Department of Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607-7170, USA
*Author for correspondence (e-mail: katzen{at}uic.edu)
Accepted 19 June 2002
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SUMMARY |
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Key words: Drosophila, Myb, Transcription factor, Replication, Mitosis, Endoreplication, Endocycle, Endoreduplication, Genomic stability
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INTRODUCTION |
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Several functional domains of the c-Myb protein have been defined (reviewed by Oh and Reddy, 1999): the sequence-specific DNA binding domain positioned near the N terminus; the transcriptional activation domain located in the middle of the protein; and a negative regulatory domain residing at a more C-terminal position (see Fig. 1). C-terminal and N-terminal sequences of the c-myb gene are missing in two independently isolated oncogenic viral Myb genes. In cultured cells, C-terminal truncation of the c-Myb protein enhances both its transforming potential and its ability to activate transcription from a reporter construct in cultured cells. A-myb and B-myb genes encode proteins that share several regions of homology with c-Myb, and are structurally similar, but not identical to the c-Myb protein (Oh and Reddy, 1999
).
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Although the finding that Dm myb plays a role in the cell cycle superficially agrees with evidence that vertebrate Myb genes are required in at least some cell types for proliferation, there are important differences. Vertebrate Myb genes are generally thought to be required for the G1/S transition and progression through S phase, whereas our analyses of mutant myb phenotypes in Drosophila have implicated Dm myb in later phases of the cell cycle. Recent studies showing that mutations in several genes known to be involved in DNA replication can lead to a block in mitosis as well as the expected G1 arrest (Pflumm and Botchan, 2001; Whittaker et al., 2000
), raise the issue of whether the Dm myb mutant phenotypes could also result from defects that occur during S phase. Data in two recently published papers have some bearing on this issue: the first shows that DMyb induces expression of the cyclin B gene in eye imaginal discs, providing support for Dm myb having a direct role in regulating the G2/M transition (Okada et al., 2002
); the second provides some indication of S-phase defects in addition to mitotic defects in null alleles of Dm myb (Manak et al., 2002
).
We have now turned to the Gal4-UAS binary system of ectopic expression to further investigate the activities of wild-type and C-terminally truncated DMyb proteins. These studies have revealed that, depending upon the type of cell cycle, DMyb can exert two opposing effects on DNA replication. Ectopic expression in developing salivary glands of C-terminally truncated DMyb (DMyb), and to a lesser extent the full-length DMyb protein, can suppress endoreduplication. By contrast, ectopic expression of either DMyb protein in diploid cells can drive cells into S phase and M phase, thereby promoting proliferation.
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MATERIALS AND METHODS |
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All other transgenic lines were generously provided by other investigators and have been previously described: UAS-RBF from Wei Du (via Bruce Edgar) (Xin et al., 2002); UAS-GFP (=UAS-GFPnls) from Bruce Edgar (Neufeld et al., 1998
); en-Gal4 (=Scer\GAL4ene16E) (FlyBase, 1999
) from Andrea Brand (Fietz et al., 1995
); sd-Gal4 (=P{GAL4}sdSG29.1) from Shelagh Campbell and isolated by Veronica Rodrigues (Roy et al., 1997
); fkh-Gal4 from Steven Beckendorf [contains the salivary gland-specific enhancers of fkh defined by Zhou et al. (Zhou et al., 2001
)]; HS-E2F,HS-DP from Bob Duronio (Follette et al., 1998
); and Actin5c-Gal4 on chromosome 3 from the Bloomington stock center (FlyBase, 1999
).
