1 mutants [Su(E1)’s]. Since thede2f1 mutant phenotype stems from the cell prolifera-tion block by dE2F2/RBF, we expected that some of theisolated suppressors would be important for dE2F2/RBF function. Indeed, we have identified mutations inl(3)mbt and B52, two genes that were previously impli-cated in dE2F2/RBF-mediated repression. We have alsoidentified two suppressors, gfzf and CG31133, whichpotentiate E2F-dependent transcription and cooperatewith the dE2F/RBF pathway. Thus, the characterizationof Su(E1)’s can provide new insights into the in vivoregulation of the dE2F/RBF pathway.

MATERIALS AND METHODS

Mutagenesis screen: Drosophila were cultured on standardmedia at 25�. A description of all alleles and fly stocks can befound in FlyBase.

For mutagenesis, 36- to 72-hr-old w; FRT 82B de2f1729/TM3Sb males were starved for 3–6 hr and fed 25 mm EMS in a 1%sugar solution for 16–18 hr. In the F1 screen, mutagenizedmales were crossed to ey-FLP; FRT 82B l(3)cl-R31/TM6B femalesand the progeny were screened for exceptional males thatcontained visible clones of de2f1 mutant cells. In the F2 screen,mutagenized males were first crossed to w; TM3 Ser/MKRS

females to balance putative suppressors of the de2f1 mutantphenotype. Then the individual FRT82B Su(E1) de2f1729/TM3or FRT82B Su(E1) de2f1729/MKRS males were crossed to threeey-FLP; FRT 82B l(3)cl-R31/TM6B females to identify potentialsuppressors of the de2f1 mutant phenotype. Isolated Su(E1)’swere retested multiple times by crossing them to ey-FLP; FRT82B l(3)cl-R31/TM6B and ey-FLP ; FRT 82B P[Ubi-GFP]/TM6Bfemales.

To map isolated Su(E1)’s, females that were w; FRT82Bde2f1729 Su(E1)/TM3 Ser were mated to males heterozygous forthe 3R chromosomal deficiencies from the Exelixis andDrosDel deficiency kits. The following deficiencies that uncovergaps present in the Exelixis and DrosDel kits were also includedin complementation tests: Df(3R)Antp-X1, Df(3R)BSC24,Df(3R)by10, Df(3R)sbd26, Df(3R)P2, Df(3R)DG2, Df(3R)Cha1a,Df(3R)Dl-BX12, Df(3R)BSC43, Df(3R)BSC56, Df(3R)Espl3,Df(3R)BSC42, Df(3R)L127, and Df(3R)B81. A recovery of ,1%of adult flies trans-heterozygous for Su(E1) and a deficiencyrelative to their heterozygous siblings were considered a failureto complement. Since Su(E1)’s were isolated in the presence ofthe de2f1729 mutation, each Su(E1) was lethal in trans toDf(3R)Exel6186, which uncovers the de2f1 gene.

To map Su(E1)-A the exon–intron junctions and ORFs ofthe following genes were sequenced: CG18682, CG11498,CG1359, CG2171, CG31025, CG9753, CG31030, CG1983,CG9747, G31029, CG15529, CG31028, CG15530, andCG15531. Genomic DNA was extracted from adult flies of w;FRT82B de2f1rm729 Su(E1)-A7d13/TM6B Tb and sequenced. Nochanges resulting in premature stop codons or affectingexon–intron junctions were found. However, we found sin-gle-nucleotide changes that do not result in the above-mentioned defects in the sequence of the following genes:CG18682, CG11498, CG31025, CG15530, CG15531, CG31028,CG31029, CG9747, and CG31030. To determine whether anyof these single-nucleotide changes were newly induced muta-tions or single-nucleotide polymorphisms, genomic DNA wasisolated from the following genotypes and used as a controlin sequencing: w; FRT82B de2f1rm729 Su(E1)5d7/TM6B Tb andw; FRT82B de2f1rm729 Su(E1)7d21/TM6,Tb. In several cases theregions of interest were sequenced using genomic DNA of thefollowing genotypes: w ; FRT82B de2f1rm729 Su(E1-A)7d13/TM3 Serand w; FRT82B de2f1rm729 Su(E1-A)7d13/TM6 Tb [ubi-GFP].Additionally, we sequenced several genes using genomicDNA isolated from flies carrying another allele of Su(E1)-A6a39. On the basis of these comparisons we concluded that allsingle-nucleotide changes that we found are SNPs of theparental chromosome and therefore are not newly inducedmutations in the above-mentioned genes.

For Su(E1)’s, Su(E1)-A7d13, CG311337d21, and gfzf 6a27, anFRT82B Su(E1) de2f11 chromosome was generated from theFRT82B Su(E1) de2f1729 chromosome by precise excision of theP element from the de2f1729 allele. Since both the de2f1729 alleleand FRT82B are marked with the rosy gene, excisions of the Pelement from the de2f1729 allele could not be followed by therosy marker. Therefore, the FRT82B Su(E1) de2f1729/D2–3 Sbmales were crossed to TM3 Ser/MKRS females and �100 rosy1

males were crossed individually to de2f17172/TM3 Sb females toselect chromosomes in which the de2f1729 allele was reverted towild type. The presence of a functional FRT82B was verified bycrossing to ey-FLP; FRT82B P[Ubi-GFP, mini-white] to visualizethe appearance of clones.

Immunofluorescence: Eye and wing imaginal discs weredissected from third instar larvae in Schneider’s insectmedium (GIBCO, Grand Island, NY) and then fixed inphosphate-buffered saline (PBS) buffer with 4% formalde-hyde. Washes were then done in PBS, 0.3% Triton X-100. Discswere then blocked in PBS, 0.1% Triton X-100, 10% normaldonkey serum for 1 hr followed by the addition of primary

80 A. M. Ambrus et al.

antibodies. Discs were incubated overnight with primary anti-bodies and then washed in PBS, 0.1% Triton X-100 three timesfor 10 min each. Discs were then incubated in blocking solutioncontaining the appropriate secondary antibodies. Washes werethen done using PBS, 0.1% Triton X-100. Discs were thenplaced into glycerol containing 0.5% propyl gallate in prepa-ration for slide mounting. Bromodeoxyuridine (BrdU)(Sigma, St. Louis) labeling of eye imaginal discs was performedby dissecting discs and incubating them in 0.3 mg/ml BrdU–Schneider’s insect medium for 30 min. Washes were then donein PBS followed by an overnight fixation in 1.5% formalde-

hyde. Washes were then done in PBS before incubating discswith DNAse [Promega (Madison, WI) RQ1] for 45 min in a 37�water bath. Then washes, blocking, and antibody incubationsfollowed as described above. Images were collected with a ZeissLSM510 confocal microscope. The primary antibodies used inthis work were mouse anti-BrdU (1:50) (BD Bioscience), ratanti-Elav (1:50) (Developmental Studies Hybridoma Bank),and rabbit anti-phospho-H3 (1:150) (Upstate). The secondaryconjugated antibodies used in this work were anti-mouse Cy3(1:100), anti-rabbit Cy5 (1:100), and anti-rat Cy3 (1:50) fromJackson Immuno Laboratories.

