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First published online 21 December 2006
doi: 10.1242/dev.02738
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1 Department of Biology, University of North Carolina, Chapel Hill, NC 27599,
USA.
2 Program in Molecular Biology and Biotechnology, University of North Carolina,
Chapel Hill, NC 27599, USA.
3 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel
Hill, NC 27599, USA.
Author for correspondence (e-mail:
duronio{at}med.unc.edu)
Accepted 14 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, E2F, pRb, Cell cycle, G1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Multiple mechanisms contribute to stable G1 quiescence, and these mechanisms
can be broadly defined as those that initiate the onset of G1 arrest and those
that maintain G1 arrest. Disruption of either or both types of regulation can
abrogate differentiation, block morphogenesis and contribute to the onset of
cancer.
The initiation of G1 arrest involves the inhibition of G1
Cyclin-Cyclin-dependent kinase (Cyc-Cdk) complexes that promote entry into S
phase. These complexes include CycD-Cdk4 and CycE-Cdk2 (Cdk2 is also known as
Cdc2c - Flybase), which are negatively regulated by the cyclin-dependent
kinase inhibitors (CKIs) p16INK4a and p27Kip1,
respectively (Sherr and Roberts,
1999). Whereas p16INK4a acts primarily as a tumor
suppressor, the induction of p27 expression is required for proper cell cycle
withdrawal and subsequent differentiation in a number of developing mammalian
tissues, including the retina, the organ of Corti and skeletal muscle
(Chen and Segil, 1999;
Chu and Lim, 2000;
Levine et al., 2000;
Lowenheim et al., 1999;
Zabludoff et al., 1998).
The maintenance of G1 arrest occurs through a distinct mechanism involving
the repression of genes necessary for S phase, which are regulated by the E2F
family of transcription factors. E2F activity is controlled mainly through
interaction with members of the retinoblastoma (pRb) tumor suppressor or
`pocket protein' family (DeGregori,
2002; Dimova and Dyson,
2005; Trimarchi and Lees,
2002). During quiescence and early G1, hypophosphorylated pocket
proteins form a complex with E2Fs that recruit co-repressors and result in the
downregulation of E2F targets. In response to growth signals, G1 Cyc-Cdk
complexes phosphorylate pocket proteins resulting in the dissociation of the
repressive pocket protein-E2F complex and the induction of transcription of S
phase genes. In lens cells, trophoblasts, keratinocytes and neural tissue, the
maintenance of cell cycle arrest is compromised by the loss of pRb, presumably
owing to an inappropriate increase in E2F activity and the consequent
activation of replication genes (Jacks et
al., 1992; MacPherson et al.,
2003; Ruiz et al.,
2004; Wu et al.,
2003).
E2F activity can also be regulated independently of pocket proteins. E2F is
a heterodimer composed of an E2f subunit and a Dp subunit that together are
necessary for binding DNA (Trimarchi and
Lees, 2002). During S phase, CycA-Cdk2 phosphorylates E2f-bound Dp
resulting in dissociation of the E2f-Dp heterodimer from DNA
(Dynlacht et al., 1994;
Dynlacht et al., 1997;
Krek et al., 1994;
Krek et al., 1995). Other
reports indicate that in mammalian cells, E2F proteins are destroyed in S-G2
via the ubiquitin-proteasome pathway
(Campanero and Flemington,
1997; Hateboer et al.,
1996; Hofmann et al.,
1996; Marti et al.,
1999). Similarly, E2f1 (E2f - Flybase) is destroyed at the G1-S
transition in Drosophila imaginal disc cells
(Asano et al., 1996;
Heriche et al., 2003;
Reis and Edgar, 2004), and
this destruction involves the ubiquitin-proteasome pathway
(Heriche et al., 2003).
Whether these modes of E2F regulation contribute substantially to gene
expression and cell cycle control during development is not known.
The cell cycles of early embryonic development display common features among a variety of animal species. In general, these cell cycles are very rapid and occur with the ubiquitous activity of key regulators such as E2F and CycE-Cdk2. In some instances (e.g. Drosophila and Xenopus), the earliest cell cycles lack measurable gap phases altogether. As development proceeds, different lineages first acquire additional cell cycle controls that result in the appearance of gap phases, and then undergo cell cycle exit and differentiation. The mechanisms contributing to specific changes in cell cycle regulation in particular tissue types during development remain incompletely understood.
