Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Seattle, WA 98109-1024, USA
Authors for correspondence (e-mail:
eisenman{at}fhcrc.org
and
bedgar{at}fhcrc.org)
Accepted 3 February 2004
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SUMMARY |
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Key words: Myc, dMyc, diminutive, Endoreplication, Polyploid, Growth, Cyclin E, Mnt, dMnt, Fat body, Salivary gland, p21, Drosophila
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Introduction |
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Myc and Mad proteins are thought to regulate these processes through
transcriptional control of genes required for growth and proliferation. Both
Myc and Mad form heterodimeric complexes with their common binding partner
Max, mediated by the basic helix-loop-helix zipper (bHLHZ) domains present in
all these proteins. Myc-Max and Mad-Max heterodimers bind DNA by recognizing
the E-box sequence CACGTG. Myc-Max heterodimers recruit the co-activators
TRRAP and p300, resulting in transcriptional activation of genes containing
promoter-proximal E-box sequences, most probably through acetylation of
histones in chromatin. By contrast, Mad-Max heterodimers recruit the Sin3
corepressor, which binds histone deacetylases to deacetylate histones and
thereby antagonize Myc/Max activity (reviewed by
Grandori et al., 2000;
Oster et al., 2002
). In
addition, Myc, by inactivating the Miz-1 transcription factor, represses some
genes (such as a subset of cyclin dependent kinase inhibitors) that negatively
regulate growth and proliferation (Seoane
et al., 2002
; Staller et al.,
2001
).
The Myc/Max/Mad network is conserved in Drosophila and comprises
dMax and single members of the Myc and Mad families, dMyc and dMnt,
respectively (Gallant et al.,
1996; Schreiber-Agus et al.,
1997
) (L. Loo, J. Secombe and R.N.E., unpublished). Both
loss-of-function and overexpression studies have demonstrated an important
role for dMyc, which is encoded by the diminutive (dm) gene,
in regulating cellular growth. Hypomorphic dm mutants are female
sterile and all mutant adults are smaller than wild type with thinner, shorter
bristles (Gallant et al.,
1996
; Johnston et al.,
1999
; Schreiber-Agus et al.,
1997
). The reduced body size of these mutants results from a
decrease in cell size and possibly cell number
(Johnston et al., 1999
).
Conversely, ectopic expression of dMyc in wing disc cell clones increases cell
size and promotes G1/S progression without altering cell doubling time.
The notion that dMyc is involved in growth is consistent with more recent
studies attempting to define Myc target genes in vertebrates and flies.
Genomic analyses to identify expression changes induced by Myc and Mad
(Boon et al., 2001;
Coller et al., 2000
;
Guo et al., 2000
;
Iritani and Eisenman, 1999
;
Neiman et al., 2001
;
Watson et al., 2002
), as well
as direct DNA binding sites for mammalian Myc
(Fernandez et al., 2003
;
Li et al., 2003
) and
Drosophila dMyc, dMax and dMnt
(Orian et al., 2003
) have, in
general, shown that Myc regulates a large and diverse set of genes whose
functions are consistent with the ability of Myc to promote growth and
proliferation. The Drosophila larva consists predominantly of
differentiated polyploid tissues that support the growth and eventual
metamorphosis of the mitotic imaginal discs and nervous system, which will
give rise to the tissues of the adult animal (see
Gilbert, 2003
). Although
growth of the imaginal discs is accomplished by cell growth coupled to
proliferation, growth of the polyploid tissues occurs in conjunction with
endoreplication, a modified cell cycle, in which DNA replication results in
polytene chromosomes. Endoreplicating cells dramatically increase in size
without dividing (reviewed by Edgar and
Orr-Weaver, 2001
). Little is known about what regulates the
endocycle in larval cells. However, it is clear both from the reproducible
final tissue ploidies (Smith and
Orr-Weaver, 1991
) and the effect of nutrient deprivation on
endoreplication (Britton and Edgar,
1998
; Britton et al.,
2002
) that environmental factors and the regulation of
developmental genes are important. Endocycles consist of distinct S and gap
phases, regulated by oscillations in Cyclin E/cdk2 activity (reviewed by
Edgar and Orr-Weaver, 2001
).
Oscillation of Cyclin E/cdk2 activity may be accomplished by the periodic
expression of Dacapo, a p27Cip/Kip homolog that inhibits Cyclin E
activity and oscillates out of phase with Cyclin E in endocycling ovarian
nurse cells (de Nooij et al.,
2000
; Lilly and Spradling,
1996
).
