Download Mosaic Genetic Screen for Suppressors of the de2f1 Mutant

Copyright Ó 2009 by the Genetics Society of America
DOI: 10.1534/genetics.109.104661
Mosaic Genetic Screen for Suppressors of the de2f1 Mutant
Phenotype 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, 2009
Accepted for publication June 6, 2009
ABSTRACT
The growth suppressive function of the retinoblastoma (pRB) tumor suppressor family is largely
attributed to its ability to negatively regulate the family of E2F transcriptional factors and, as a result, to
repress E2F-dependent transcription. Deregulation of the pRB pathway is thought to be an obligatory event
in most types of cancers. The large number of mammalian E2F proteins is one of the major obstacles that
complicate their genetic analysis. In Drosophila, the E2F family consists of only two members. They are
classified as an activator (dE2F1) and a repressor (dE2F2). It has been previously shown that proliferation of
de2f1 mutant cells is severely reduced due to unchecked activity of the repressor dE2F2 in these cells. We
report here a mosaic screen utilizing the de2f1 mutant phenotype to identify suppressors that overcome the
dE2F2/RBF-dependent proliferation block. We have isolated l(3)mbt and B52, which are known to be
required for dE2F2 function, as well as genes that were not previously linked to the E2F/pRB pathway such
as Doa, gfzf, and CG31133. Inactivation of gfzf, Doa, or CG31133 does not relieve repression by dE2F2. We
have shown that gfzf and CG31133 potentiate E2F-dependent activation and synergize with inactivation of
RBF, suggesting that they may act in parallel to dE2F. Thus, our results demonstrate the efficacy of the
described screening strategy for studying regulation of the dE2F/RBF pathway in vivo.
T
HE family of E2F transcription factors and retinoblastoma (pRB) family tumor suppressor proteins
play a pivotal role in control of cell proliferation(reviewed
in Cayirlioglu and Duronio 2001; Trimarchi and
Lees 2002; Blais and Dynlacht 2004; Dimova and
Dyson 2005; Degregori and Johnson 2006). Although
E2F is involved in a variety of cellular activities, the best
understood function of E2F is to regulate transcription
of genes at the G1/S transition. Among E2F targets are
genes that encode regulators of S-phase entry and
components of the DNA replication machinery. In
mammals, there are eight E2F genes. E2F-1 through
E2F-6 function as heterodimers with a DP subunit, while
E2F-7 and E2F-8 do not require DP to bind to DNA. In
spite of structural similarities among E2F proteins, E2F1, E2F-2, and E2F-3a are predominately involved in
activation of gene expression while the group E2F-3b,
E2F-4, E2F-5, E2F-6, E2F-7, and E2F-8 behave as
repressors. In quiescent cells, when activity of cyclindependent kinases (cdks) is low, repressor E2Fs are
complexed with the pRB family members (also called
pocket proteins) and repress expression of E2F regulated genes. The prevailing mechanism of the repression
is thought to be through the direct recruitment of
1
These authors contributed equally to this work.
Corresponding author: Department of Biochemistry and Molecular
Genetics, University of Illinois, MBRB 2352, MC 669, 900 S. Ashland
Ave., Chicago, IL 60607. E-mail: [email protected]
2
Genetics 183: 79–92 (September 2009)
histone deacetylases, histone methylases, and other
corepressor complexes by pocket proteins to E2F
regulated promoters. Upon entry into the cell cycle,
mitogenic stimulation leads to an increase in the activity
of G1 cdks, which phosphorylate pRB family members
and disrupt their interaction with E2Fs. This coincides
with displacement of the repressor E2Fs, appearance of
free activator E2Fs on the promoters of E2F responsive
genes, and induction of the E2F transcriptional program. The critical role of the E2F/pRB network in cell
proliferation is underscored by obligatory inactivation
of pRB control in almost all cancers (Hanahan and
Weinberg 2000). The prevailing view is that the tumor
suppressor property of pRB is to constrain E2F activity.
However, our knowledge of the various tiers of regulation by which pRB has the capacity to block cell proliferation in the context of a multicellular organism is still
very limited. Thus, understanding how the growth
suppressive function of pRB/E2F can be overridden is
important to fully understand the importance of the pRB
pathway in cancer and during normal development.
Drosophila represents an ideal model system to
address this question. This is primarily due to the ability
to carry out high-throughput genetic screens to unveil
novel functional interactors of the E2F/pRB pathway in
an unbiased way. Importantly, the pRB/E2F network is
highly conserved in Drosophila, yet the families are
much smaller than in mice and humans. The Drosophila
genome encodes an activator, dE2F1, and a repressor,
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A. M. Ambrus et al.
dE2F2, their heterodimeric partner, dDP, and two pocket
proteins, RBF1 and RBF2 (reviewed in Cayirlioglu and
Duronio 2001; Dimova and Dyson 2005; van den
Heuvel and Dyson 2008). Studies of the phenotypes of
de2f and rbf mutant and transgenic animals have provided clear insights into the critical roles of these
genes 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 in
mammals, dE2F1 is a potent inducer of S-phase entry.
Overexpression of de2f1 drives postmitotic cells of the
eye 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 cell
cycles (Du and Dyson 1999; Firth and Baker 2005).
Analysis of single and double de2f mutant animals
revealed that de2f1 and de2f2 act antagonistically during
larval development. This conclusion is based on the
observation that the phenotype of de2f1 de2f2 double
mutant animals is less severe than the phenotype of
de2f1 mutants (Frolov et al. 2001). Cell proliferation
and E2F-dependent transcription are severely reduced
in de2f1 mutant animals. These defects are suppressed
by a concomitant mutation of de2f2. The pattern of cell
proliferation is largely normal and repression of several
examined E2F targets is relieved in de2f1 de2f2 double
mutants. These results indicate that unchecked de2f2
provides a significant contribution to the de2f1 mutant
phenotype and therefore, the lack of cell proliferation
in de2f1 mutants can be potentially viewed as a readout
of the de2f2 activity.
Here, we present the results of a mosaic genetic
screen aimed at identifying suppressors that rescue
proliferation in de2f1 mutants [Su(E1)’s]. Since the
de2f1 mutant phenotype stems from the cell proliferation block by dE2F2/RBF, we expected that some of the
isolated suppressors would be important for dE2F2/
RBF function. Indeed, we have identified mutations in
l(3)mbt and B52, two genes that were previously implicated in dE2F2/RBF-mediated repression. We have also
identified two suppressors, gfzf and CG31133, which
potentiate E2F-dependent transcription and cooperate
with the dE2F/RBF pathway. Thus, the characterization
of Su(E1)’s can provide new insights into the in vivo
regulation of the dE2F/RBF pathway.
MATERIALS AND METHODS
Mutagenesis screen: Drosophila were cultured on standard
media at 25°. A description of all alleles and fly stocks can be
found in FlyBase.