Drosophila embryos provide an excellent experimental system to
address this issue because they execute a stereotyped,
developmentally-controlled cell cycle program that is well-characterized
(Lee and Orr-Weaver, 2003)
(Fig. 1I). The first 13 cycles
are rapid S-M cycles driven by ubiquitous maternal factors
(Foe and Alberts, 1983). The
first gap phase, G2, appears at the blastoderm stage during cell cycle 14
because of degradation of maternal string (stg) mRNA and
protein (Edgar and Datar,
1996). stg encodes a Cdc25-type phosphatase that removes
the inhibitory phosphates from Cdk1 (Cdc2 - Flybase) to allow entry into
mitosis (Edgar and O'Farrell,
1989). After gastrulation begins, a pulse of zygotic transcription
of stg in late G2 triggers the entry into mitosis during cycles 14,
15 and 16 (Edgar et al., 1994;
Edgar and O'Farrell, 1990). In
these so-called post-blastoderm division cycles there is no G1 phase, and S
phase begins immediately after mitosis. G1 phase first appears during cell
cycle 17, after which some cells (e.g. in the epidermis) remain arrested in
G117 whereas others (e.g. in the midgut) re-enter S phase from
G117 and begin endoreduplication cycles.
The regulation of stg establishes a paradigm for developmental
control of the Drosophila embryonic cell cycle. The transition from
ubiquitous, maternally-provided stg to regulated, zygotic expression
of stg accounts for both the introduction of the first G2 phase and
subsequent G2-M cell cycle regulation. This paradigm also applies to the
introduction of G1-S regulation in cell cycle 17. Because Cyclin E is required
for S phase in Drosophila
(Knoblich et al., 1994), the
change in activity of CycE-Cdk2 from ubiquitous (cycles 1-16) to cell
cycle-regulated accounts for both the introduction of G1 phase in cycle 17 and
subsequent regulation of the G1-S transition
(Duronio and O'Farrell, 1995;
Richardson et al., 1993;
Sauer et al., 1995). This
transition is achieved in part by zygotic transcription of dacapo
(dap), which encodes the single Drosophila p27-like CKI
(de Nooij et al., 1996;
Lane et al., 1996).
dap transcription is controlled by a complex cis-acting regulatory
region that responds to developmental inputs that induce Dap production during
cycle 16 (Liu et al., 2002;
Meyer et al., 2002b). This
results in the inhibition of CycE-Cdk2 and the appearance of G1 in cycle 17.
Consequently, dap-mutant epidermal cells do not enter
G117, but instead enter S17 immediately after the
completion of M16 and undergo an ectopic cell division cycle
(de Nooij et al., 1996;
Lane et al., 1996).
The maintenance of a stable G117 arrest in the embryonic
epidermis requires the function of Rbf1 (Rbf - Flybase), a Drosophila
pRb homolog (Du et al.,
1996a). Rbf1 negatively regulates the activity of E2f1. In
Drosophila, E2f1 is necessary for the expression of replication
factor genes including Cyclin E, although these genes are also
regulated by additional factors such as Dref
(Duronio et al., 1998;
Duronio et al., 1995;
Hirose et al., 1993;
Royzman et al., 1997;
Sawado et al., 1998;
Yamaguchi et al., 1996).
Rbf1-mutant embryos develop normally through cycle 17, and the
epidermal cells are able to initiate G117 owing to the activity of
Dap. However, some Rbf1-mutant epidermal cells fail to maintain G1
arrest and ultimately re-enter the cell cycle because of inappropriate
expression of E2f1-target genes including Cyclin E
(Du and Dyson, 1999). The
developmental inputs and mechanisms that result in Rbf1 repressor function and
the downregulation of replication genes are unknown. Here we show that,
surprisingly, the initial downregulation of the E2f1-target gene RnrS
prior to G117 does not require Rbf1 or Dap. Instead, loss of
RnrS expression occurs coincident with the onset of S phase-coupled
destruction of E2f1 protein, which may provide a mechanism for pRb-independent
regulation of E2F activity.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Duronio et al., 1995;
Lane et al., 1996;
McEwen et al., 2000;
Sigrist and Lehner, 1997;
Spradling et al., 1995;
Whittaker et al., 2000). y
w; stg7B/TM3 e as well as UAS Rbf-280/TM3, UAS
Rbf1, and Rbf114 FRT14A-B/FM7 were
kindly provided by Patrick O'Farrell and Wei Du, respectively
(Du and Dyson, 1999;
Edgar and O'Farrell, 1989;
Xin et al., 2002).
dap4454/CyO wg-lacZ, Df(1)biD3/FM7Actin-GFP,
dupa1/CyO wg-lacZ, dupa3/CyO wg-lacZ,
CycEAR95/CyO wg-lacZ, and Rbf114 FRT
14A-B/FM7 Actin-GFP were constructed for this study.