Because of the relationship of dMyc function to cell growth in mitotic cells, we have now investigated the roles of dMyc, as well as dMnt, in larval endoreplicating tissues and explored their potential functions in regulation of both growth and DNA endoreplication.
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Materials and methods |
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Deletion alleles of dm were generated by imprecise P-element excision, in flies carrying P{w+mGT=GT1}dmBG02383 alone or in trans to P{w+mGT=GT1}dmBG00605. Deletions in the dm gene were identified by PCR and confirmed by sequencing. To verify that the observed phenotypes were caused by the deletion in dm4, dm4/FM7i, Act-GFP females were crossed to males homozygous for a transgene in which the tubulin promoter drives dMyc expression (a kind gift from Peter Gallant). Progeny were scored for the presence of viable dm4 males. dm1-14-2 retains all of the dm gene and 32 bp of P{w+mGT=GT1}dmBG02383 P-element sequence and is phenotypically wild type in all of our assays.
Western blotting
One hundred larvae per sample were homogenized and boiled in SDS sample
buffer and run on 8% SDS-PAGE gels. Monoclonal antibodies were used to detect
dMyc (P4C4-B10, undiluted) (Prober and
Edgar, 2000) and actin (1:10,000, Sigma), which were visualized
using HRP-conjugated secondary antibodies (Zymed) and SuperSignal ECL
(Pierce).
Immunocytochemistry
Dissected larvae were fixed in 4% paraformaldehyde/PBS or 70% ethanol/PBS.
Washes and antibody incubations were performed in 0.1% Tween-20 in PBS or 0.1%
Triton and 0.1% BSA in PBS. Primary antibodies used were mouse anti-dMyc
(P4C4B10, undiluted) (Prober and Edgar,
2000), mouse anti-dMnt (P5D6B8, undiluted) (L. Loo, J. Secombe and
R.N.E., unpublished), mouse anti-BrdU (Becton-Dickinson, 1:100), mouse
anti-Nop1p, for detection of fibrillarin, (1:600)
(Aris and Blobel, 1988
) and
guinea pig anti-Cyclin E (1:1600) (T. Orr-Weaver). Alexa- (Molecular Probes)
or FITC-conjugated (Zymed) secondary antibodies were used. DNA was visualized
with 4',6-Diamidine-2-phenylindole (DAPI) or propidium iodide. Larvae
were fed 5-bromodeoxyuridine (BrdU) at 100 µg/ml.
Ectopic expression
Ectopic expression of UAS-regulated transgenes was driven by ADH or Ptc
Gal4 drivers (Brand and Perrimon,
1993) or by using the Flp/Gal4 method
(Neufeld et al., 1998
;
Pignoni and Zipursky, 1997
),
with or without a 1 hour 37°C heat shock during the first larval
instar.
Quantification of DNA content
A Delta Vision microscope (Applied Precision) was used to take 0.2-0.5
µm sections through nuclei in the posterior salivary gland or fat body. The
fluorescent intensity of single nuclei was calculated using the ImageQuant
software package (Applied Precision).
RT-PCR
For RT-PCR analysis of dm mutants, RNA was isolated from larvae
using TRIzol® Reagent (Invitrogen). PCR (94°C for 3 minutes; 25 cycles
of 94°C for 1 minute, 60°C for 1 minute and 72°C for 2 minutes;
72°C for 10 minutes) was performed to detect dMyc sequence, across the
junction of exons two and three (primers:
5'-GAGCAACAACAGGCCATCGATATAG-3',
5'-CCTTCAGACTGGATCGTTTGCG-3') and within exon three
(5'-TGTGCAGATGAGGAAATCGATGTCG-3',
5'-TGCGTCACTTTGTTATTGACTCCC-3'), and CaMKII
(5'-CAGTGGCGACTTTGATGGATACAC-3',
5'-TGTAGCACTTTCATTAACATGTGC-3'). CaMKII was chosen as a control
because overexpression of dMyc does not change its expression
(Orian et al., 2003).
For RT-PCR from isolated tissues, RNA was isolated and reverse transcribed using the Cells-to-cDNA kit (Ambion). Forty-five cycles of PCR was performed to detect dMyc sequence, across the junction of exons two and three, and Rp49, using the above conditions.
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Results |
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dMyc and dMnt are expressed in larval endocycling tissues in complementary patterns
We next used antibody staining to investigate the expression patterns for
dMyc and dMnt during larval stages. In several endoreplicating tissues,
including the salivary gland (Fig.