For mutagenesis, 36- to 72-hr-old w; FRT 82B de2f1729/TM3
Sb 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, mutagenized
males were crossed to ey-FLP; FRT 82B l(3)cl-R31/TM6B females
and the progeny were screened for exceptional males that
contained 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 mutant
phenotype. Then the individual FRT82B Su(E1) de2f1729/TM3
or FRT82B Su(E1) de2f1729/MKRS males were crossed to three
ey-FLP; FRT 82B l(3)cl-R31/TM6B females to identify potential
suppressors of the de2f1 mutant phenotype. Isolated Su(E1)’s
were retested multiple times by crossing them to ey-FLP; FRT
82B l(3)cl-R31/TM6B and ey-FLP ; FRT 82B P[Ubi-GFP]/TM6B
females.
To map isolated Su(E1)’s, females that were w; FRT82B
de2f1729 Su(E1)/TM3 Ser were mated to males heterozygous for
the 3R chromosomal deficiencies from the Exelixis and
DrosDel deficiency kits. The following deficiencies that uncover
gaps present in the Exelixis and DrosDel kits were also included
in 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 deficiency
relative to their heterozygous siblings were considered a failure
to complement. Since Su(E1)’s were isolated in the presence of
the de2f1729 mutation, each Su(E1) was lethal in trans to
Df(3R)Exel6186, which uncovers the de2f1 gene.
To map Su(E1)-A the exon–intron junctions and ORFs of
the following genes were sequenced: CG18682, CG11498,
CG1359, CG2171, CG31025, CG9753, CG31030, CG1983,
CG9747, G31029, CG15529, CG31028, CG15530, and
CG15531. Genomic DNA was extracted from adult flies of w;
FRT82B de2f1rm729 Su(E1)-A7d13/TM6B Tb and sequenced. No
changes resulting in premature stop codons or affecting
exon–intron junctions were found. However, we found single-nucleotide changes that do not result in the abovementioned defects in the sequence of the following genes:
CG18682, CG11498, CG31025, CG15530, CG15531, CG31028,
CG31029, CG9747, and CG31030. To determine whether any
of these single-nucleotide changes were newly induced mutations or single-nucleotide polymorphisms, genomic DNA was
isolated from the following genotypes and used as a control
in sequencing: w; FRT82B de2f1rm729 Su(E1)5d7/TM6B Tb and
w; FRT82B de2f1rm729 Su(E1)7d21/TM6,Tb. In several cases the
regions of interest were sequenced using genomic DNA of the
following genotypes: w ; FRT82B de2f1rm729 Su(E1-A)7d13/TM3 Ser
and w; FRT82B de2f1rm729 Su(E1-A)7d13/TM6 Tb [ubi-GFP].
Additionally, we sequenced several genes using genomic
DNA isolated from flies carrying another allele of Su(E1)A6a39. On the basis of these comparisons we concluded that all
single-nucleotide changes that we found are SNPs of the
parental chromosome and therefore are not newly induced
mutations in the above-mentioned genes.
For Su(E1)’s, Su(E1)-A7d13, CG311337d21, and gfzf 6a27, an
FRT82B Su(E1) de2f11 chromosome was generated from the
FRT82B Su(E1) de2f1729 chromosome by precise excision of the
P element from the de2f1729 allele. Since both the de2f1729 allele
and FRT82B are marked with the rosy gene, excisions of the P
element from the de2f1729 allele could not be followed by the
rosy marker. Therefore, the FRT82B Su(E1) de2f1729/D2–3 Sb
males were crossed to TM3 Ser/MKRS females and 100 rosy1
males were crossed individually to de2f17172/TM3 Sb females to
select chromosomes in which the de2f1729 allele was reverted to
wild type. The presence of a functional FRT82B was verified by
crossing to ey-FLP; FRT82B P[Ubi-GFP, mini-white] to visualize
the appearance of clones.
Immunofluorescence: Eye and wing imaginal discs were
dissected from third instar larvae in Schneider’s insect
medium (GIBCO, Grand Island, NY) and then fixed in
phosphate-buffered saline (PBS) buffer with 4% formaldehyde. Washes were then done in PBS, 0.3% Triton X-100. Discs
were then blocked in PBS, 0.1% Triton X-100, 10% normal
donkey serum for 1 hr followed by the addition of primary
Screen for Suppressors of the de2f1 Mutant Phenotype
81
Figure 1.—Suppression of the de2f1 mutant phenotype in adult eyes and wing imaginal discs. (A–J) Mitotic recombination
was induced in the eyes of heterozygous
animals by ey-FLP. Wild-type chromosomes
carry the mini-white gene and therefore
wild-type clones (red) can be distinguished
from homozygous mutant tissue (white).
(A and B) Adult eyes of ey-FLP; FRT42D/
FRT42D P[mini-white] (wild type) and eyFLP; FRT42D dDP a3/FRT42D P[mini-white]
(DP). Arrow indicates the location of the
dDP clone in B. (C and D) dDP mutant tissue in the cell lethal background. Adult
eyes of ey-FLP; FRT42D /FRT42D l(2)clR111 P[mini-white] (wild-type CL) and eyFLP ; FRT42D dDP a3/FRT42D l(2)cl-R111
P[mini-white] (DP CL) are shown. (E) No
de2f1 mutant tissue can be seen on the cell
lethal background in ey-FLP; FRT42D
de2f1729 /FRT42D l(3)cl-R31 P[mini-white].
Thus, de2f1 mutant tissue is not able to contribute to the size of the fly eye and the
wild-type tissue is not able to compensate
for this lack of tissue, due to the cell lethal
background, which is why this eye appears
smaller 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 proliferation of de2f1 mutant cells as evidenced by
the presence of double mutant tissue
(white). (K–Q) Wing imaginal discs were
dissected from wandering third-instar larvae. Clones of wild-type cells, identified
by the presence of GFP (green), and homozygous mutant cells, identified by the
absence of GFP (green), were induced by
Ubx-FLP. DAPI is shown in blue. (K) Control experiment in the wing disc showing
clones of cells homozygous for wild-type
chromosomes. Without any mutations
there is approximately an equal distribution of GFP (green)-positive tissue and tissue lacking GFP (green). (L) Almost no
de2f1729 mutant tissue, identified by the lack
of GFP (green), can be seen. Arrows point
to representative clones of de2f1 mutant
cells. 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), and
Doa6d2 (J) is able to rescue proliferation of de2f1 mutant cells as evidenced by the presence of more double mutant tissue than tissue
generated by de2f1 single mutant cells (L). Double mutant tissue is identified by the lack of GFP (green). (K9–Q9) The same corresponding images as K–Q showing only GFP (green).