Rbf114 germ line clones were generated as described
(Du and Dyson, 1999).
stg7B dap4454 double-mutant embryos were
unambiguously identified using Dap antibody staining and by the altered
morphology caused by the stg G214 arrest phenotype.
RNA in situ hybridization and BrdU labeling
Embryos were dechorionated, fixed in 1:1 4% formaldehyde in PBS:heptane for
25 minutes, and devitellinized with methanol. For BrdU labeling, dechorionated
embryos were permeabilized with octane, then pulse-labeled with 1 mg/ml BrdU
for either 5 minutes or 15 minutes in Schneider's Drosophila medium
prior to fixation. Embryos were stored in methanol at -20°C.
In situ hybridization with digoxigenin-labeled antisense RNA probes was
performed as described (Kearney et al.,
2004). Fluorescent detection of hybrids (FISH) was achieved with
the TSA Fluorescence System (Perkin Elmer) using a 30-60 minute incubation in
TSA-Cy3 or TSA-Fluorescein. For all triple fluorescent staining (i.e. FISH,
anti-protein, anti-BrdU) except E2f1 or Dap plus FISH, embryos were first
processed for FISH, then for immunodetection of proteins, and finally for BrdU
detection by acid denaturation of chromosomes
(Schubiger and Palka, 1987).
For E2f1 or Dap detection plus FISH, the TSA Fluorescence System was first
used for immunodetection of E2f1 or Dap, and then the embryos were fixed for
30 minutes in 4% formaldehyde to quench the peroxidase prior to FISH and BrdU
detection.
Immunostaining
Embryos were rehydrated with PBS-0.1% Tween20 (PBS-T) and incubated with
primary antibodies overnight at 4°C. Primary antibodies used were: mouse
anti-BrdU monoclonal antibody (1:100, Becton Dickinson), rabbit anti-E2f1
(1:500 or 1:1000, gift of Maki Asano)
(Asano et al., 1996), rabbit
anti-phospho-tyrosine (1:100, Upstate), rat anti-phospho-tyrosine (1:50 or
1:100, R and D Systems), rabbit anti-ß-galactosidase (1:200, Chemicon),
mouse anti-phospho-Ser10-histone H3 (1:2000, Upstate), rabbit anti-GFP
(1:2000, Abcam) and rabbit anti-Dap (1:600)
(Lane et al., 1996). Secondary
antibodies used were: goat anti-mouse Oregon Green (1:1000, Molecular Probes),
goat anti-mouse-Cy5 (1:500, Jackson), goat anti-mouse-Cy3 (1:500, Jackson),
goat anti-rabbit-Cy2 (1:500, Jackson), goat anti-rabbit rhodamine (1:1000,
Molecular Probes), donkey anti-rat-Cy5 (1:500, Jackson), and goat
anti-rabbit-Cy5 (1:500, Abcam). For detection of E2f1 and Dap, the TSA
Fluorescence System (Perkin Elmer) was used with a biotin-conjugated
anti-rabbit secondary antibody (1:1000, Chemicon). Stained embryos were
mounted with Fluoromount-G (Southern Biotech) and visualized with either a
Nikon Eclipse E800 microscope or a Zeiss LSM 510 scanning confocal
microscope.
Co-immunoprecipitations and western blotting
Immunoprecipitations were performed with extracts from 0-4 hour and 5-8
hour w1118 embryos as described
(Peifer et al., 1993), and
analyzed by SDS-PAGE (7.5% precast gel, Biorad) and western blotting with
mouse anti-Rbf1 (DX-3, 1:4) (Du et al.,
1996a), rabbit anti-E2f1 (see above) and mouse anti-Dp (YUN1-3
1:4) (Du et al., 1996b).
Secondary antibodies were ECL-sheep anti-mouse HRP (1:5000) and ECL-donkey
anti-rabbit HRP (1:5000) from Amersham Biosciences.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Stevaux et al., 2002). This
suggests that embryonic Rbf1 activity is regulated post-translationally. We
therefore hypothesized that Rbf1 is hyperphosphorylated and thus inactivated
until cycle 16 by constitutive G1 Cyclin-Cdk activity, resulting in ubiquitous
expression of E2f1-target genes (Fig.
1I). To test this, we utilized a mutant version of Rbf1 (Rbf-280)
containing mutations in four Cdk consensus sites that cannot be inhibited by
the activity of G1 Cyclin-Cdk complexes such as CycE-Cdk2
(Xin et al., 2002). UAS
Rbf-280 was expressed with two strong drivers that are active during
cycles 14-16, prd-Gal4 and arm-Gal4-VP16. E2f1 activity was
monitored by in situ hybridization with a probe derived from the small subunit
of ribonucleotide reductase (RnrS), a well-established E2f1-target
gene (Duronio et al., 1995).