2), the proventriculus and gastric cecae (data not shown), dMyc
and dMnt display inverse patterns of expression. dMyc expression persists in
the distal region of the third instar salivary gland, whereas dMnt expression
is limited to the proximal region (Fig.
2). In both cases, DAPI stained nuclei are visible throughout. The
distal region of the salivary gland undergoes more endoreplication than the
proximal region (Berendes and Ashburner,
1978), resulting in approximate final ploidies of 2048C and 1024C
(Hammond and Laird, 1985
).
Because of this defined difference in local endoreplication, we chose to focus
our studies on this tissue. BrdU labeling revealed that the cells that cycle
last in the salivary gland are those at the distal region of the gland (data
not shown), which both express dMyc longer and attain the highest final
ploidy. The fact that sustained expression of dMyc correlates with this extra
endoreplication suggests that dMyc may be linked to endoreplication. Larvae
that were stained for both dMyc expression and BrdU incorporation showed that
most cells that express dMyc are also labeled with BrdU and thus are actively
endoreplicating (data not shown). Conversely, dMnt is expressed in proximal
salivary gland cells that have exited the endocycle.
|
dMyc is required for DNA endoreplication
To determine whether dMyc is required for endoreplication, we compared
nuclear size of endocycling cells in dm4 mutant and
control animals. Larval tissues were fixed and stained with DAPI to visualize
the DNA. At approximately the time of hatching (24 hours AED), the nuclei of
dm4 mutant salivary gland, fat body, and gut tissue
appeared similar to those of control tissue
(Fig. 3A and data not shown).
However, by 48 hours AED, nuclei of mutant tissue were noticeably smaller than
control nuclei and the difference was even more pronounced at 72 hours AED
(Fig. 3A). In addition, the
overall size of the dm4 mutant salivary gland and gut was
smaller than control (Fig. 3A),
consistent with a reduction in cell size.
|
Ectopic expression of dMyc and dMnt have opposing effects in endoreplication
Endogenous expression patterns suggest opposing roles for dMyc and dMnt in
larval endoreplication (Fig.
2A). To test this hypothesis, we used two techniques to
ectopically express dMyc or dMnt UAS-regulated transgenes in endoreplicating
larval cells. The first employed the Gal4/UAS system
(Brand and Perrimon, 1993) to
express dMyc or dMnt in salivary gland and fat body, under the control of
Ptc-Gal4 or Adh-Gal4, respectively. Second, we used the FLP/Gal4 system to
induce expression of dMyc and dMnt in random clones of cells throughout the
larva, which are identifiable by co-expression of nuclear-localized GFP
(Neufeld et al., 1998
;
Pignoni and Zipursky, 1997
).
Although this system is regulated by the heat-shock promoter, heat
shock-independent activation of Gal4 occurs in a small number of cells in
various tissues, including fat body and gut, before the onset of larval growth
and endoreplication (Britton et al.,
2002
). Because these heat shock-independent events occur early,
they result in transgene expression throughout larval development. Using both
of these techniques, we found that ectopic dMyc and dMnt had opposite effects.
dMyc drove ectopic endoreplication and resulted in larger cells with a higher
ploidy than normal in fat body (Fig.
4A-C) and salivary gland (not shown) cells, whereas ectopic dMnt
limited the number of endocycles, leading to smaller fat body
(Fig. 4D-F) and salivary gland
cells (not shown) with decreased final ploidy.
|
|
dMyc influences nucleolar size
During our analysis of larval cells expressing ectopic dMyc, we observed
what appeared to be abnormally large nucleoli. One of the hallmarks of
vertebrate Myc overexpression in tumor cells is enlarged nucleoli
(Abrams et al., 1982;
Hann et al., 1983
). To examine
this, we stained cells with an antibody to fibrillarin
(Aris and Blobel, 1988
), which
is involved in rRNA processing and ribosome assembly
(Tollervey et al., 1993
) and
has been reported to be a transcriptional target of dMyc
(Orian et al., 2003
) and
vertebrate Myc (Coller et al.,
2000
). Anti-fibrillarin staining of fat body overexpressing dMyc
confirmed that nucleoli were abnormally large and expressed increased amounts
of fibrillarin (Fig. 4H-K).
This suggests that one mechanism by which dMyc drives growth and
endoreplication is to increase the rate of ribosome biogenesis.