antibodies. Discs were incubated overnight with primary antibodies and then washed in PBS, 0.1% Triton X-100 three times
for 10 min each. Discs were then incubated in blocking solution
containing the appropriate secondary antibodies. Washes were
then done using PBS, 0.1% Triton X-100. Discs were then
placed into glycerol containing 0.5% propyl gallate in preparation for slide mounting. Bromodeoxyuridine (BrdU)
(Sigma, St. Louis) labeling of eye imaginal discs was performed
by dissecting discs and incubating them in 0.3 mg/ml BrdU–
Schneider’s insect medium for 30 min. Washes were then done
in PBS followed by an overnight fixation in 1.5% formalde-
hyde. Washes were then done in PBS before incubating discs
with DNAse [Promega (Madison, WI) RQ1] for 45 min in a 37°
water bath. Then washes, blocking, and antibody incubations
followed as described above. Images were collected with a Zeiss
LSM510 confocal microscope. The primary antibodies used in
this work were mouse anti-BrdU (1:50) (BD Bioscience), rat
anti-Elav (1:50) (Developmental Studies Hybridoma Bank),
and rabbit anti-phospho-H3 (1:150) (Upstate). The secondary
conjugated antibodies used in this work were anti-mouse Cy3
(1:100), anti-rabbit Cy5 (1:100), and anti-rat Cy3 (1:50) from
Jackson Immuno Laboratories.
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A. M. Ambrus et al.
TABLE 1
Summary of the screen for suppressors of the de2f1 mutant phenotype
Screen
F1
F2
Chromosomes
screened
Suppressors
isolated
Suppressors
retested
Strong
suppressors
Weak
suppressors
18,000
40,000
75
244
8
244
2
84
6
160
RNAi, Western blot analysis, transient transfections, and
real-time PCR in Drosophila tissue culture cells: For RNAi
treatments 2.5 3 104 S2R1 cells per well were plated in a
24-well plate containing 400 ml of Schnieder’s insect medium
with 10% fetal bovine serum (Sch 1 FBS). (Schnieder’s insect medium was from GIBCO and FBS was from Atlanta
Biologicals). Cells were allowed to adhere to the plate for 1 hr
before being washed with medium. Then 12.5 mg of each
appropriate dsRNA in 200 ml medium were added to each
appropriate well. After 3 hr, 400 ml of Sch 1 FBS were added to
each well. After 4 days, appropriate wells were treated with
dE2F1 dsRNA following the above procedure for 18 hr. Then
200 ml of cells per well were replated in duplicate in a 24-well
plate containing 200 ml Sch 1 FBS. Cells were allowed to
adhere for 1 hr before a transfection solution consisting of
appropriate DNA and a transfection agent, FuGENE HD
(Roche Diagnostics, Indianapolis), in 200 ml Sch was added
to 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 before
assaying for luciferase and b-galactosidase activity. Luciferase
assays were performed on a BIO-TEK Clarity Microplate
Luminometer. b-Galactosidase assays were performed on a
Labsystems Multiskan MS Plate Reader. Quantitative real-time
PCR on RNA isolated from S2 cells and primers were as
previously described (Rasheva et al. 2006). For Western blot
analysis, 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) buffer
and frozen for 5 min at 80°. Proteins were resolved by sodium
dodecyl sulfate–10% polyacrylamide gel electrophoresis.
Guinea pig polyclonal anti-dE2F1 (Bosco et al. 2001) and
mouse anti-tubulin antibodies were used for Western blot
analysis.
RESULTS
A suppressor screen for genes that relieve the de2f2dependent block to cell proliferation: Genetic mosaic
screens based on mitotic recombination have proved to
be particularly effective in identifying genes that regulate cell proliferation. The ey-FLP/FRT technique provides a high level of mitotic recombination, thus allowing
for the generation of homozygous mutant tissue in the
eye with high efficiency. Since the wild-type chromosome
carries the white gene (P[mini-white]), the homozygous
mutant tissue can be easily distinguished by the lack of
the white eye color marker in the adult eye (Figure 1A).
Thus, the ey-FLP/FRT-based screen is an attractive approach for identifying novel genes that are important for
dE2F2/RBF function in a high-throughput manner.
Additionally, we sought a genetic strategy that meets
the following criteria: does not rely on overexpression of
de2f or rbf genes and is sufficiently sensitive. The de2f1
mutant phenotype meets these criteria. Clones of de2f1
mutant cells are extremely small due to a strong proliferation block in these cells (Brook et al. 1996; Neufeld
et al. 1998). Since proliferation of de2f1 mutant cells can
be rescued by inactivation of de2f2, patches of de2f1 de2f2
double mutant tissue can be found in the adult eye
(Ambrus et al. 2007). An example of a clone in which
both de2f1 and de2f2 are functionally inactivated by a dDP
mutation is shown in Figure 1B. Therefore, we expected
that mutations that compromise the dE2F2/RBF function should act as suppressors of the de2f1 mutant
phenotype [Su(E1)’s] and could be identified by the
appearance of clones of de2f1 Su(E1) double mutant cells
in the adult eye.
To widen the observable range of suppression of the
de2f1 mutant phenotype, we adopted an ey-FLP/FRT cell
lethal system in which most of the homozygous wild-type
tissue is eliminated due to the presence of a recessive cell
lethal mutation on a wild-type chromosome (Newsome
et al. 2000). When clones of E2F-deficient cells were
induced with the ey-FLP cell lethal technique using a dDP
mutation, large patches of the mutant tissue could be
easily identified in all animals (Figure 1, C and D).
Importantly, even with the ey-FLP/FRT cell lethal technique, no white tissue, representing de2f1 mutant cells,
can be seen in the eye (Figure 1E). Thus, the small size
of clones of de2f1 mutant cells is a highly consistent
phenotype, which is sensitive to inactivation of de2f2. We
concluded that the de2f1 mutant phenotype might be
suitable for a suppressor mosaic genetic screen to identify
novel genes that affect the dE2F2/RBF function.
To test our screening strategy we initially performed
an F1 screen. Isogenic FRT82B de2f1729/TM3 males were
mutagenized with EMS and crossed to ey-FLP; FRT 82B
l(3)cl-R31/TM6B females. The male F1 progeny of these
flies were scored for the appearance of white patches
indicating the presence of a potential Su(E1), which
allows for the recovery of de2f1 mutant tissue. Such
exceptional males were crossed to females carrying the
third chromosomal balancers and then the male progeny were backcrossed to the ey-FLP; FRT 82B l(3)cl-R31/
TM6B females to confirm the suppression of the de2f1
mutant phenotype. A total of 18,000 chromosomes were
screened, and 75 F1 males having white patches of the
de2f1 Su(E1) double mutant tissue were selected. However, only 8 mutants were successfully retested (Table 1).