Figure 1.—Suppression of the de2f1 mu-tant phenotype in adult eyes and wing ima-ginal discs. (A–J) Mitotic recombinationwas induced in the eyes of heterozygousanimals by ey-FLP. Wild-type chromosomescarry the mini-white gene and thereforewild-type clones (red) can be distinguishedfrom homozygous mutant tissue (white).(A and B) Adult eyes of ey-FLP; FRT42D/FRT42D P[mini-white] (wild type) and ey-FLP; FRT42D dDPa3/FRT42D P[mini-white](DP). Arrow indicates the location of thedDP clone in B. (C and D) dDP mutant tis-sue in the cell lethal background. Adulteyes of ey-FLP; FRT42D /FRT42D l(2)cl-R111 P[mini-white] (wild-type CL) and ey-FLP ; FRT42D dDP a3/FRT42D l(2)cl-R111

P[mini-white] (DP CL) are shown. (E) Node2f1 mutant tissue can be seen on the celllethal background in ey-FLP; FRT42Dde2f1729 /FRT42D l(3)cl-R31 P[mini-white].Thus, de2f1 mutant tissue is not able to con-tribute to the size of the fly eye and thewild-type tissue is not able to compensatefor this lack of tissue, due to the cell lethalbackground, which is why this eye appearssmaller than the eyes in other panels. (F–J)Mutation of suppressors l(3)mbt3d33 (F),Su(E1)-A7d13 (G), CG311337d21 (H), gfzf 6a27

(I), and Doa6d2 (J) is able to rescue prolifer-ation of de2f1 mutant cells as evidenced bythe presence of double mutant tissue(white). (K–Q) Wing imaginal discs weredissected from wandering third-instar lar-vae. Clones of wild-type cells, identifiedby the presence of GFP (green), and ho-mozygous mutant cells, identified by theabsence of GFP (green), were induced byUbx-FLP. DAPI is shown in blue. (K) Con-trol experiment in the wing disc showingclones of cells homozygous for wild-typechromosomes. Without any mutationsthere is approximately an equal distribu-tion of GFP (green)-positive tissue and tis-sue lacking GFP (green). (L) Almost node2f1729 mutant tissue, identified by the lackof GFP (green), can be seen. Arrows pointto representative clones of de2f1 mutantcells. Note the small size of the clones.Folds in the tissue do not have a GFP signal

and are identified by asterisks (*). (M–Q) Mutation of suppressors l(3)mbt3d33 (F), Su(E1)-A7d13 (G), CG311337d21 (H), gfzf 6a27 (I), andDoa6d2 (J) is able to rescue proliferation of de2f1 mutant cells as evidenced by the presence of more double mutant tissue than tissuegenerated by de2f1 single mutant cells (L). Double mutant tissue is identified by the lack of GFP (green). (K9–Q9) The same cor-responding images as K–Q showing only GFP (green).

Screen for Suppressors of the de2f1 Mutant Phenotype 81

RNAi, Western blot analysis, transient transfections, andreal-time PCR in Drosophila tissue culture cells: For RNAitreatments �2.5 3 104 S2R1 cells per well were plated in a24-well plate containing 400 ml of Schnieder’s insect mediumwith 10% fetal bovine serum (Sch 1 FBS). (Schnieder’s in-sect medium was from GIBCO and FBS was from AtlantaBiologicals). Cells were allowed to adhere to the plate for 1 hrbefore being washed with medium. Then 12.5 mg of eachappropriate dsRNA in 200 ml medium were added to eachappropriate well. After 3 hr, 400 ml of Sch 1 FBS were added toeach well. After 4 days, appropriate wells were treated withdE2F1 dsRNA following the above procedure for 18 hr. Then200 ml of cells per well were replated in duplicate in a 24-wellplate containing 200 ml Sch 1 FBS. Cells were allowed toadhere for 1 hr before a transfection solution consisting ofappropriate DNA and a transfection agent, FuGENE HD(Roche Diagnostics, Indianapolis), in 200 ml Sch was addedto each well. After�30 hr cells were lysed in 1 mm EDTA, 100 mm

NaCl, 10 mm Tris-HCl (pH 7.8), and 0.25% NP-40 beforeassaying for luciferase and b-galactosidase activity. Luciferaseassays were performed on a BIO-TEK Clarity MicroplateLuminometer. b-Galactosidase assays were performed on aLabsystems Multiskan MS Plate Reader. Quantitative real-timePCR on RNA isolated from S2 cells and primers were aspreviously described (Rasheva et al. 2006). For Western blotanalysis, 5–10 3 106 cells were lysed in 250 ml of 100 mm NaCl,10 mm tris-HCl, 1 mm EDTA, and 0.25% NP-40 (pH 7.8) bufferand frozen for 5 min at�80�. Proteins were resolved by sodiumdodecyl sulfate–10% polyacrylamide gel electrophoresis.Guinea pig polyclonal anti-dE2F1 (Bosco et al. 2001) andmouse anti-tubulin antibodies were used for Western blotanalysis.

RESULTS

A suppressor screen for genes that relieve the de2f2-dependent block to cell proliferation: Genetic mosaicscreens based on mitotic recombination have proved tobe particularly effective in identifying genes that regu-late cell proliferation. The ey-FLP/FRT technique pro-vides a high level of mitotic recombination, thus allowingfor the generation of homozygous mutant tissue in theeye with high efficiency. Since the wild-type chromosomecarries the white gene (P[mini-white]), the homozygousmutant tissue can be easily distinguished by the lack ofthe white eye color marker in the adult eye (Figure 1A).Thus, the ey-FLP/FRT-based screen is an attractive ap-proach for identifying novel genes that are important fordE2F2/RBF function in a high-throughput manner.Additionally, we sought a genetic strategy that meetsthe following criteria: does not rely on overexpression ofde2f or rbf genes and is sufficiently sensitive. The de2f1mutant phenotype meets these criteria. Clones of de2f1

mutant cells are extremely small due to a strong pro-liferation block in these cells (Brook et al. 1996; Neufeld

et al. 1998). Since proliferation of de2f1 mutant cells canbe rescued by inactivation of de2f2, patches of de2f1 de2f2double mutant tissue can be found in the adult eye(Ambrus et al. 2007). An example of a clone in whichboth de2f1 and de2f2 are functionally inactivated by a dDPmutation is shown in Figure 1B. Therefore, we expectedthat mutations that compromise the dE2F2/RBF func-tion should act as suppressors of the de2f1 mutantphenotype [Su(E1)’s] and could be identified by theappearance of clones of de2f1 Su(E1) double mutant cellsin the adult eye.