Cells overexpressing dMyc undergo a normal endocycle
The endocycle consists of discrete periods of DNA synthesis separated by
gap phases. dMyc might increase the rate of endoreplication by eliminating the
gap phase or by speeding up the G/S cycle. Both salivary gland and fat body
overexpressing dMyc contain a subset of cells that fail to incorporate BrdU
(Table 1 and
Fig. 5A-D), although their
nuclear size indicates previous endoreplication. From this we conclude that
dMyc-overexpressing cells display discrete S-phases separated by a gap
phase.
|
dMyc growth effects are tightly linked to the cell cycle
Our studies of endoreplicating tissues, as well as our earlier work on
mitotic imaginal disc cells (Johnston et
al., 1999), have demonstrated that ectopically expressed dMyc
drives both cellular growth and endocycle progression. To further study the
mechanism by which this occurs, we asked whether ectopically expressed dMyc
could overcome endoreplication arrest imposed by the expression of specific
transgenes. We were particularly interested in whether induction of growth and
endoreplication by ectopic dMyc could be uncoupled. Ptc-Gal4, which is
predominantly expressed in the salivary gland throughout larval development,
was used to drive expression of different transgenes. The salivary glands of
wandering third instar larvae were analyzed by staining with DAPI and
anti-fibrillarin antibodies to visualize DNA and nucleoli, respectively.
Continuous ectopic expression of Cyclin E blocks endoreplication and salivary
gland growth (Follette et al.,
1998
; Weiss et al.,
1998
) (Fig. 6A,B),
as does ectopic expression of vertebrate p21, a cyclin-dependent kinase
inhibitor (Fig. 6A,C). It is
thought that continuous high levels of active CyclinE/cdk2 complexes generated
by Cyclin E overexpression block the `resetting' of origins of replication,
and thus S-phase initiation, in endoreplicating cells
(Follette et al., 1998
).
Overexpression of p21 inhibits the activity of Cyclin E/cdk2 complexes, which
are required to progress through the G/S transition. When dMyc was
co-expressed with Cyclin E or p21, a slight increase in DNA and fibrillarin
staining was observed (compare Fig.
6I,M with Fig. 6J,N
and Fig. 6K,O), with little
increase in the overall size of the salivary gland (compare
Fig. 6F with 6B and
Fig. 6G with 6C).
Quantification of DAPI fluorescence by deconvolution microscopy indicated that
co-expression of dMyc with Cyclin E or p21 increased the nuclear DNA content
approximately twofold. By contrast, the nuclear DNA content of control
salivary glands was
25 times higher. These results suggest that ectopic
dMyc is insufficient to drive significant endoreplication or cell growth when
the cell cycle itself is arrested.
|
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Discussion |
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Analysis of a new dm mutant allele
To determine whether there is a requirement for dMyc in driving
endoreplication, we isolated dm4, a null allele of
dm. The failure of dm4 mutants to grow beyond the
second instar (Fig. 1B)
indicates that dMyc is required for growth at the organismal level. We presume
that maternally deposited dm gene products, or other maternal
products, are sufficient for development of the embryo. The fact that maternal
dm transcripts and protein are undetectable by the time of hatching
suggests that dMyc is not required for the initiation of larval growth and may
not be required for completion of embryogenesis, although it is possible that
a small amount of residual maternal dMyc supports the growth of
dm4 mutant larvae prior to their arrest. The massive
growth that takes place during larval development is tightly coupled to the
endoreplication that takes place in all larval tissues except the imaginal
discs and nervous system (Galloni and
Edgar, 1999; Smith and
Orr-Weaver, 1991
). Our finding that dMyc and dMnt are expressed in
distinct groups of cells in these tissues is suggestive of roles in promoting
(dMyc) and limiting (dMnt) endoreplication
(Fig. 2) and suggests that the
failure of dm4 mutants to grow is the result of loss of
dMyc in endocycling tissues.
Larval growth involves both cytoplasmic growth and DNA endoreplication
(Edgar and Orr-Weaver, 2001).
The reduced rate of BrdU incorporation in early larval tissues and the failure
of dm4 mutant nuclei to grow suggest that these mutants
undergo reduced DNA replication. The fact that mutant cells and larvae are
smaller than age-matched controls indicates that that there is also a growth
defect. Thus, directly or indirectly, dMyc is required for both growth and DNA
replication during endoreplication (Fig.
3).
Overexpression of dMyc and dMnt have opposite effects on endoreplicating cells
Consistent with our finding that dMyc loss of function negatively affects
endoreplication and growth, overexpression of dMyc drives both cellular growth
and DNA replication (Fig.