To improve the recovery of suppressors, we performed
an F2 screen in which mutagenized FRT82B de2f1729
chromosomes were first balanced. Then FRT82B
Screen for Suppressors of the de2f1 Mutant Phenotype
83
Figure 2.—Design of the F2 mosaic screen to
recover suppressors of the de2f1 mutant phenotype.
de2f1729/TM3 or FRT82B de2f1729/MKRS males carrying
a potential Su(E1) were crossed individually to the eyFLP; FRT 82B l(3)cl-R31/TM6B females (Figure 2). Of
44,600 individual crosses 40,000 crosses produced
progeny and 244 mutant lines exhibited a suppression
of the de2f1 mutant phenotype (examples are shown in
Figure 1, F–J). Males of each of the 244 lines were backcrossed twice to females carrying the balancer chromosomes to replace the ey-FLP-carrying X chromosome
with a wild-type X chromosome. Additionally, these
males were retested to confirm consistency of the suppression of the de2f1 mutant phenotype.
To determine whether the five Su(E1)’s shown in
Figure 1, F–J, are able to rescue the de2f1 mutant
phenotype in tissues other than the eye, we induced
clones of de2f1 Su(E1) double mutant cells in the third
instar larval wing imaginal disc, using Ubx-FLP. The UbxFLP/FRT technique provides a high level of mitotic
recombination in multiple tissues. In the wing imaginal
disc, the homozygous wild-type tissue can be easily
distinguished from homozygous mutant tissue by the
presence of the GFP signal (Figure 1K). As expected,
patches of clones of de2f1 mutant cells are limited to
approximately one to five cells and therefore very little
de2f1 homozygous mutant tissue (GFP negative) is
found in the wing disc (Figure 1L). In contrast, much
larger clones of de2f1 Su(E1) double mutant cells can be
recovered (Figure 1, M–Q). Thus, examined Su(E1)’s
are able to at least partially rescue the strong proliferation defects of de2f1 mutant cells in multiple
tissues.
Initial classification of suppressors: Next, we tested
whether the suppressors rescue the proliferation defects
of de2f1 mutant cells without the aid of the cell lethal
mutation, l(3)cl-R31, on the wild-type chromosome. The
ability of isolated mutations to rescue cell proliferation
of de2f1 mutant cells in these settings provided a
rigorous test of the strength of suppression since both
wild-type and the mutant cells compete against each
other in equal conditions. All of the 252 Su(E1)’s that
were isolated in the F1 and F2 screens were crossed to eyFLP; FRT82B P[Ubi-GFP ; w1] females and the eyes of the
progeny were examined for the appearance of clones of
mutant cells. A total of 166 mutants were characterized
as weak suppressors while 86 suppressors exerted a
robust effect on the de2f1 mutant phenotype giving rise
to visible white patches of de2f1 mutant tissue in the eye
(Table 2). Among these 86 lines, five mutants produced
a very strong rough eye phenotype with occasional scars
indicating that these suppressors are likely to affect
developmental pathways and therefore were not considered further.
Mapping and complementation analysis: We used a
lethality potentially associated with Su(E1)’s in an initial
complementation analysis. Although the lethal mutations are not necessarily in each case Su(E1)’s that are
responsible for the phenotype, such an approach allows
for quick mapping of lethal Su(E1)’s. However, the
presence of a lethal de2f1 mutation on each of the
Su(E1)-containing chromosomes complicates complementation and mapping analyses. Each Su(E1) is lethal
in trans to each other due to the presence of the de2f1729
mutation. Similarly, this precludes a standard mapping
by meiotic recombination onto a chromosome with
multiple recessive markers, since the presence of a
Su(E1) can be determined only in the background of
the de2f1 mutation. As an alternative way to estimate how
many genes were represented by the 81 strongest
Su(E1)’s, we tested these lines in trans to a combined
Exelixis and DrosDel deficiency kit that uncovers the
right arm of chromosome 3 (for details see materials
and methods). Forty-nine lines fully complemented
the deficiency kit, indicating that either corresponding
Su(E1)’s are viable or they are located within the limited
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A. M. Ambrus et al.
TABLE 2
Genes isolated in the screen for suppressors of the de2f1 mutant phenotype
Suppressor/gene name
l(3)mbt
Doa
gfzf/CG31329
B52
CG31133
belle
Su(E1)-A
Su(E1)-B
Su(E1)-C
Unannotated single hits
Su(E1)-D
Su(E1)-E
Su(E1)-F
Su(E1)-G
Su(E1)-H
Su(E1)-I
Su(E1)-J
Su(E1)-K
Su(E1)-L
Su(E1)-M
Su(E1)-N
Su(E1)-O
Alleles
Map position
6a14, 4d30, 3d23, 5d8,
4d16, 5d6, 6a31, 3d33
6d2, 6a21, 5d19, 6a52, 5d7
97F1
6a27
7d7, 5d21, 3d16, 3d24
7d21
7d19
7d13, 6a39
4d26, 6a11
1d24, 3d28
84C6–84D1
87F7
95E5
85A2–5
99D5–E2
89A5–8
86E11–87B11
6a47
3d25
6b9
6a3
6a50
6d9
6a8
6b12
6b19
6b31
4d22
6a19
84C8–D9
84D9–E11
85F10–16
87F10–14
87F14–88A14
89E11–90C1
94A9–B2
94B2–B5
95B1–5
95E5–F8
99A5
99A5–B2
98F2
Annotation
Associated with the MMB/DREAM
repressor complex
LAMMER protein kinase,
phosphorylates SR proteins
including B52
FLYWCH zinc finger protein
SR protein splicing factor
Unknown protein
DEAD-box RNA helicase
Two B52 alleles, B527d7 and B525d21, are described in Rasheva et al. (2006).
Mutant allele belle7d19 is described in Ambrus et al. (2007).
intervals between the breakpoints of the deficiencies.
Each of the remaining 32 mutants were lethal in trans to
a particular deficiency (Table 2). To determine whether
the group of weak mutants contained additional alleles,
we crossed weak mutants to identified deficiencies. In
this way, 7 additional mutant alleles were identified.
Publicly available known mutations and P-element insertions in the relevant regions uncovered by the deficiencies were tested for complementation to determine
the identities of Su(E1)’s. This allowed us to identify new
alleles of l(3)mbt, Doa, gfzf, B52, and CG31133 as suppressors of the de2f1 mutant phenotype. The mapping results
are 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 interval
between 97E2 and 98A7. Each of these alleles was lethal
in trans to the deficiency Df(3R)ED6265. One of the
genes uncovered by the deficiency is l(3)mbt, which
encodes a protein associated with the dE2F2/RBF
repressor complex DREAM/MMB (Lewis et al. 2004).
Each of the seven suppressors was lethal in trans to the
nonsense allele l(3)mbtE2 and to the P-element insertion
in l(3)mbt, f02565. Additionally, all seven were lethal in
trans to a deficiency, Df(3R)D605, which uncovers
l(3)mbt. This suggests that this group of Su(E1)’s is allelic
to l(3)mbt.