UAS Rbf-280 expression with prd-Gal4 resulted in the
precocious termination of RnrS expression in alternating segments
(Fig. 1A,C). Utilizing
fluorescent detection of RnrS transcripts and BrdU labeling, we
confirmed that the precocious termination occurs during cycle 15
(Fig. 1D). A similar but more
widespread result was observed using the ubiquitous arm-Gal4-VP16
driver (Fig. 1E,G). Little
change in RnrS expression was observed after expressing wild-type
Rbf1 (Fig. 1B,F), indicating
that the precocious termination of RnrS expression is specific to UAS
Rbf-280. These results suggest that Rbf-280 can bypass the normal
mechanism of Rbf1 control in the early embryo, and are consistent with the
idea that Rbf1 is hyperphosphorylated and thus inactivated by constitutive
Cyclin-Cdk activity in the early embryo to permit expression of replication
factor genes such as RnrS.
|
;
Xin et al., 2002). Therefore,
our interpretation of the Rbf-280 results predicts that Rbf1-E2f1 complexes
will not be present during early embryogenesis, and that these complexes will
be detected only after the introduction of G1 control at 
7 hours of
development. Consistent with this hypothesis, E2f1 and Rbf1 co-precipitate
from 5-8 hour (cycles 16-17) but not from 0-4 hour (prior to cycle 16) embryo
extracts (Fig. 1H). Dp
co-precipitates with Rbf1 in both cases
(Fig. 1H). The Rbf1-Dp
interaction in 0- to 4-hour-old embryos is likely to represent the recently
described Myb-MuvB-dREAM complex that contains E2f2-Dp-Rbf and which acts to
repress many genes involved in developmental processes other than cell cycle
progression (Korenjak et al.,
2004; Lewis et al.,
2004). We have been unable to detect hyperphosphorylated Rbf1 by
reduced mobility on SDS-PAGE gels, as is commonly performed with mammalian
pRb. Nevertheless, our results suggest that in early embryogenesis (cycles
1-16), Rbf1 is present in a hyperphosphorylated, inactive form that is not
bound to E2f1.
The initial termination of E2f1-target gene expression does not require CycE-Cdk2 inhibition
In wild-type embryonic epidermis, the expression of E2f1 targets is
terminated prior to G117, and Rbf1 is required to maintain
repression of E2f1 targets during G117
(Du and Dyson, 1999;
Duronio and O'Farrell, 1994;
Richardson et al., 1993).
Since our data imply that Rbf1 is hyperphosphorylated in the early embryo, we
hypothesized that prior to the introduction of G117, Rbf1 is
converted to a hypophosphorylated form that binds E2f1 and terminates
E2f1-target gene expression. A possible mechanism for the conversion of Rbf1
to a hypophosphorylated form is the inhibition of G1 Cyclin-Cdk complexes,
specifically CycD-Cdk4 and CycE-Cdk2, which in vertebrates are known to
phosphorylate pRb (Dyson,
1998). Since the regulation of RnrS expression in the
epidermis of both CycD- and Cdk4-mutant embryos is normal,
the modulation of CycD-Cdk4 activity may not be part of the mechanism
(Emmerich et al., 2004;
Meyer et al., 2002a). By
contrast, CycE-Cdk2, which can phosphorylate and inhibit Rbf1
(Du et al., 1996a), is
inhibited just prior to the introduction of G117 by the
developmentally-regulated induction of dap transcription during cycle
16 (de Nooij et al., 1996;
Lane et al., 1996)
(Fig. 1I). If the inhibition of
CycE-Cdk2 activity by Dap is necessary for the accumulation of
hypophosphorylated Rbf1 and the consequent suppression of E2f1 targets, then
in dap mutants RnrS expression would not be terminated
properly. However, we found that RnrS expression is downregulated in
the epidermis of dap mutants prior to the completion of
S16, just as it is in wild-type embryos
(Fig. 2A,B). Moreover, the
termination of RnrS expression occured even though the epidermal
cells of dap-mutant embryos enter an ectopic S17
(Fig. 2C,D).
|
; Sigrist and Lehner,
1997). Similar to dap mutants, epidermal cells in
fzr mutants fail to exit the cell cycle and inappropriately enter an
ectopic S17, which is likely to be driven by CycE-Cdk2
(Sigrist and Lehner, 1997). In
spite of this, RnrS expression was seen to be properly downregulated
in fzr mutants (Fig.