To widen the observable range of suppression of thede2f1 mutant phenotype, we adopted an ey-FLP/FRTcelllethal system in which most of the homozygous wild-typetissue is eliminated due to the presence of a recessive celllethal mutation on a wild-type chromosome (Newsome

et al. 2000). When clones of E2F-deficient cells wereinduced with the ey-FLP cell lethal technique using a dDPmutation, large patches of the mutant tissue could beeasily identified in all animals (Figure 1, C and D).Importantly, even with the ey-FLP/FRT cell lethal tech-nique, no white tissue, representing de2f1 mutant cells,can be seen in the eye (Figure 1E). Thus, the small sizeof clones of de2f1 mutant cells is a highly consistentphenotype, which is sensitive to inactivation of de2f2. Weconcluded that the de2f1 mutant phenotype might besuitable for a suppressor mosaic genetic screen to identifynovel genes that affect the dE2F2/RBF function.

To test our screening strategy we initially performedan F1 screen. Isogenic FRT82B de2f1729/TM3 males weremutagenized with EMS and crossed to ey-FLP; FRT 82Bl(3)cl-R31/TM6B females. The male F1 progeny of theseflies were scored for the appearance of white patchesindicating the presence of a potential Su(E1), whichallows for the recovery of de2f1 mutant tissue. Suchexceptional males were crossed to females carrying thethird chromosomal balancers and then the male prog-eny were backcrossed to the ey-FLP; FRT 82B l(3)cl-R31/TM6B females to confirm the suppression of the de2f1mutant phenotype. A total of 18,000 chromosomes werescreened, and 75 F1 males having white patches of thede2f1 Su(E1) double mutant tissue were selected. How-ever, only 8 mutants were successfully retested (Table 1).To improve the recovery of suppressors, we performedan F2 screen in which mutagenized FRT82B de2f1729

chromosomes were first balanced. Then FRT82B

TABLE 1

Summary of the screen for suppressors of the de2f1 mutant phenotype

ScreenChromosomes

screenedSuppressors

isolatedSuppressors

retestedStrong

suppressorsWeak

suppressors

F1 �18,000 75 8 2 6F2 �40,000 244 244 84 160


de2f1729/TM3 or FRT82B de2f1729/MKRS males carryinga potential Su(E1) were crossed individually to the ey-FLP; FRT 82B l(3)cl-R31/TM6B females (Figure 2). Of44,600 individual crosses �40,000 crosses producedprogeny and 244 mutant lines exhibited a suppressionof the de2f1 mutant phenotype (examples are shown inFigure 1, F–J). Males of each of the 244 lines were back-crossed twice to females carrying the balancer chromo-somes to replace the ey-FLP-carrying X chromosomewith a wild-type X chromosome. Additionally, thesemales were retested to confirm consistency of the sup-pression of the de2f1 mutant phenotype.

To determine whether the five Su(E1)’s shown inFigure 1, F–J, are able to rescue the de2f1 mutantphenotype in tissues other than the eye, we inducedclones of de2f1 Su(E1) double mutant cells in the thirdinstar larval wing imaginal disc, using Ubx-FLP. The Ubx-FLP/FRT technique provides a high level of mitoticrecombination in multiple tissues. In the wing imaginaldisc, the homozygous wild-type tissue can be easilydistinguished from homozygous mutant tissue by thepresence of the GFP signal (Figure 1K). As expected,patches of clones of de2f1 mutant cells are limited toapproximately one to five cells and therefore very littlede2f1 homozygous mutant tissue (GFP negative) isfound in the wing disc (Figure 1L). In contrast, muchlarger clones of de2f1 Su(E1) double mutant cells can berecovered (Figure 1, M–Q). Thus, examined Su(E1)’sare able to at least partially rescue the strong prolif-eration defects of de2f1 mutant cells in multipletissues.

Initial classification of suppressors: Next, we testedwhether the suppressors rescue the proliferation defectsof de2f1 mutant cells without the aid of the cell lethalmutation, l(3)cl-R31, on the wild-type chromosome. Theability of isolated mutations to rescue cell proliferationof de2f1 mutant cells in these settings provided a

rigorous test of the strength of suppression since bothwild-type and the mutant cells compete against eachother in equal conditions. All of the 252 Su(E1)’s thatwere isolated in the F1 and F2 screens were crossed to ey-FLP; FRT82B P[Ubi-GFP ; w1] females and the eyes of theprogeny were examined for the appearance of clones ofmutant cells. A total of 166 mutants were characterizedas weak suppressors while 86 suppressors exerted arobust effect on the de2f1 mutant phenotype giving riseto visible white patches of de2f1 mutant tissue in the eye(Table 2). Among these 86 lines, five mutants produceda very strong rough eye phenotype with occasional scarsindicating that these suppressors are likely to affectdevelopmental pathways and therefore were not con-sidered further.