Doa: Another group included five Su(E1)’s: 6d2, 6a21,
5d19, 6a52, and 5d7. Each mutant of this group failed to
complement Df(3R)Exel6210. Testing for complementation with known mutations and P-element insertions in
these regions identified an allele of the Darkener of apricot
(Doa) gene, Doa01705b, which failed to complement each
of five Su(E1)’s. Similar results were obtained with two
other Doa alleles, DoaKG09056 and Doa3. This suggests that
this group of Su(E1)’s consists of mutations in the Doa
gene. Doa mutations were initially isolated as suppressors of the weak white-apricot allele (Rabinow et al. 1993).
B52: The B52 gene encodes an SR protein that is
required for the correct splicing of the de2f2 pre-mRNA.
Accordingly, mutations in B52 were shown to rescue
proliferation 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 complement
the deficiency uncovering the B52 gene Df(3R)Exel6169
and the B52s2249 mutant allele.
CG31133: A Su(E1), 7d21, was mapped to the region
95E5–F8, which is uncovered by the deficiencies
Df(3R)Exel6198 and Df(3R)ED6187. To further narrow
down the position of 7d21, we generated two smaller
deletions, Df(3R)7d21.I and Df(3R)7d21.II, within this
interval according to the method described in Parks
et al. (2004). In this technique, a deletion is generated
Screen for Suppressors of the de2f1 Mutant Phenotype
85
Figure 3.—The SMW in clones of de2f1
Su(E1) double mutant cells. Eye imaginal
discs were dissected from third-instar larvae. Clones of wild-type cells, identified
by the presence of GFP (green), and homozygous mutant cells, identified by the
absence of GFP (green) were induced by
ey-FLP. Genotypes are shown, the morphogenetic furrow (MF) is marked by an arrowhead, and posterior is to the right. (A–J)
Eye imaginal discs were labeled with BrdU
(red) to visualize cells in S phase. (A) Wildtype disc showing only BrdU incorporation. (B and C) Suppressors l(3)mbt3d33
(B) and Su(E1)-A7d13 (C) strongly rescue
the SMW in de2f1 mutant cells as evidenced
by the entry into and exit from the SMW
in double mutant cells at approximately
the same time as adjacent wild-type cells.
(D–F) In contrast, suppressors CG311337d21
(D), gfzf6a27 (E), and Doa6d2 (F) only weakly
rescue the SMW in de2f1 mutant cells as
evidenced by BrdU incorporation beginning and ending several columns more
posterior in double mutant cells than in
adjacent wild-type cells. (G–J) BrdU incorporation in clones of single mutant suppressors Su(E1)-A7d13 (G), CG311337d21
(H), and gfzf 6a27 (I) shows no SMW defects
as evidenced by the entry into and exit
from the SMW in single mutant cells at approximately the same time as adjacent wildtype cells. Doa KG09056 single mutant clones
show a slightly delayed SMW entry ( J),
but the delay is less extreme than the delay
in de2f1729 Doa6d2 double mutant cells (F).
(B9–J9) The same corresponding images
as B–J showing only BrdU (red). Clones
of mutant cells are outlined.
between two FRT-bearing insertions in the presence of
the FLP recombinase. We utilized a pair of XP insertions, d01966 and d05015, to generate Df(3R)7d21.I
while Df(3R)7d21.II was recovered using a pair of XP
insertions, d09860 and d05397. 7d21 was found to be
lethal in trans to Df(3R)7d21.I, but not to Df(3R)7d21.II.
Among publicly available mutations in genes uncovered
by Df(3R)7d21.I only insertion f07858 failed to complement 7d21. f07858 is an insertion in the gene CG31133,
which encodes a protein of unknown function. To
unambiguously confirm that a mutation in CG31133 is
responsible for the rescue of the de2f1 mutant phenotype, we recombined CG31133 f07858 with the de2f1729
mutant allele and found that clones of de2f1729
CG31133f07858 double mutant cells can be recovered in
the eye (data not shown). This suggests that CG31133 is
a novel suppressor of the de2f1 mutant phenotype.
GST-containing FLYWCH zinc-finger protein: A lone
Su(E1) 6a27 failed to complement two deficiencies
Df(3R)ED5221 and Df(3R)ED7665, which uncover the
interval 84C4–E10. Candidate mutations in this interval
were tested for their ability to complement 6a27. 6a27
was lethal in trans to an EP insertion EY08448. EY0848 is
inserted into the GST-containing FLYWCH zinc-finger
protein (gfzf ) gene, which is located within a second
intron of CG2656. Thus, EY08448 disrupts both gfzf and
CG2656. To determine which of the two genes is affected
by 6a27, we used the KG08427 and e00303 P-element
insertions in a complementation test with 6a27. In
KG08427 and in e00303, the P element is inserted into
the first and the second exons of CG2656, respectively.
Hence, the P-element insertions are upstream of the gfzf
gene and therefore, gfzf function is unlikely to be
affected by either KG08427 or e00303. 6a27 was found
to be fully viable in trans to KG08427 and e00303. In
contrast, 6a27 failed to complement two gfzf mutant
alleles, gfzf 2 and gfzf cz811 . gfzf cz811 contains a deletion at
residue F469, producing a frameshift to a stop codon at
residue 476 (Provost et al. 2006). Thus, 6a27 fails to
exclusively complement mutations in the gfzf gene, but
is fully viable in trans to mutant alleles of CG2656. These
data suggest that 6a27 is a novel mutant allele of gfzf.
Su(E1)-A: Two Su(E1)’s, 7d13 and 6a39, were lethal in
trans to Df(3R)Exel6214, which deletes the region of
86
A. M. Ambrus et al.
Figure 4.—Mitotic marker phospho-H3 in
clones of de2f1 Su(E1) double mutant cells. Eye
imaginal discs were dissected from third-instar
larvae. Clones of wild-type cells, identified by
the presence of GFP (green), and homozygous
mutant cells, identified by the absence of GFP
(green), were induced by ey-FLP. Genotypes are
shown, and posterior is to the right. (A–I) Eye
imaginal discs were labeled with anti-phosphoH3 (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 progress through mitosis as evidenced by phosphoH3 (magenta)-positive cells in clones of mutant
tissue.
99D5–E2. According to FlyBase (Drosophila genome
sequence R5.17), there are 17 predicted genes within
this interval. We tested available mutant alleles of
CG15531, CG7951, CG2184, CG31027, and CG9747
and found that each fully complements 7d13 and
6a39, indicating that these 5 genes do not correspond
to Su(E1)-A. We have sequenced the ORFs and intron–
exon junctions of the remaining 12 genes as well as
CG15531 and CG9747 (for details see materials and
methods). No newly induced mutations in these genes
were found. There are a couple of possible explanations
to account for this. First, lethality associated with Su(E1)-A
might be unrelated to the suppressor effect. This seems
unlikely given that we isolated two independent alleles,
7d13 and 6a39, which were mapped to the same genomic
interval. Another explanation is that these alleles carry
mutations in a regulatory region of a gene. Such mutations can potentially affect the level of transcription or
translation but would be missed in our analysis. Finally, it is
possible that there is another currently unannotated gene
within this genomic interval that corresponds to Su(E1)-A.