2E). Thus, although unrestricted CycE-Cdk2 activity can prevent
the initiation of G117 in epidermal cells, E2f1-target gene
expression is still terminated at the appropriate time. These data suggest
that either the inhibition of CycE-Cdk2 does not result in the accumulation of
hypophosphorylated Rbf1, or that a different mechanism is involved in the
initial termination of E2f1-target gene expression.
Cell cycle-regulated destruction of E2f1 protein in the embryonic epidermis
One possible mechanism for the inhibition of E2f1 activity is the
destruction of E2f1 protein. In both the eye and wing imaginal discs, E2f1
protein is destroyed at the G1-S transition and reaccumulates during G2 and M
phase (Asano et al., 1996;
Heriche et al., 2003;
Reis and Edgar, 2004). We
therefore postulated that E2f1 destruction during S phase of the
post-blastoderm cell cycles contributes to the termination of E2f1-target gene
expression in the epidermis. To examine this, we visualized E2f1 protein
abundance by immunofluorescence in embryos that were pulse-labeled with BrdU
(Fig. 3).
E2f1 protein is present throughout the embryo during early syncytial cycles
1-13 (data not shown). Notably, unlike imaginal disc cells, nuclear E2f1 was
detected during S phase of cycles 13 and 14
(Fig. 3A,B). E2f1 protein
accumulated to high levels in the nucleus during G214
(Fig. 3C), and then rapidly
diminished as cells entered S15
(Fig. 3D). This effect is
post-transcriptional, as E2f1 transcripts are ubiquitous during cycle 15
(Duronio et al., 1995),
suggesting that E2f1 protein is destroyed upon entry into S phase. In
addition, the lack of S phase destruction of E2f1 in S13 and
S14 suggests that zygotic gene expression, most of which begins
during cycle 14, is necessary for the coupling of E2f1 destruction with S
phase beginning in cycle 15.
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). E2f1
protein was also downregulated during S phase in these cells
(Fig. 3G,H, arrows). These data
indicate that E2f1 protein abundance is inversely correlated with S phase
during the post-blastoderm cell division cycles.
To determine the timing of E2f1 destruction more precisely, we compared
E2f1 abundance with the pattern of BrdU incorporation as well as with
phospho-histone H3 staining, which detects condensed mitotic chromosomes
(Fig. 4). As reported for wing
imaginal cells (Reis and Edgar,
2004), E2f1 protein was found to be abundant during mitosis. E2f1
was nuclear in early prophase prior to nuclear envelope breakdown
(Fig. 4A, arrowhead). In
metaphase and anaphase, E2f1 protein appeared more diffuse, most probably
because of nuclear envelope breakdown (Fig.
4A, large and small arrows, respectively). E2f1 was present in
newly formed daughter cells, suggesting that it is not destroyed by the APC/C
during mitosis (Fig. 4A, double
arrow). A high level of E2f1 protein was observed in cells in early S phase,
characterized by uniform BrdU incorporation throughout the nucleus
(Fig. 4B, large arrow). In
mid-S phase, where BrdU incorporation was less uniform, there was a
significant reduction in E2f1 protein (Fig.
4B, small arrow). By late S phase, where the more punctuate BrdU
incorporation pattern marks late-replicating heterochromatin, there was very
little E2f1 protein present (Fig.
4B, arrowhead). These data are consistent with the destruction of
E2f1 protein after the initiation of S phase, and differ slightly from
previous results in imaginal discs where no overlap between E2f1 staining and
BrdU-labeling was detected (Heriche et
al., 2003; Reis and Edgar,
2004). This difference may be due to the short embryonic cell
cycle that lacks a G1 phase, as opposed to the canonical G1-S-G2-M disc
cycles.
E2f1 staining in E2f1-mutant embryos was indistinguishable from
wild type until S14 (data not shown), suggesting that maternal
protein persists until S14. E2f1-mutant embryos did
contain a detectable amount of E2f1 protein in G214, but this was
less than in sibling controls (see Fig. S1A,B in the supplementary material),
indicating that zygotic E2f1 synthesis is responsible for a portion of the
E2f1 protein found in G214. Zygotic RnrS mRNA rapidly
accumulated in the epidermis during cycle 14, and then begin to decline during
cycle 15 such that by the beginning of S16 these mRNAs were of very
low abundance (Fig. 1D,
arrowhead; Fig. 2A; see Fig. S2
in the supplementary material). This dynamic pattern of expression was not
altered in E2f1-mutant embryos (see Fig. S1E,F in the supplementary
material) (Duronio et al.,
1995). These data suggest that maternal E2f1 is sufficient to
induce early, zygotic transcription of E2f1 targets, and are consistent with
the hypothesis that S phase-coupled destruction of E2f1 protein contributes to
the decline of E2f1-regulated transcripts during cycle 15.