Mapping and complementation analysis: We used alethality potentially associated with Su(E1)’s in an initialcomplementation analysis. Although the lethal muta-tions are not necessarily in each case Su(E1)’s that areresponsible for the phenotype, such an approach allowsfor quick mapping of lethal Su(E1)’s. However, thepresence of a lethal de2f1 mutation on each of theSu(E1)-containing chromosomes complicates comple-mentation and mapping analyses. Each Su(E1) is lethalin trans to each other due to the presence of the de2f1729

mutation. Similarly, this precludes a standard mappingby meiotic recombination onto a chromosome withmultiple recessive markers, since the presence of aSu(E1) can be determined only in the background ofthe de2f1 mutation. As an alternative way to estimate howmany genes were represented by the 81 strongestSu(E1)’s, we tested these lines in trans to a combinedExelixis and DrosDel deficiency kit that uncovers theright arm of chromosome 3 (for details see materials

and methods). Forty-nine lines fully complementedthe deficiency kit, indicating that either correspondingSu(E1)’s are viable or they are located within the limited

Figure 2.—Design of the F2 mosaic screen torecover suppressors of the de2f1 mutant pheno-type.


intervals between the breakpoints of the deficiencies.Each of the remaining 32 mutants were lethal in trans toa particular deficiency (Table 2). To determine whetherthe group of weak mutants contained additional alleles,we crossed weak mutants to identified deficiencies. Inthis way, 7 additional mutant alleles were identified.Publicly available known mutations and P-element in-sertions in the relevant regions uncovered by the defi-ciencies were tested for complementation to determinethe identities of Su(E1)’s. This allowed us to identify newalleles of l(3)mbt, Doa, gfzf, B52, and CG31133 as suppres-sors of the de2f1 mutant phenotype. The mapping resultsare summarized in Table 2 and described below.

l(3)mbt: Seven suppressors (6a14, 4d30, 3d23, 5d8,4d16, 5d6, 6a31, and 3d33) were mapped to the intervalbetween 97E2 and 98A7. Each of these alleles was lethalin trans to the deficiency Df(3R)ED6265. One of thegenes uncovered by the deficiency is l(3)mbt, whichencodes a protein associated with the dE2F2/RBFrepressor complex DREAM/MMB (Lewis et al. 2004).Each of the seven suppressors was lethal in trans to thenonsense allele l(3)mbtE2 and to the P-element insertionin l(3)mbt, f02565. Additionally, all seven were lethal intrans to a deficiency, Df(3R)D605, which uncoversl(3)mbt. This suggests that this group of Su(E1)’s is allelicto l(3)mbt.

Doa: Another group included five Su(E1)’s: 6d2, 6a21,5d19, 6a52, and 5d7. Each mutant of this group failed tocomplement Df(3R)Exel6210. Testing for complementa-tion with known mutations and P-element insertions inthese regions identified an allele of the Darkener of apricot(Doa) gene, Doa01705b, which failed to complement eachof five Su(E1)’s. Similar results were obtained with twoother Doa alleles, DoaKG09056 and Doa3. This suggests thatthis group of Su(E1)’s consists of mutations in the Doagene. Doa mutations were initially isolated as suppres-sors of the weak white-apricot allele (Rabinow et al. 1993).

B52: The B52 gene encodes an SR protein that isrequired for the correct splicing of the de2f2 pre-mRNA.Accordingly, mutations in B52 were shown to rescueproliferation of de2f1 mutant cells (Rasheva et al. 2006).Two new mutations in the B52 gene, 3d16 and 3d25,were isolated. Both 3d16 and 3d25 failed to complementthe deficiency uncovering the B52 gene Df(3R)Exel6169and the B52s2249 mutant allele.

CG31133: A Su(E1), 7d21, was mapped to the region95E5–F8, which is uncovered by the deficienciesDf(3R)Exel6198 and Df(3R)ED6187. To further narrowdown the position of 7d21, we generated two smallerdeletions, Df(3R)7d21.I and Df(3R)7d21.II, within thisinterval according to the method described in Parks

et al. (2004). In this technique, a deletion is generated

TABLE 2

Genes isolated in the screen for suppressors of the de2f1 mutant phenotype

Suppressor/gene name Alleles Map position Annotation

l(3)mbt 6a14, 4d30, 3d23, 5d8,4d16, 5d6, 6a31, 3d33

97F1 Associated with the MMB/DREAMrepressor complex

Doa 6d2, 6a21, 5d19, 6a52, 5d7 98F2 LAMMER protein kinase,phosphorylates SR proteinsincluding B52

gfzf/CG31329 6a27 84C6–84D1 FLYWCH zinc finger proteinB52 7d7, 5d21, 3d16, 3d24 87F7 SR protein splicing factorCG31133 7d21 95E5 Unknown proteinbelle 7d19 85A2–5 DEAD-box RNA helicaseSu(E1)-A 7d13, 6a39 99D5–E2Su(E1)-B 4d26, 6a11 89A5–8Su(E1)-C 1d24, 3d28 86E11–87B11Unannotated single hits

Su(E1)-D 6a47 84C8–D9Su(E1)-E 3d25 84D9–E11Su(E1)-F 6b9 85F10–16Su(E1)-G 6a3 87F10–14Su(E1)-H 6a50 87F14–88A14Su(E1)-I 6d9 89E11–90C1Su(E1)-J 6a8 94A9–B2Su(E1)-K 6b12 94B2–B5Su(E1)-L 6b19 95B1–5Su(E1)-M 6b31 95E5–F8Su(E1)-N 4d22 99A5Su(E1)-O 6a19 99A5–B2

Two B52 alleles, B527d7 and B525d21, are described in Rasheva et al. (2006).Mutant allele belle7d19 is described in Ambrus et al. (2007).


between two FRT-bearing insertions in the presence ofthe FLP recombinase. We utilized a pair of XP inser-tions, d01966 and d05015, to generate Df(3R)7d21.Iwhile Df(3R)7d21.II was recovered using a pair of XPinsertions, d09860 and d05397. 7d21 was found to belethal in trans to Df(3R)7d21.I, but not to Df(3R)7d21.II.Among publicly available mutations in genes uncoveredby Df(3R)7d21.I only insertion f07858 failed to comple-ment 7d21. f07858 is an insertion in the gene CG31133,which encodes a protein of unknown function. Tounambiguously confirm that a mutation in CG31133 isresponsible for the rescue of the de2f1 mutant pheno-type, we recombined CG31133 f07858 with the de2f1729

mutant allele and found that clones of de2f1729

CG31133f07858 double mutant cells can be recovered inthe eye (data not shown). This suggests that CG31133 isa novel suppressor of the de2f1 mutant phenotype.

GST-containing FLYWCH zinc-finger protein: A loneSu(E1) 6a27 failed to complement two deficienciesDf(3R)ED5221 and Df(3R)ED7665, which uncover theinterval 84C4–E10. Candidate mutations in this intervalwere tested for their ability to complement 6a27. 6a27

was lethal in trans to an EP insertion EY08448. EY0848 isinserted into the GST-containing FLYWCH zinc-fingerprotein (gfzf ) gene, which is located within a secondintron of CG2656. Thus, EY08448 disrupts both gfzf andCG2656. To determine which of the two genes is affectedby 6a27, we used the KG08427 and e00303 P-elementinsertions in a complementation test with 6a27. InKG08427 and in e00303, the P element is inserted intothe first and the second exons of CG2656, respectively.Hence, the P-element insertions are upstream of the gfzfgene and therefore, gfzf function is unlikely to beaffected by either KG08427 or e00303. 6a27 was foundto be fully viable in trans to KG08427 and e00303. Incontrast, 6a27 failed to complement two gfzf mutantalleles, gfzf 2 and gfzf cz811 . gfzf cz811 contains a deletion atresidue F469, producing a frameshift to a stop codon atresidue 476 (Provost et al. 2006). Thus, 6a27 fails toexclusively complement mutations in the gfzf gene, butis fully viable in trans to mutant alleles of CG2656. Thesedata suggest that 6a27 is a novel mutant allele of gfzf.