Phenotypic analysis: For our initial analysis, we
studied the phenotypes of five above-mentioned
Su(E1)’s by examining markers of cell proliferation in
clones of de2f1 Su(E1) double mutant cells in the eye
imaginal disc since the pattern of cell proliferation is
well characterized. In the larval eye imaginal disc, the
pattern of cell divisions is defined by the position of the
morphogenetic furrow (MF), which traverses from
posterior to anterior during development. We labeled
the eye disc with BrdU to visualize cells in S phase. In the
wild-type disc, cells are asynchronously dividing anterior
to 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 highly
synchronous manner to form a tight stripe of BrdUpositive cells called the second mitotic wave (SMW).
Posterior to the SMW cells exit the cell cycle and commit
to neuronal differentiation. Unlike wild-type cells, de2f1
mutant cells fail to enter the SMW (Du 2000). This
defect is due to the presence of de2f2 since de2f1 de2f2
double mutant cells and dDP single mutant cells enter
the SMW normally although with a slightly reduced rate
of S-phase progression (Frolov et al. 2005; Ambrus et al.
2007). Therefore, we categorized Su(E1)’s by the extent
to which they restore the SMW in de2f1 mutant cells. We
Screen for Suppressors of the de2f1 Mutant Phenotype
87
Figure 5.—Neuronal marker ELAV in
clones of de2f1 Su(E1) double mutant cells.
Eye imaginal discs were dissected from
third-instar larvae. Clones of wild-type
cells, identified by the presence of GFP
(green), and homozygous mutant cells,
identified by the absence of GFP (green),
were induced by ey-FLP. Genotypes are
shown, and posterior is to the right. Cells
were stained with the neuronal marker
ELAV (red). (A–D) The onset of neuronal 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) single
mutant cells initiates at the same time as
in the adjacent wild-type cells. (E and I)
There is a slight delay in the onset of neuronal differentiation in de2f1729 Doa6d2 (E)
double mutant cells and in DoaKG09056 (I)
single mutant cells. (A9–I9) The same corresponding images as A–I showing only
ELAV (red). Clones of mutant cells are outlined.
chose to analyze 3d33, 6d2, 6a27, 7d21, and 7d13, which
represent mutant alleles of l(3)mbt, Doa, gfzf, CG31133,
and Su(E1)-A, respectively. Clones of double mutant
cells carrying a Su(E1) and the de2f1729 allele were
induced with ey-FLP. Homozygous mutant tissue was
distinguished by the lack of GFP. From this analysis we
found that Su(E1)’s restored the SMW in de2f1 mutant
cells to varying extents. Mutant alleles of l(3)mbt and
Su(E1)-A provided a stronger rescue of the SMW in de2f1
mutant cells as evident by the appearance of BrdUpositive de2f1 l(3)mbt3d33 and de2f1 Su(E1)-A7d13 double
mutant cells in the SMW at about the same time as in the
adjacent wild-type cells (Figure 3, B and C). However, we
note that the intensity of BrdU labeling was somewhat
reduced in de2f1 Su(E1)-A7d13 mutant cells. In contrast to
l(3)mbt and Su(E1)-A, de2f1 CG311337d21 double mutant
cells entered the SMW several columns more posterior
than wild-type cells (Figure 3D), which is indicative of
a delay of entry into the S phase. Additionally, BrdU
incorporation persisted several columns more posterior
in the double mutant cells compared to the adjacent
wild-type tissue. This indicates that it takes longer than
normal for de2f1 CG311337d21 double mutant cells to
progress through the S phase, suggesting that the S
phase is likely to be slowed. The defects in the SMW were
less noticeable in the case of the gfzf 6a27 mutant allele
(Figure 3E). Although occasionally we observed clones of
de2f1 gfzf 6a27 double mutant cells in which entry into the
SMW occurred relatively on time, in most cases, the
BrdU-positive mutant cells were situated farther posterior. Finally, de2f1 Doa6d2 double mutant cells were only
sporadically labeled with BrdU in the SMW. However, any
de2f1 Doa6d2 double mutant cells that did incorporate
BrdU were always found to be significantly more posterior than BrdU incorporating adjacent wild-type cells
(Figure 3F). Taken together these results indicate that
Su(E1)’s differ among each other by the extent of rescue
of the timing of entry into and exit from the SMW in de2f1
mutant cells. Clones of de2f1 l(3)mbt and de2f1 Su(E1)-A
mutant cells enter and exit the SMW relatively on time
while de2f1 gfzf 6a27, de2f1 CG311337d21, and de2f1 Doa6d2
exhibit a delay in the entry into and/or exit from the
SMW.
The extent of the defects in the SMW may reflect the
ability of each Su(E1) to rescue the cell cycle arrest in
de2f1 mutant cells. Alternatively, Su(E1) mutant cells
88
A. M. Ambrus et al.
Figure 6.—Effect of Su(E1)’s on E2F-dependent transcription in tissue culture cells. (A)
Depletion of dE2F1 represses expression of a
PCNA-luc reporter. Codepletion of L(3)MBT, or
dE2F2, or RBF1 (R1) and RBF2 (R2), together
with dE2F1, derepresses expression of the E2F reporter. S2R1 cells were incubated with either
control or experimental double-stranded RNA
(dsRNA) as indicated. Cells were then incubated
for an additional day with either control (NS) or
dE2F1 dsRNA before being cotransfected with a
PCNA-luc reporter and a b-Gal expression plasmid in duplicates. (B) Codepletion of L(3)MBT
or dE2F2 (E2) with dE2F1 (E1) derepresses expression of the endogenous PCNA gene. S2 cells
were incubated with either control (NS) or corresponding dsRNAs as indicated. After 4 days,
the steady-state level of the PCNA mRNA was
determined by real-time reverse transcription–
PCR in triplicates. (C) The basal level of expression of a PCNA-luc reporter is elevated in cells
depleted of either GFZF or CG31133 proteins.
S2R1 cells were incubated with control (NS)
or corresponding dsRNAs as indicated. After
4 days, cells were transfected with a PCNA-luc reporter in duplicates. (D) S2R1 cells were incubated with control (NS) or corresponding
dsRNAs as indicated. After 4 days cells were lysed
and the level of dE2F1 was determined by Western blot analysis. Tubulin was used as a loading
control. (E) Codepletion of GFZF or CG31133
with RBF1 (R1) and RBF2 (R2) synergistically activates a PCNA-luc reporter. After 4 days, cells
were cotransfected with a PCNA-luc reporter
and a b-Gal expression plasmid in duplicates.
themselves could have an aberrant SMW. To discriminate between these two possibilities we examined BrdU
incorporation in the SMW of clones of Su(E1)-A,
CG31133, gfzf, and Doa single mutant cells. As shown
in Figure 3, G–J, Su(E1)-A, CG31133, or gfzf single
mutant cells enter and exit the SMW at about the same
time as the adjacent wild-type cells, while the SMW entry
is slightly delayed in Doa single mutant cells. However,
this delay is not as extreme as that observed in de2f1 Doa
double mutant cells (Figure 3F). We infer from these
results that the defects in the SMW of clones of de2f1729
Su(E1) mutant cells likely reflect the ability of each
Su(E1) to rescue the SMW in de2f1729 mutant cells.