Destruction of E2f1 protein is S phase-dependent
The correlation between E2f1 disappearance and BrdU labeling suggests that
either cell cycle progression into S phase or DNA synthesis per se triggers
E2f1 destruction. To test if entry into S phase is required for the
destruction of E2f1, we analyzed E2f1 protein levels in stg mutants,
which arrest in G214 (Edgar and
O'Farrell, 1990). E2f1 accumulated to a high level in the
epidermis of stg-mutant embryos
(Fig. 5A). Aminoserosa cells,
which in wild-type embryos permanently exit the cell cycle in G214,
also accumulated high levels of E2f1 (Fig.
3E-H, arrowheads). In Cyclin E mutants, E2f1 protein was
not destroyed in the thoracic cells that normally enter a seventeenth division
cycle (Fig. 3G,H), because
these cells do not enter S phase (data not shown). These data suggest that the
destruction of E2f1 in the epidermis requires entry into S phase.
To test if DNA synthesis is required for E2f1 destruction, we analyzed
double parked (dup)-mutant embryos. dup encodes
Drosophila Cdt1, a component of the prereplication complex (pre-RC)
that is required for eukaryotic DNA synthesis. dup-mutant embryos
develop normally through cycle 15, and then display impaired DNA replication
in S16 causing cell cycle arrest and embryonic lethality
(Garner et al., 2001;
Whittaker et al., 2000).
S16 is absent in dupa1-null mutants, whereas
dupa3 hypomorphic mutants display weak BrdU incorporation
during a prolonged and partial S16
(Fig. 5B,C)
(Garner et al., 2001).
dupa1 mutants accumulated high levels of E2f1 in the
epidermis, suggesting that DNA synthesis is necessary for E2f1 destruction
(Fig. 5B). Interestingly,
dupa3 mutants also accumulated high levels of E2f1 even
though these epidermal cells were capable of incorporating some BrdU
(Fig. 5C). This suggests that
efficient progression through S phase is necessary to trigger E2f1 destruction
and/or that Dup plays a more direct role in E2f1 destruction.
|
|
).
Not all Rbf1-mutant epidermal cells re-enter S phase, suggesting that
other inputs modulate the cell cycle response to Rbf1 loss. These may include
cell-by-cell differences in the amount of E2f1, as we observed that cells with
the most E2f1 were usually the same ones that entered S phase inappropriately.
This is consistent with previous observations that transgene-mediated high
level E2f1-Dp expression can drive most of the G117 epidermal cells
into S phase (Duronio et al.,
1996). These data indicate that Rbf1 is not required for
the initial termination of E2f1-target gene expression, but rather for
sustained termination and stable G1 arrest.
Dap expression promotes conversion of Rbf1 to a repressor
Although our data suggest that E2f1-target genes are controlled
independently of Rbf1 prior to cycle 17, they do not define the mechanism by
which Rbf1 is converted to a repressor during G117. To address this
issue, we re-evaluated the inhibition of CycE-Cdk2 activity by Dap. We
hypothesized that developmentally-controlled Dap expression in cycle 16 does
indeed convert Rbf1 into a repressor, but that Rbf1 is not required for the
initial shut down of RnrS because other mechanisms, such as E2f1
destruction in cycles 15 and 16, are sufficient. Rather, Rbf1 is required to
prevent the reactivation of E2f1-target genes as E2f1 protein reaccumulates
during G216 and G117.
The phenotype of stg mutants allowed us to test this hypothesis.
Previous experiments revealed that E2f1-target gene expression terminates on
schedule in stg mutants even though stg-mutant epidermal
cells arrest in G214 (Duronio
and O'Farrell, 1994). This is an indication of a
developmentally-timed event that occurs independently of cell cycle
progression. The high level of E2f1 protein in stg-mutant epidermal
cells (Fig. 5), which never
enter S phase, would at first seem to be at odds with this result. However,
developmentally controlled Dap expression in a stg mutant may inhibit
CycE-Cdk2 and result in the accumulation of hypophosphorylated Rbf1 and the
downregulation of E2f1 targets (Fig.