Su(E1)-A: Two Su(E1)’s, 7d13 and 6a39, were lethal intrans to Df(3R)Exel6214, which deletes the region of

Figure 3.—The SMW in clones of de2f1Su(E1) double mutant cells. Eye imaginaldiscs were dissected from third-instar lar-vae. Clones of wild-type cells, identifiedby the presence of GFP (green), and ho-mozygous mutant cells, identified by theabsence of GFP (green) were induced byey-FLP. Genotypes are shown, the morpho-genetic furrow (MF) is marked by an arrow-head, and posterior is to the right. (A–J)Eye imaginal discs were labeled with BrdU(red) to visualize cells in S phase. (A) Wild-type disc showing only BrdU incorpora-tion. (B and C) Suppressors l(3)mbt3d33

(B) and Su(E1)-A7d13 (C) strongly rescuethe SMW in de2f1 mutant cells as evidencedby the entry into and exit from the SMWin double mutant cells at approximatelythe same time as adjacent wild-type cells.(D–F) In contrast, suppressors CG311337d21

(D), gfzf6a27 (E), and Doa6d2 (F) only weaklyrescue the SMW in de2f1 mutant cells asevidenced by BrdU incorporation begin-ning and ending several columns moreposterior in double mutant cells than inadjacent wild-type cells. (G–J) BrdU incor-poration in clones of single mutant sup-pressors Su(E1)-A7d13 (G), CG311337d21

(H), and gfzf 6a27 (I) shows no SMW defectsas evidenced by the entry into and exitfrom the SMW in single mutant cells at ap-proximately the same time as adjacent wild-type cells. Doa KG09056 single mutant clonesshow a slightly delayed SMW entry ( J),but the delay is less extreme than the delayin de2f1729 Doa6d2 double mutant cells (F).(B9–J9) The same corresponding imagesas B–J showing only BrdU (red). Clonesof mutant cells are outlined.


99D5–E2. According to FlyBase (Drosophila genomesequence R5.17), there are 17 predicted genes withinthis interval. We tested available mutant alleles ofCG15531, CG7951, CG2184, CG31027, and CG9747and found that each fully complements 7d13 and6a39, indicating that these 5 genes do not correspondto Su(E1)-A. We have sequenced the ORFs and intron–exon junctions of the remaining 12 genes as well asCG15531 and CG9747 (for details see materials and

methods). No newly induced mutations in these geneswere found. There are a couple of possible explanationsto account for this. First, lethality associated with Su(E1)-Amight be unrelated to the suppressor effect. This seemsunlikely given that we isolated two independent alleles,7d13 and 6a39, which were mapped to the same genomicinterval. Another explanation is that these alleles carrymutations in a regulatory region of a gene. Such muta-tions can potentially affect the level of transcription ortranslation but would be missed in our analysis. Finally, it ispossible that there is another currently unannotated genewithin this genomic interval that corresponds to Su(E1)-A.

Phenotypic analysis: For our initial analysis, westudied the phenotypes of five above-mentioned

Su(E1)’s by examining markers of cell proliferation inclones of de2f1 Su(E1) double mutant cells in the eyeimaginal disc since the pattern of cell proliferation iswell characterized. In the larval eye imaginal disc, thepattern of cell divisions is defined by the position of themorphogenetic furrow (MF), which traverses fromposterior to anterior during development. We labeledthe eye disc with BrdU to visualize cells in S phase. In thewild-type disc, cells are asynchronously dividing anteriorto the MF and then become transiently arrested in G1

within the MF (Figure 3A). Upon emerging from the G1

arrest in the MF, cells enter the last S phase in a highlysynchronous manner to form a tight stripe of BrdU-positive cells called the second mitotic wave (SMW).Posterior to the SMW cells exit the cell cycle and committo neuronal differentiation. Unlike wild-type cells, de2f1mutant cells fail to enter the SMW (Du 2000). Thisdefect is due to the presence of de2f2 since de2f1 de2f2double mutant cells and dDP single mutant cells enterthe SMW normally although with a slightly reduced rateof S-phase progression (Frolov et al. 2005; Ambrus et al.2007). Therefore, we categorized Su(E1)’s by the extentto which they restore the SMW in de2f1 mutant cells. We

Figure 4.—Mitotic marker phospho-H3 inclones of de2f1 Su(E1) double mutant cells. Eyeimaginal discs were dissected from third-instarlarvae. Clones of wild-type cells, identified bythe presence of GFP (green), and homozygousmutant cells, identified by the absence of GFP(green), were induced by ey-FLP. Genotypes areshown, and posterior is to the right. (A–I) Eyeimaginal discs were labeled with anti-phospho-H3 (magenta) to mark cells in mitosis. (A)Wild-type disc showing only phospho-H3. (B–I)de2f1729 l(3)mbt3d33 (B), de2f1729 Su(E1)-A7d13 (C),de2f1729 CG311337d21 (D), de2f1729 gfzf 6a27 (E),Su(E1)-A7d13 (F), de2f1729 CG311337d21 (G),de2f1729 gfzf 6a27 (H), and DoaKG09056 (I) cells pro-gress through mitosis as evidenced by phospho-H3 (magenta)-positive cells in clones of mutanttissue.