4A-C,G). By contrast, overexpression of the Drosophila
ortholog of Mad, dMnt (L. Loo, J. Secombe and R.N.E., unpublished), blocks
cellular growth and DNA replication (Fig.
4D-F). These results are consistent with a model in which dMyc and
dMnt act antagonistically, with dMnt binding and repressing the genes required
for endoreplication that dMyc activates. We find that dMnt is normally highly
expressed in the third instar salivary gland and other tissues that have
exited the endocycle, indicating that dMnt-mediated gene repression may be
necessary for this transition. However, dMnt, the only Mad family ortholog in
Drosophila, is non-essential. dMnt null mutants develop normally into
adults with modestly increased body weights and shorter lifespans (L. Loo, J.
Secombe and R.N.E., unpublished). Although the increased body weight of dMnt
mutants is consistent with negative regulation of growth by dMnt, we have not
observed altered endoreplication in mutants (L. Loo, J. Secombe, unpublished),
suggesting that negative regulators of endoreplication other than dMnt must
exist.
dMyc influences growth and S phase entry in endoreplicating cells
A role for dMyc in regulating both organismal size and the size of
mitotically dividing cells has been previously reported
(Johnston et al., 1999). We
show that in endoreplicating cells ectopic expression of dMyc results in
increased cytoplasmic and nuclear volume, as well as in enlarged nucleoli, as
detected by increased anti-fibrillarin staining
(Fig. 4H-K). Fibrillarin has
been implicated as a Myc target gene in both vertebrate and
Drosophila cells (Coller et al.,
2000
; Orian et al.,
2003
) and its augmented expression is consistent with the notion
that dMyc/Myc promotes ribosome biogenesis
(Tollervey et al., 1993
). The
pitchoune gene, which encodes a putative RNA-helicase localized to
the nucleolus, is also induced by ectopic dMyc, and pit null mutants
have a severe larval growth defect similar to dm4 mutants
(Zaffran et al., 1998
). The
mammalian ortholog of pit, MrDB (DDX18), has been identified as a
direct target of c-Myc (Grandori et al.,
1996
). In addition, many other known and suspected targets of the
Myc family are involved in this process
(Boon et al., 2001
;
Guo et al., 2000
;
Neiman et al., 2001
;
Orian et al., 2003
).
S-phase of the endocycle is initiated by the activity of Cyclin E/cdk2
(Follette et al., 1998) but
endoreplication can be blocked by continuous ectopic expression of Cyclin E or
the human cdk inhibitor p21 (Follette et
al., 1998
; Weiss et al.,
1998
) (Fig. 6). It
is thought that Cyclin E levels must drop after each S-phase and then increase
again prior to the next S-phase to allow reinitiation of DNA replication. In
mitotic cells, this prevents more than one round of DNA replication from
occurring during each cell cycle. In endoreplicating cells it results in
discrete S-phases separated by a gap phase. Ectopic p21 is likely to inhibit
the activity of cdk2 even in the presence of Cyclin E
(de Nooij et al., 1996
). The
extra endocycles driven by ectopic dMyc appear to be normal, in that there are
discrete periods of DNA replication and Cyclin E appears to oscillate
(Fig. 5E-H). As ectopic dMyc
induces cells to accumulate high levels of DNA earlier in development
(Fig. 4G), we presume that
S-phases and Cyclin E oscillations occur more frequently than normal. It is
also possible that the S-phases are shorter and that Cyclin E peaks at higher
levels when ectopic dMyc is present. When co-expressed with ectopic
unregulated Cyclin E or p21, dMyc drives very little endoreplication
(Fig. 6), suggesting that the
cell cycle control exerted by oscillating Cyclin E/cdk2 activity is downstream
of dMyc function. Consistent with this, ectopic dMyc can
post-transcriptionally increase Cyclin E levels in wing discs
(Prober and Edgar, 2000
) and
studies in mammalian cells suggest that Myc can indirectly induce Cyclin E
expression (Steiner et al.,
1995
). Microarray analysis did not identify Cyclin E as a
transcriptional target of dMyc (Orian et
al., 2003
), suggesting that the transcriptional oscillation of
Cyclin E is not directly regulated by dMyc. Thus, dMyc is unable to drive
endoreplication in the absence of normal cdk activity. Although the level of
fibrillarin staining was not quantified, dMyc appears to drive somewhat more
nucleolar growth than DNA accumulation when co-expressed with Cyclin E or p21
(Fig. 6B-O), indicating that
dMyc may be able to drive a limited amount of nucleolar growth in the absence
of DNA replication.