To further characterize the effect of Su(E1)’s on the
SMW in de2f1 mutant cells we used phospho-H3 as a
mitotic marker (Figure 4). In wild-type eye discs, the
stripe of phospho-H3-positive cells posterior to the MF
marks cells of the SMW (Figure 4A). Importantly, we
found phospho-H3-positive double mutant and single
mutant cells in the SMW (Figure 4, B–I). Expression of
ELAV, a marker of neuronal differentiation, is induced
in double and single mutant cells although de2f1 Doa
double mutant and Doa single mutant cells exhibited a
slight delay in the onset of ELAV expression (Figure 5).
This could be a consequence of a delay in exit from the
SMW. In no cases did we find cells that coexpress phosphoH3 and ELAV. This suggests that de2f1 Su(E1) double
mutants finish the SMW and commit to differentiation.
l(3)mbt affects expression of an E2F reporter in cell
culture-based assays differently than gfzf or CG31133:
Having established that the loss of l(3)mbt restores the
timing of the S-phase entry in the SMW in de2f1 mutant
cells, while mutations in gfzf and CG31133 do not, we
wished to determine whether inactivation of these genes
differentially affects E2F-dependent transcription in
dE2F1-deficient cells. We employed RNA interference
(RNAi) in Drosophila S2R1 tissue culture cells to deplete
dE2F1, dE2F2, RBF, L(3) malignant brain tumor
(MBT), GFZF, and CG31133 proteins and then transiently transfected these cells with a reporter containing
the endogenous PCNA promoter fused to a luciferase
gene. The PCNA gene is a direct transcriptional target of
Screen for Suppressors of the de2f1 Mutant Phenotype
dE2F and has been previously validated as an accurate
readout of dE2F transcriptional activity (Frolov et al.
2001). As expected, the E2F reporter is strongly repressed in dE2F1-depleted cells (Figure 6A). This is due
to the presence of dE2F2 because the repression was
alleviated when dE2F1 and dE2F2 were inactivated
simultaneously. Accordingly, depletion of RBF proteins,
which are required for repression by dE2F2, also relieved repression of the reporter (Figure 6A). Thus,
expression of the E2F reporter in dE2F1 single- and in
dE2F1 dE2F2 double-depleted cells essentially recapitulates the response of the endogenous PCNA gene in
mutant animals or in tissue culture cells (Frolov et al.
2005). Inactivation of GFZF or CG31133 had no effect
on the E2F reporter in dE2F1-deficient cells, as evidenced by the reporter remaining fully repressed in
these cells (Figure 6A). In contrast, depletion of
L(3)MBT relieved the repression of the reporter in
dE2F1-deficient cells to the same extent as inactivation
of dE2F2 (Figure 6A). To test whether repression of an
endogenous PCNA gene in dE2F1-depleted cells is
alleviated by codepletion of L(3)MBT, we used realtime RT–PCR to monitor the steady-state PCNA mRNA
level. As has been previously shown, PCNA expression is
reduced in dE2F1-depleted cells (Frolov et al. 2005).
Consistent with the results in transient transfections, the
repression of PCNA was relieved in dE2F1 L(3)MBT
double-deficient cells to a level comparable to that of
dE2F1 dE2F2-depleted cells (Figure 6B). Thus, inactivation of L(3)MBT alleviates the repression of an E2F
target in dE2F1-depleted cells as efficiently as removal
of dE2F2, indicating that L(3)MBT is needed for dE2F2
to repress.
Next, we determined the effect of depletion of
L(3)MBT, CG3133, GFZF, and DOA on the basal level
of expression of the E2F reporter. Interestingly, although
depletion of GFZF or CG31133 does not relieve dE2F2mediated repression, we found that the E2F reporter is
consistently elevated in cells lacking GFZF or CG31133
(Figure 6C). This effect cannot be attributed to an
elevation of dE2F1 protein since dE2F1 levels are unaltered when any of the Su(E1)’s are depleted in cell
culture by RNAi (Figure 6D). To determine whether
these effects are mediated through pocket proteins we
determined the epistatic effect of depletion of GFZF and
CG31133 relative to RBF proteins. As expected, depletion of RBF proteins elevates expression of the E2F
reporter (Figure 6E), which is likely due to release of an
inhibitory effect of RBFs on endogenous dE2F1. Codepletion of L(3)MBT does not potentiate the effect of
inactivation of RBF proteins. This is consistent with the
finding that L(3)MBT and RBF are present together in
the same repressor complex (Lewis et al. 2004). Unlike
L(3)MBT, codepletion of GFZF and RBFs or CG31133
and RBFs resulted in strong additive effects on activation
of the E2F reporter as compared to targeting either of
them alone (Figure 6E). These observations support the
89
idea that inactivation of GFZF or CG31133 elevates
activation of the E2F reporter, but this occurs in an
RBF-independent manner.
DISCUSSION
Although a critical role of the pRB/E2F pathway in
normal cell proliferation and in cancer has been firmly
established, our understandings of what aspects of
pRB/E2F function are important in vivo and how
precisely the pathway is regulated during development
are still very limited. As a way to begin to address
these important questions, we have designed a mosaic
genetic screen in Drosophila to isolate genes that when
mutated overcome the dE2F2/RBF-dependent block
to cell proliferation in de2f1 mutant cells. Several
genetic modifier screens have been previously performed 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 screens
provided critical contributions to the understanding
of mechanisms underlying actions of pRB and
E2F. However, such screens were designed to exclusively isolate haploinsufficient modifiers in the background of ectopically expressed rbf, de2f, and cyclin E
genes, thus limiting the spectrum of potential modifiers. The screening strategy described in this work
avoids complications associated with protein overexpression since the cell cycle block in de2f1 mutant cells
is caused by the endogenously present de2f2. Additionally, the FLP/FRT technique allows for the isolation of
recessive suppressors. These are the two major advantages of our approach from the above-mentioned
screens.