chose to analyze 3d33, 6d2, 6a27, 7d21, and 7d13, whichrepresent mutant alleles of l(3)mbt, Doa, gfzf, CG31133,and Su(E1)-A, respectively. Clones of double mutantcells carrying a Su(E1) and the de2f1729 allele wereinduced with ey-FLP. Homozygous mutant tissue wasdistinguished by the lack of GFP. From this analysis wefound that Su(E1)’s restored the SMW in de2f1 mutantcells to varying extents. Mutant alleles of l(3)mbt andSu(E1)-A provided a stronger rescue of the SMW in de2f1mutant cells as evident by the appearance of BrdU-positive de2f1 l(3)mbt3d33 and de2f1 Su(E1)-A7d13 doublemutant cells in the SMW at about the same time as in theadjacent wild-type cells (Figure 3, B and C). However, wenote that the intensity of BrdU labeling was somewhatreduced in de2f1 Su(E1)-A7d13 mutant cells. In contrast tol(3)mbt and Su(E1)-A, de2f1 CG311337d21 double mutantcells entered the SMW several columns more posteriorthan wild-type cells (Figure 3D), which is indicative ofa delay of entry into the S phase. Additionally, BrdUincorporation persisted several columns more posteriorin the double mutant cells compared to the adjacentwild-type tissue. This indicates that it takes longer thannormal for de2f1 CG311337d21 double mutant cells to

progress through the S phase, suggesting that the Sphase is likely to be slowed. The defects in the SMW wereless noticeable in the case of the gfzf 6a27 mutant allele(Figure 3E). Although occasionally we observed clones ofde2f1 gfzf 6a27 double mutant cells in which entry into theSMW occurred relatively on time, in most cases, theBrdU-positive mutant cells were situated farther poste-rior. Finally, de2f1 Doa6d2 double mutant cells were onlysporadically labeled with BrdU in the SMW. However, anyde2f1 Doa6d2 double mutant cells that did incorporateBrdU were always found to be significantly more poste-rior than BrdU incorporating adjacent wild-type cells(Figure 3F). Taken together these results indicate thatSu(E1)’s differ among each other by the extent of rescueof the timing of entry into and exit from the SMW in de2f1mutant cells. Clones of de2f1 l(3)mbt and de2f1 Su(E1)-Amutant cells enter and exit the SMW relatively on timewhile de2f1 gfzf 6a27, de2f1 CG311337d21, and de2f1 Doa6d2

exhibit a delay in the entry into and/or exit from theSMW.

The extent of the defects in the SMW may reflect theability of each Su(E1) to rescue the cell cycle arrest inde2f1 mutant cells. Alternatively, Su(E1) mutant cells

Figure 5.—Neuronal marker ELAV inclones of de2f1 Su(E1) double mutant cells.Eye imaginal discs were dissected fromthird-instar larvae. Clones of wild-typecells, identified by the presence of GFP(green), and homozygous mutant cells,identified by the absence of GFP (green),were induced by ey-FLP. Genotypes areshown, and posterior is to the right. Cellswere stained with the neuronal markerELAV (red). (A–D) The onset of neuro-nal differentiation in de2f1729 l(3)mbt3d33

(A), de2f1729 Su(E1)-A7d13 (B), de2f1729

CG311337d21 (C), and de2f1729 gfzf 6a27 (D)double mutant cells and Su(E1)-A7d13 (F),CG311337d21 (G), and gfzf 6a27 (H) singlemutant cells initiates at the same time asin the adjacent wild-type cells. (E and I)There is a slight delay in the onset of neu-ronal differentiation in de2f1729 Doa6d2 (E)double mutant cells and in DoaKG09056 (I)single mutant cells. (A9–I9) The same cor-responding images as A–I showing onlyELAV (red). Clones of mutant cells are out-lined.


themselves could have an aberrant SMW. To discrimi-nate between these two possibilities we examined BrdUincorporation in the SMW of clones of Su(E1)-A,CG31133, gfzf, and Doa single mutant cells. As shownin Figure 3, G–J, Su(E1)-A, CG31133, or gfzf singlemutant cells enter and exit the SMW at about the sametime as the adjacent wild-type cells, while the SMW entryis slightly delayed in Doa single mutant cells. However,this delay is not as extreme as that observed in de2f1 Doadouble mutant cells (Figure 3F). We infer from theseresults that the defects in the SMW of clones of de2f1729

Su(E1) mutant cells likely reflect the ability of eachSu(E1) to rescue the SMW in de2f1729 mutant cells.

To further characterize the effect of Su(E1)’s on theSMW in de2f1 mutant cells we used phospho-H3 as amitotic marker (Figure 4). In wild-type eye discs, thestripe of phospho-H3-positive cells posterior to the MFmarks cells of the SMW (Figure 4A). Importantly, wefound phospho-H3-positive double mutant and singlemutant cells in the SMW (Figure 4, B–I). Expression ofELAV, a marker of neuronal differentiation, is induced

in double and single mutant cells although de2f1 Doadouble mutant and Doa single mutant cells exhibited aslight delay in the onset of ELAV expression (Figure 5).This could be a consequence of a delay in exit from theSMW. In no cases did we find cells that coexpress phospho-H3 and ELAV. This suggests that de2f1 Su(E1) doublemutants finish the SMW and commit to differentiation.

l(3)mbt affects expression of an E2F reporter in cellculture-based assays differently than gfzf or CG31133:Having established that the loss of l(3)mbt restores thetiming of the S-phase entry in the SMW in de2f1 mutantcells, while mutations in gfzf and CG31133 do not, wewished to determine whether inactivation of these genesdifferentially affects E2F-dependent transcription indE2F1-deficient cells. We employed RNA interference(RNAi) in Drosophila S2R1 tissue culture cells to depletedE2F1, dE2F2, RBF, L(3) malignant brain tumor(MBT), GFZF, and CG31133 proteins and then tran-siently transfected these cells with a reporter containingthe endogenous PCNA promoter fused to a luciferasegene. The PCNA gene is a direct transcriptional target of

Figure 6.—Effect of Su(E1)’s on E2F-depen-dent transcription in tissue culture cells. (A)Depletion of dE2F1 represses expression of aPCNA-luc reporter. Codepletion of L(3)MBT, ordE2F2, or RBF1 (R1) and RBF2 (R2), togetherwith dE2F1, derepresses expression of the E2F re-porter. S2R1 cells were incubated with eithercontrol or experimental double-stranded RNA(dsRNA) as indicated. Cells were then incubatedfor an additional day with either control (NS) ordE2F1 dsRNA before being cotransfected with aPCNA-luc reporter and a b-Gal expression plas-mid in duplicates. (B) Codepletion of L(3)MBTor dE2F2 (E2) with dE2F1 (E1) derepresses ex-pression of the endogenous PCNA gene. S2 cellswere incubated with either control (NS) or corre-sponding dsRNAs as indicated. After 4 days,the steady-state level of the PCNA mRNA wasdetermined by real-time reverse transcription–PCR in triplicates. (C) The basal level of expres-sion of a PCNA-luc reporter is elevated in cellsdepleted of either GFZF or CG31133 proteins.S2R1 cells were incubated with control (NS)or corresponding dsRNAs as indicated. After4 days, cells were transfected with a PCNA-luc re-porter in duplicates. (D) S2R1 cells were incu-bated with control (NS) or correspondingdsRNAs as indicated. After 4 days cells were lysedand the level of dE2F1 was determined by West-ern blot analysis. Tubulin was used as a loadingcontrol. (E) Codepletion of GFZF or CG31133with RBF1 (R1) and RBF2 (R2) synergistically ac-tivates a PCNA-luc reporter. After 4 days, cellswere cotransfected with a PCNA-luc reporterand a b-Gal expression plasmid in duplicates.