The Drosophila insulin signaling pathway is also essential for
growth. Mutations in the receptor InR and downstream components of the
pathway, including Dp110, a PI3 kinase homolog, cause larval growth defects
(Chen et al., 1996;
Weinkove et al., 1999
). We
found that when PI3 kinase signaling was blocked by ectopic expression of p60,
dMyc was still able to induce a significant amount of cellular growth and DNA
replication (Fig. 6). This
suggests either that dMyc is downstream of PI3 kinase signaling or that dMyc
and dDp110 represent independent pathways that are both essential for growth.
Recent studies have found that Dp110 or InR overexpression did not result in
increased dMyc transcription (B.E. and L. Li, unpublished) and that activated
Ras increased dMyc levels and PI3 kinase activity via independent effector
pathways (Prober and Edgar,
2002
), suggesting that dMyc transcription is not downstream of the
insulin signaling pathway. In addition, although ectopic expression of either
dMyc or Dp110 leads to increased cell growth, the increase in nuclear size is
more pronounced in response to dMyc whereas the increase in cytoplasmic volume
is more pronounced in response to Dp110
(Saucedo and Edgar, 2002
),
further supporting the idea that dMyc and Dp110 regulate growth and
endoreplication independently.
Defining dMyc pathways for growth and replication
dMyc overexpression augments cell growth in mitotic wing disc cells by
shortening the mass doubling time. Such cells display a decrease in the length
of G1 and a compensatory increase in the length of G2/M, resulting in a
division time equal to that of control cells
(Johnston et al., 1999). They
retain their normal ploidy and show little effect on the length of S phase. We
show here that in endoreplicating cells, dMyc drives both cellular growth and
DNA replication. What is the relationship of dMyc function to these processes?
dMyc transcriptionally activates a wide range of genes involved in ribosome
biogenesis, translation and metabolism, suggesting that the relationship of
dMyc to growth is likely to be very direct. The absence of an effect of dMyc
on S phase length and cell division rate in mitotic cells argues that perhaps
the only role of dMyc is to regulate cell growth. Interestingly, the division
rate of dMyc-overexpressing mitotic cells is increased by introduction of
String, which accelerates G2/M, resulting in the generation of a larger number
of cells. Perhaps in endoreplicating cells, which lack G2/M entirely, dMyc
simply increases the growth rate thereby shortening the G1-S transition and
leading to a higher rate of S phase entry. Because such cells are incapable of
division, the net effect observed is larger cells with increased ploidy. In
this model, dMyc is thought to augment endoreplication indirectly, through its
promotion of growth. While this paper was in preparation Maines et al.
(Maines et al., 2004
) reported
that dMyc is required during oogenesis for somatic and germ cell growth and
endoreplication, but not for proliferation prior to the onset of
endoreplication. The finding that dMyc mutant follicle cells exhibit reduced
growth prior to endoreplication suggests that the defect in endoreplication
may be secondary to the defect in cellular growth.
Alternatively, dMyc might affect endoreplication more directly. Although
both mammalian and Drosophila Myc target genes are predominantly
growth related, a smaller number of gene targets are involved in cell cycle
control and DNA replication (Fernandez et
al., 2003; Orian et al.,
2003
; Staller et al.,
2001
). Importantly, dMyc does not increase transcript levels of
Cyclin E or the Drosophila E2F1 transcription factor, the only known
limiting factors for endocycles in endoreplicating tissues
(Orian et al., 2003
). Our
finding that the effect of dMyc on growth is attenuated when the cell cycle is
blocked by continuous expression of Cyclin E or p21
(Fig. 6) indicates that
dMyc-induced growth is tightly coupled to DNA replication, at least in
endoreplicating cells. The large number and diversity of the genes identified
as likely targets of Myc genes indicates that Myc activity impinges on a broad
range of cellular functions that must be highly coordinated for proper cell
behavior. Interestingly Myc overexpression has been reported to lead to
endoreplication and polyploidy in human kertinocytes
(Gandarillas et al., 2000
).
Furthermore, in murine fibroblasts treated with colcemid, Myc overexpression
leads to abrogation of the G2/M checkpoint and marked polyploidy
(Li and Dang, 1999
). These
results suggest that Myc function is involved in controlling S-phase entry and
G2/M in diverse vertebrate cell types. The Drosophila endoreplicating
cell system should provide a good model for better defining the precise role
of Myc in coordinating growth and cell cycle.
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ACKNOWLEDGMENTS |
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Footnotes |
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