The genetic screen reported here identified genes
that are known to be required for dE2F2 function as well
as novel genes that were not previously linked to the
pRB/E2F pathway. Of the complementation groups
isolated in the screen, l(3)mbt and B52 have been
previously 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 component of a native dE2F/RBF complex Drosophila RBF
E2F and MYB/Myb-MuvB (DREAM/MMB), although in
a substoichimetric ratio (Lewis et al. 2004). Mutations in
l(3)mbt give rise to malignant transformations in the
larval brain as well as disruption of synchronous cell
divisions in early embryos (Yohn et al. 2003; Trojer and
Reinberg 2008). l(3)mbt encodes a protein with three
MBT domains that are structurally similar to other
chromatin-binding domains such as the chromodomain. Its mammalian homolog L3MBTL1 is a transcriptional repressor that is thought to promote the higherorder chromatin structure by simultaneously binding to
two adjacent nucleosomes and moving nucleosomes
closer together. Such binding is dependent on the
presence of mono- and dimethylation of histone H4 at
90
A. M. Ambrus et al.
lysine 20, which is commonly found in facultative
heterochromatin (Trojer et al. 2007). L3MBTL1 associates with pRB and is needed for repression of E2Fregulated genes c-myc and cyclin E in mammalian cells
(Trojer et al. 2007). Interestingly, depletion of
L(3)MBT in Drosophila tissue culture cells results in
derepression of genes that are normally kept silent by
dE2F2/RBF in asynchronously dividing cells (Lewis
et al. 2004; Lu et al. 2007). In this respect, isolation of
l(3)mbt as a suppressor of the de2f1 mutant phenotype
described here is significant because it provides the first
genetic evidence for the importance of l(3)mbt in de2f2dependent cell cycle arrest in vivo. This conclusion is
further supported by the finding that L(3)MBT is
needed for dE2F2 to repress in dE2F1-deficient cells.
Finally, isolation of l(3)mbt serves as a proof of principle
that genes that interface directly with the dE2F/RBF
module can be identified as suppressors of the de2f1
mutant phenotype.
In addition to l(3)mbt, three genes, rbf2, Chromatin
assembly factor 1 subunit (Caf1/p55), and rpd3, reside on
3R and encode components of the DREAM/MMB
complex. However, rpd3 is not required for the repression 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 primarily
involved in E2F-dependent repression in embryos, but
not in imaginal discs (Stevaux et al. 2005), and
therefore it is not surprising that mutant alleles of
rpd3 and rbf2 were not isolated in this screen. The lack of
Caf1/p55 mutations among Su(E1)’s is somewhat puzzling given that inactivation of CAF1/p55 abrogates
dE2F2-mediated repression (Taylor-Harding et al.
2004). One possibility is that the CAF1/p55 function
itself is needed during cell proliferation due to its role in
DNA replication. Alternatively, since no mutant alleles
in Caf1/p55 have been described to date it is not known
whether mutations in Caf1/p55 are lethal. If the loss of
Caf1/p55 does not result in lethality, then mutant alleles
of Caf1/p55 would be apparently missed in our initial
mapping approach using lethality as a complementation test.
Among Su(E1)’s we have isolated multiple mutant
alleles of the Doa gene. Doa has been implicated in a
variety of cellular functions including differentiation 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 best
known for its role in the regulation of alternative
splicing through phosphorylation of multiple SR proteins, among which are TRA, TRA2, and B52 (Nikolakaki et al. 2002). Since B52 is required for splicing of
de2f2 pre-mRNA and B52 is a Su(E1) (Rasheva et al.
2006), we considered the possibility that mutation of
Doa rescues the de2f1 mutant phenotype through its
regulation of B52. So far we have not found evidence
to support this model. Inactivation of Doa does not
alleviate repression of endogenous dE2F2 targets in eye
discs or in tissue culture cells. Additionally, depletion of
DOA has no effect on dE2F2-dependent repression in
transient transfection assays. Finally, there was no
difference in the dE2F2 level in clones of Doa mutant
cells or when DOA was depleted by RNAi in S2 cells
(data not shown). Thus, it is possible that although DOA
phosphorylates B52 in an in vitro kinase assay, B52 may
not be an endogenous substrate of DOA. Alternatively,
regulation of B52 by DOA may be tissue specific or
regulation of B52 by means of phosphorylation in vivo
could be directed by multiple SR protein kinases.
In contrast to depletion of L(3)MBT (this work) and
B52 (Rasheva et al. 2006), depletion of GFZF and
CG31133 does not compromise dE2F2/RBF-mediated
repression, but instead we found that expression of the
E2F reporter is elevated in these cells. Since the E2F
reporter is sensitive to the level of endogenous E2F
activity, this raises the possibility that inactivation of
GFZF or CG31133 may potentiate dE2F1 activity
through, for example, the release of RBF inhibition of
dE2F1. Such an explanation seems unlikely since
codepletion of RBF together with GFZF or CG31133
exerts an additive effect on the E2F reporter, suggesting
that the two Su(E1)’s likely act in parallel to the RBF/E2F
pathway. Finally, since both gfzf and CG31133 were
isolated in the background of a de2f1 mutation, this
disfavors the interpretation that they act through
dE2F1. We suggest that CG31133 and GFZF may directly
or indirectly cooperate with dE2F1 on a subset of target
genes. How precisely GFZF and CG31133 elevate the
expression of the E2F reporter is not known and further
experiments will be needed to dissect the molecular
mechanism of this effect.
Interestingly, the S-phase entry and exit in the SMW
are delayed and the S phase appears to be extended in
de2f1 gfzf and de2f1 CG31133 double mutant cells. In
another example, rescue of the de2f1 mutant phenotype
by a mutation in the gene belle is not accompanied by the
full restoration of the SMW in de2f1 belle double mutant
cells (Ambrus et al. 2007). Similarly, the loss of belle does
not 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) compromise dE2F2-mediated repression while de2f1 l(3)mbt
and de2f1 B52 double mutant cells enter and exit the
SMW relatively on time. Thus, it is tempting to suggest
that relief of dE2F2-mediated repression is needed for
de2f1 mutant cells to rescue the correct timing of the
SMW. Since prior to the S phase entry in the SMW cells
is transiently arrested in G1 within the MF, this may
explain why de2f1 mutant cells in the SMW are particularly sensitive to the reduced level of E2F targets. This
idea is in agreement with the results of experiments
in mammalian cells with a dominant negative form of
E2F, which suggested that E2F-dependent activation is
needed for cell cycle reentry from quiescence (Rowland
Screen for Suppressors of the de2f1 Mutant Phenotype
and Bernards 2006). Future studies of other isolated
suppressors of the de2f1 mutant phenotype are likely to
provide novel insights into the growth suppressive
function of dE2F2/RBF in vivo.
We are grateful to the Developmental Studies Hybridoma Bank
(University of Iowa) and the Bloomington Stock Center for fly stocks
and antibodies. We thank D. Knight, K. Marsh, R. Suckling, and
H. Summersgill for technical assistance and N. Dyson, A. Katzen,
G. Ramsey, and M. Truscott for critical discussions. This work was supported by grant GM079774 from the National Institutes of Health to
M.V.F. and by predoctoral fellowship 0815661G from the American
Heart Association to A.M.A.
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Communicating editor: J. Tamkun