dE2F and has been previously validated as an accuratereadout of dE2F transcriptional activity (Frolov et al.2001). As expected, the E2F reporter is strongly re-pressed in dE2F1-depleted cells (Figure 6A). This is dueto the presence of dE2F2 because the repression wasalleviated when dE2F1 and dE2F2 were inactivatedsimultaneously. Accordingly, depletion of RBF proteins,which are required for repression by dE2F2, also re-lieved repression of the reporter (Figure 6A). Thus,expression of the E2F reporter in dE2F1 single- and indE2F1 dE2F2 double-depleted cells essentially recapit-ulates the response of the endogenous PCNA gene inmutant animals or in tissue culture cells (Frolov et al.2005). Inactivation of GFZF or CG31133 had no effecton the E2F reporter in dE2F1-deficient cells, as evi-denced by the reporter remaining fully repressed inthese cells (Figure 6A). In contrast, depletion ofL(3)MBT relieved the repression of the reporter indE2F1-deficient cells to the same extent as inactivationof dE2F2 (Figure 6A). To test whether repression of anendogenous PCNA gene in dE2F1-depleted cells isalleviated by codepletion of L(3)MBT, we used real-time RT–PCR to monitor the steady-state PCNA mRNAlevel. As has been previously shown, PCNA expression isreduced in dE2F1-depleted cells (Frolov et al. 2005).Consistent with the results in transient transfections, therepression of PCNA was relieved in dE2F1 L(3)MBTdouble-deficient cells to a level comparable to that ofdE2F1 dE2F2-depleted cells (Figure 6B). Thus, inactiva-tion of L(3)MBT alleviates the repression of an E2Ftarget in dE2F1-depleted cells as efficiently as removalof dE2F2, indicating that L(3)MBT is needed for dE2F2to repress.

Next, we determined the effect of depletion ofL(3)MBT, CG3133, GFZF, and DOA on the basal levelof expression of the E2F reporter. Interestingly, althoughdepletion of GFZF or CG31133 does not relieve dE2F2-mediated repression, we found that the E2F reporter isconsistently elevated in cells lacking GFZF or CG31133(Figure 6C). This effect cannot be attributed to anelevation of dE2F1 protein since dE2F1 levels are un-altered when any of the Su(E1)’s are depleted in cellculture by RNAi (Figure 6D). To determine whetherthese effects are mediated through pocket proteins wedetermined the epistatic effect of depletion of GFZF andCG31133 relative to RBF proteins. As expected, deple-tion of RBF proteins elevates expression of the E2Freporter (Figure 6E), which is likely due to release of aninhibitory effect of RBFs on endogenous dE2F1. Code-pletion of L(3)MBT does not potentiate the effect ofinactivation of RBF proteins. This is consistent with thefinding that L(3)MBT and RBF are present together inthe same repressor complex (Lewis et al. 2004). UnlikeL(3)MBT, codepletion of GFZF and RBFs or CG31133and RBFs resulted in strong additive effects on activationof the E2F reporter as compared to targeting either ofthem alone (Figure 6E). These observations support the

idea that inactivation of GFZF or CG31133 elevatesactivation of the E2F reporter, but this occurs in anRBF-independent manner.

DISCUSSION

Although a critical role of the pRB/E2F pathway innormal cell proliferation and in cancer has been firmlyestablished, our understandings of what aspects ofpRB/E2F function are important in vivo and howprecisely the pathway is regulated during developmentare still very limited. As a way to begin to addressthese important questions, we have designed a mosaicgenetic screen in Drosophila to isolate genes that whenmutated overcome the dE2F2/RBF-dependent blockto cell proliferation in de2f1 mutant cells. Severalgenetic modifier screens have been previously per-formed in Drosophila to dissect the pRB/E2F pathway(for examples see Staehling-Hampton et al. 1999;Lane et al. 2000; Weng et al. 2003). These screensprovided critical contributions to the understandingof mechanisms underlying actions of pRB andE2F. However, such screens were designed to exclu-sively isolate haploinsufficient modifiers in the back-ground of ectopically expressed rbf, de2f, and cyclin Egenes, thus limiting the spectrum of potential modi-fiers. The screening strategy described in this workavoids complications associated with protein overex-pression since the cell cycle block in de2f1 mutant cellsis caused by the endogenously present de2f2. Addition-ally, the FLP/FRT technique allows for the isolation ofrecessive suppressors. These are the two major advan-tages of our approach from the above-mentionedscreens.

The genetic screen reported here identified genesthat are known to be required for dE2F2 function as wellas novel genes that were not previously linked to thepRB/E2F pathway. Of the complementation groupsisolated in the screen, l(3)mbt and B52 have beenpreviously implicated in dE2F2-mediated repression(Lewis et al. 2004; Rasheva et al. 2006; Lu et al. 2007).L(3)MBT has been biochemically purified as a compo-nent of a native dE2F/RBF complex Drosophila RBFE2F and MYB/Myb-MuvB (DREAM/MMB), although ina substoichimetric ratio (Lewis et al. 2004). Mutations inl(3)mbt give rise to malignant transformations in thelarval brain as well as disruption of synchronous celldivisions in early embryos (Yohn et al. 2003; Trojer andReinberg 2008). l(3)mbt encodes a protein with threeMBT domains that are structurally similar to otherchromatin-binding domains such as the chromodo-main. Its mammalian homolog L3MBTL1 is a transcrip-tional repressor that is thought to promote the higher-order chromatin structure by simultaneously binding totwo adjacent nucleosomes and moving nucleosomescloser together. Such binding is dependent on thepresence of mono- and dimethylation of histone H4 at


lysine 20, which is commonly found in facultativeheterochromatin (Trojer et al. 2007). L3MBTL1 asso-ciates with pRB and is needed for repression of E2F-regulated genes c-myc and cyclin E in mammalian cells(Trojer et al. 2007). Interestingly, depletion ofL(3)MBT in Drosophila tissue culture cells results inderepression of genes that are normally kept silent bydE2F2/RBF in asynchronously dividing cells (Lewis

et al. 2004; Lu et al. 2007). In this respect, isolation ofl(3)mbt as a suppressor of the de2f1 mutant phenotypedescribed here is significant because it provides the firstgenetic evidence for the importance of l(3)mbt in de2f2-dependent cell cycle arrest in vivo. This conclusion isfurther supported by the finding that L(3)MBT isneeded for dE2F2 to repress in dE2F1-deficient cells.Finally, isolation of l(3)mbt serves as a proof of principlethat genes that interface directly with the dE2F/RBFmodule can be identified as suppressors of the de2f1mutant phenotype.