1I) (Meyer et al.,
2002b). We therefore simultaneously examined Dap and RnrS
expression in stg mutants. In the epidermis of stg mutants
at the normal time of cycle 15 (i.e. after gastrulation and germ band
extension), RnrS transcripts were abundant and Dap protein was not
detected (Fig. 7A). Later, as
Dap protein accumulated, RnrS expression decreased
(Fig. 7B). To test whether loss
of RnrS expression in stg-mutant embryos required Dap, we
analyzed stg dap double-mutant embryos shortly after the time when
Dap is first induced. RnrS was not suppressed in those cells of
stg dap double-mutant embryos that corresponded to cells with high
levels of Dap protein in stg single-mutant sibling embryos
(Fig. 7C,D, bracket). These
data are consistent with our model that the inhibition of CycE-Cdk2 by
developmentally-controlled Dap expression results in the accumulation of
Rbf1-E2f1 repressor complexes. However, as the stg dap mutant embryos
aged, RnrS expression was eventually lost in many epidermal cells
(Fig. 7E). This also occurred
in the aminoserosa, which contains cells that have exited the cell cycle in
G214 (Fig. 7D,
asterisk). These data imply the existence of Rbf1-indepenent mechanisms to
extinguish E2f1-target gene expression. Perhaps, when cells exit the cycle,
Dap-mediated Rbf1 activation terminates E2f1-target gene transcription while
additional mechanisms dramatically decrease mRNA stability.
|
).
dupa1-mutant epidermal cells failed to downregulate
RnrS at the time of S16, and dupa3
mutants continued to express RnrS during the prolonged and partial
S16 (see Fig. S3A-C in the supplementary material). This may be
explained by the high level of E2f1 protein that accumulates in dup
mutants (Fig. 5). However, Dap
protein accumulates during cycle 16 in dup mutants (data not shown),
and this should result in the downregulation of E2f1 targets as in
stg mutants. Rbf-280 expression using the prd-Gal4 driver
suppressed the ectopic RnrS expression in dupa1
mutants, suggesting that E2f1 could still be repressed by hypophosphorylated
Rbf1 in dup mutants (see Fig. S3D-G in the supplementary material).
At later stages, RnrS transcripts eventually began to decline in
dup mutants (data not shown). We suggest that in dup
mutants, Rbf1 is still converted into an active, hypophosphorylated form in
response to Dap expression, but that the termination of E2f1-dependent
transcription occurs slowly because of the abnormally high level of E2f1
protein. |
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Heriche et al.,
2003; Reis and Edgar,
2004). E2f1 destruction first occurs during S15, at the
same time that E2f1-regulated transcripts such as RnrS begin to
decline. Because E2f1 functions as a transcriptional activator, and because we
show that Rbf1 is not required for the initial decline in RnrS
transcripts, we propose that the loss of E2f1 protein contributes to the
initial termination of replication factor gene expression. Rbf1 is first
required during development for the maintenance of G117 arrest and
the continued repression of E2f1-target genes. Our data suggest that Rbf1 is
converted to a repressor after the developmentally-induced expression of Dap,
most likely because the consequent inhibition of CycE-Cdk2 results in the
accumulation of hypophosphorylated Rbf1. Dap expression accompanies the
downregulation of Cyclin E transcription, and each of these
mechanisms of inhibition of CycE-Cdk2 contributes to G1 arrest.
The high level of E2f1 protein in G117 epidermal cells may
permit the formation of E2f1-Rbf1 complexes necessary to actively and stably
repress replication factor genes during G1 arrest
(Frolov and Dyson, 2004), and
also provides a simple explanation for why the loss of Rbf1 function results
in the ectopic expression of E2f1 targets
(Du and Dyson, 1999). After
hatching, and in response to the first instar larvae beginning to feed, the
epidermal cells start to endoreduplicate. Thus, the accumulation of Rbf1-E2f1
complexes during G1 arrest may prepare cells for rapid production of
replication factors and efficient re-entry into the cell cycle upon activation
of G1 Cyclin-Cdk complexes after growth stimulation.
RnrS expression is lost in E2f1 zygotic mutant embryos,
but not until cell cycle 17 (Duronio et
al., 1995). One interpretation of this result is that maternal
stores of E2f1 are sufficient for the early induction of replication gene
expression in the post-blastoderm divisions. Consistent with this, maternal
E2f1 protein persists into cycle 14, coincident with the commencement of
zygotic transcription of E2f1 targets such as RnrS. In addition,
mutation of the E2f1-binding sites in the regulatory region of the
Pcna gene (mus209 - Flybase) is sufficient to abolish
zygotic Pcna expression (Thacker
et al., 2003). However, our data do not demonstrate a requirement
for E2f1 for early zygotic RnrS expression, and E2f1 may be only one
of several factors necessary for early zygotic expression of genes encoding
replication factors (Hirose et al.,
1993; Sawado et al.,
1998; Yamaguchi et al.,
1996). For instance, the transcription of Cyclin E
requires E2f1 in embryonic endocycles, but also occurs independently of E2f1
via tissue-specific enhancer elements such as those operating in the CNS
(Duronio and O'Farrell, 1995;
Jones et al., 2000). Thus, any
control of replication factor gene expression by E2f1 abundance may be
modulated by other transcription factors, or bypassed entirely in certain cell
types by E2f1-independent modes of expression.