In addition to l(3)mbt, three genes, rbf2, Chromatinassembly factor 1 subunit (Caf1/p55), and rpd3, reside on3R and encode components of the DREAM/MMBcomplex. However, rpd3 is not required for the re-pression of dE2F2/RBF-regulated genes (Korenjak

et al. 2004; Lewis et al. 2004; Taylor-Harding et al.2004; Georlette et al. 2007) while rbf2 is primarilyinvolved in E2F-dependent repression in embryos, butnot in imaginal discs (Stevaux et al. 2005), andtherefore it is not surprising that mutant alleles ofrpd3 and rbf2 were not isolated in this screen. The lack ofCaf1/p55 mutations among Su(E1)’s is somewhat puz-zling given that inactivation of CAF1/p55 abrogatesdE2F2-mediated repression (Taylor-Harding et al.2004). One possibility is that the CAF1/p55 functionitself is needed during cell proliferation due to its role inDNA replication. Alternatively, since no mutant allelesin Caf1/p55 have been described to date it is not knownwhether mutations in Caf1/p55 are lethal. If the loss ofCaf1/p55 does not result in lethality, then mutant allelesof Caf1/p55 would be apparently missed in our initialmapping approach using lethality as a complementa-tion test.

Among Su(E1)’s we have isolated multiple mutantalleles of the Doa gene. Doa has been implicated in avariety of cellular functions including differentia-tion and cell cycle progression (Yun et al. 2000;Bettencourt-Dias et al. 2004; Bjorklund et al. 2006).The Doa gene encodes a protein kinase that is bestknown for its role in the regulation of alternativesplicing through phosphorylation of multiple SR pro-teins, among which are TRA, TRA2, and B52 (Nikola-

kaki et al. 2002). Since B52 is required for splicing ofde2f2 pre-mRNA and B52 is a Su(E1) (Rasheva et al.2006), we considered the possibility that mutation ofDoa rescues the de2f1 mutant phenotype through itsregulation of B52. So far we have not found evidenceto support this model. Inactivation of Doa does not

alleviate repression of endogenous dE2F2 targets in eyediscs or in tissue culture cells. Additionally, depletion ofDOA has no effect on dE2F2-dependent repression intransient transfection assays. Finally, there was nodifference in the dE2F2 level in clones of Doa mutantcells or when DOA was depleted by RNAi in S2 cells(data not shown). Thus, it is possible that although DOAphosphorylates B52 in an in vitro kinase assay, B52 maynot be an endogenous substrate of DOA. Alternatively,regulation of B52 by DOA may be tissue specific orregulation of B52 by means of phosphorylation in vivocould be directed by multiple SR protein kinases.

In contrast to depletion of L(3)MBT (this work) andB52 (Rasheva et al. 2006), depletion of GFZF andCG31133 does not compromise dE2F2/RBF-mediatedrepression, but instead we found that expression of theE2F reporter is elevated in these cells. Since the E2Freporter is sensitive to the level of endogenous E2Factivity, this raises the possibility that inactivation ofGFZF or CG31133 may potentiate dE2F1 activitythrough, for example, the release of RBF inhibition ofdE2F1. Such an explanation seems unlikely sincecodepletion of RBF together with GFZF or CG31133exerts an additive effect on the E2F reporter, suggestingthat the two Su(E1)’s likely act in parallel to the RBF/E2Fpathway. Finally, since both gfzf and CG31133 wereisolated in the background of a de2f1 mutation, thisdisfavors the interpretation that they act throughdE2F1. We suggest that CG31133 and GFZF may directlyor indirectly cooperate with dE2F1 on a subset of targetgenes. How precisely GFZF and CG31133 elevate theexpression of the E2F reporter is not known and furtherexperiments will be needed to dissect the molecularmechanism of this effect.

Interestingly, the S-phase entry and exit in the SMWare delayed and the S phase appears to be extended inde2f1 gfzf and de2f1 CG31133 double mutant cells. Inanother example, rescue of the de2f1 mutant phenotypeby a mutation in the gene belle is not accompanied by thefull restoration of the SMW in de2f1 belle double mutantcells (Ambrus et al. 2007). Similarly, the loss of belle doesnot relieve dE2F2-mediated repression. In contrast,mutations of l(3)mbt (this work and Lewis et al. 2004;Lu et al. 2007) and B52 (Rasheva et al. 2006) compro-mise dE2F2-mediated repression while de2f1 l(3)mbtand de2f1 B52 double mutant cells enter and exit theSMW relatively on time. Thus, it is tempting to suggestthat relief of dE2F2-mediated repression is needed forde2f1 mutant cells to rescue the correct timing of theSMW. Since prior to the S phase entry in the SMW cellsis transiently arrested in G1 within the MF, this mayexplain why de2f1 mutant cells in the SMW are partic-ularly sensitive to the reduced level of E2F targets. Thisidea is in agreement with the results of experimentsin mammalian cells with a dominant negative form ofE2F, which suggested that E2F-dependent activation isneeded for cell cycle reentry from quiescence (Rowland


and Bernards 2006). Future studies of other isolatedsuppressors of the de2f1 mutant phenotype are likely toprovide novel insights into the growth suppressivefunction of dE2F2/RBF in vivo.

We are grateful to the Developmental Studies Hybridoma Bank(University of Iowa) and the Bloomington Stock Center for fly stocksand antibodies. We thank D. Knight, K. Marsh, R. Suckling, andH. Summersgill for technical assistance and N. Dyson, A. Katzen,G. Ramsey, and M. Truscott for critical discussions. This work was sup-ported by grant GM079774 from the National Institutes of Health toM.V.F. and by predoctoral fellowship 0815661G from the AmericanHeart Association to A.M.A.

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Communicating editor: J. Tamkun


Mosaic Genetic Screen for Suppressors of the de2f1 Mutant ... · Duronio 2001; Dimova and Dyson...

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