Mechanisms of cell cycle-regulated E2f1 destruction
Our data suggest that E2f1 destruction is coupled to DNA synthesis.
CycE-Cdk2 has been suggested as a possible cell cycle input for E2f1
destruction in imaginal cells because it is activated at the G1-S transition
when E2f1 is destroyed (Heriche et al.,
2003; Reis and Edgar,
2004). However, CycE-Cdk2 is continuously active during the
embryonic post-blastoderm cell cycles, whereas E2f1 is destroyed only during S
phase (Sauer et al., 1995).
Thus, CycE-Cdk2 is unlikely to be the only signal, and actively replicating
DNA may provide a necessary input into E2f1 destruction. This model is
consistent with our observation that E2f1 destruction occurs after DNA
synthesis begins, resulting in cells that are positive for both E2f1 and BrdU
incorporation in early interphase.
Previous studies have suggested that mammalian E2f1 is degraded by the
ubiquitin-proteasome pathway (Campanero and
Flemington, 1997; Hateboer et
al., 1996; Hofmann et al.,
1996; Marti et al.,
1999; Ohta and Xiong,
2001). In this pathway, E3 ubiquitin ligases bind to and mediate
the ubiquitylation of specific proteins. The SCF class of cullin-dependent E3
ligases has been implicated in E2F1 destruction
(Marti et al., 1999). In
Drosophila, evidence from genetic and cell biology studies suggest
that SCFSLMB mediates E2f1 destruction at the G1-S transition in
wing imaginal disc cells (Heriche et al.,
2003). Although there is no evidence implicating a specific E3
ligase in the destruction of embryonic E2f1, there are interesting parallels
with recent experiments describing the destruction of Cdt1/Dup. Like E2f1,
Cdt1/Dup is degraded at the G1-S transition and cannot be detected during S
phase (Thomer et al., 2004).
In vertebrates, Cdt1 destruction is mediated by two independent and apparently
redundant mechanisms: direct Cdk2 phosphorylation that targets Cdt1 to
SCFSKP2, and binding of PCNA to the Cdt1/Dup amino-terminus that
targets Cdt1 to Cul4DDB1 (Arias
and Walter, 2006; Nishitani et
al., 2006; Senga et al.,
2006). This latter result is consistent with a recent study
indicating that Drosophila Dup hyperaccumulates in cells where DNA
synthesis is attenuated (May et al.,
2005). Thus, more than one E3 ubiquitin ligase may participate in
E2f1 destruction (Ohta and Xiong,
2001). Determining the molecular mechanism of E2f1 destruction
should permit us to directly test whether prevention of E2f1 destruction would
affect replication factor gene expression in the embryo.
pRb-independent E2F regulation and early animal development
E2F is necessary for the development of worms, flies and mice
(DeGregori, 2002). Remarkably,
however, pRb is not needed for the entirety of mouse embryonic development
(Wu et al., 2003). This could
in part be due to redundancy with other pRb family members, such as p107 and
p130 (Dannenberg et al., 2004).
Alternatively, a pRb-independent mechanism of regulating E2F activity may
control S phase gene expression and cell cycle progression during early
mammalian development. This idea is supported by experiments modeling the cell
cycles of early vertebrate development in cell culture using murine embryonic
stem cells (White et al.,
2005). These pluripotent cells have a cell cycle composed mostly
of S phase that is characterized by ubiquitous Cdk activity and the absence of
CKIs (Faast et al., 2004;
Savatier et al., 1996;
Stead et al., 2002). As in the
Drosophila embryo, E2F-regulated transcripts are also ubiquitous even
though pRb family members are expressed
(Savatier et al., 1994;
Stead et al., 2002).
Differentiation requires the lengthening of G1 and the negative regulation of
Cdk2 activity, which is accomplished both by increases in the level of CKIs
and by the downregulation of Cyclin E1 expression via inhibition of E2F
(White et al., 2005). Thus,
evolutionarily conserved regulatory mechanisms operating in early development
may mediate the conversion from rapid cell cycles driven by intrinsic cues to
slower, more highly regulated cycles that are influenced by extrinsic
developmental and environmental cues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/467/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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