Preparation and fluorescent staining of imaginal discs and salivary glands
Animals were raised at 24°C unless otherwise specified. For heat shock induction of HS-E2F; HS-DP, animals were incubated at 37°C for 30 minutes once every 12 or 24 hours, as noted. Salivary glands and imaginal discs were dissected from larvae (either wandering third instar or timed in hours after egg deposition, AED, if so noted) and fixed in 4% paraformaldehyde in PBS and 0.1% Triton-X for 30 minutes at room temperature. Immunostaining was performed as previously described (Audibert et al., 1996; Theurkauf, 1994
) with the following dilutions for primary antibodies: 1:700 for the polyclonal rabbit antibody against the DMyb DNA-binding domain (Jackson et al., 2001
); 1:1000 for PH3 (Upstate Biotech); and 1:5 for Cyclin B (mouse monoclonal supernatant, F2F4, Developmental Studies Hybridoma Bank of the University of Iowa). For BrdU (5-bromo-2-deoxyuridine) labeling, dissected imaginal discs or salivary glands were incubated in Schneiders media (Gibco) containing 1 mg/ml BrdU for 30 minutes (discs) or 1 hour (salivary glands). Afterwards, they were fixed as above, washed three times, denatured in 2N HCl and neutralized in 100 mM sodium tetraborate. Samples were blocked with bovine serum albumin (BSA) and incubated with mouse anti-BrdU monoclonal antibody (Sigma) at 1:20. Secondary antibodies conjugated to either FITC or rhodamine (Boehringer Mannheim) were used at recommended dilutions. After immunostaining, samples were treated with DAPI at 0.5 µg/ml for 10 minutes, rinsed and mounted in Vectashield (Vector).
To identify apoptotic cells, discs were stained with Acridine Orange or the TUNEL assay. For the former, wing discs were dissected in 5 µg/ml Acridine Orange solution, rinsed in PBS, and then mounted on slides for viewing. For the latter, we used the ApopTag kit (Intergen) and followed the protocol described by White et al. (White et al., 1996).
Samples were imaged either by confocal microscopy (Zeiss LSM 550) or by wide-field microscopy (Zeiss Axioplan2) using a Princeton Instruments Micromax cooled CCD camera.
Quantitation of DNA content
We followed the protocol described by A. Weiss and colleagues (Weiss et al., 1998) for quantitation. All salivary glands were dissected from climbing stage third instar larvae that were aged to
120 hours AED. For each genotype, a total of 35-65 salivary gland nuclei from four to six salivary glands were measured. Then the ratios for each salivary gland nucleus to the average fat body nucleus for each genotype (derived from 20-30 nuclei) were determined and the mean ratio and standard deviations were calculated.
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RESULTS |
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In initial experiments, we found that driving ubiquitous ectopic expression of either DMyb protein during development was detrimental. For example, when UAS-DMyb expression was driven by Actin5c-Gal4 at 25°C, less than 1% of the animals survived to adulthood. Viability improved when temperatures were lowered, with about half of the pupae emerging as adults at 21°C and more than 80% emerging at 18°C (the Gal4-UAS system of expression shows temperature sensitivity, driving higher levels of expression at higher temperatures) (Greenspan, 1997; Morimura et al., 1996
). By comparison, when UAS-
DMyb was driven by Actin5c-Gal4, the result was 100% lethality at all three temperatures, demonstrating that
DMyb is a more potent effector than full-length DMyb, as predicted.
Ectopic expression of DMyb induced proliferation in imaginal disc cells
We then focused on the consequences of ectopic DMyb expression in cells of the larval imaginal discs from wandering third instar larvae. Two Gal4 drivers were used for these experiments: engrailed (en)-Gal4, which drives expression of UAS-reporter constructs in the posterior compartment of each imaginal disc (Fig. 2A), and scalloped (sd)-Gal4, which drives expression throughout the wing pouch (Fig. 2C). An antibody against the DMyb protein (Jackson et al., 2001) detected increased levels of DMyb protein in the appropriate regions of the discs when the UAS-DMyb constructs were ectopically expressed via the Gal4 drivers (Fig. 2B and not shown). When DMyb expression was driven by either Gal4 driver, wing discs were malformed, appearing to be overgrown (or bulging) in the areas where DMyb was ectopically expressed (Fig. 2E and not shown). Ectopic expression of
DMyb caused similar, albeit often stronger, morphological effects on the wing discs (Fig. 2B,F).
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To determine whether ectopic DMyb activity is capable of suppressing endoreduplication in larval tissues that normally enter into an endocycle, we used two Gal4 lines that drive expression in salivary glands, fkh-Gal4 and sd-Gal4. fkh-Gal4 uses the salivary gland-specific enhancers of fork head (fkh) (Zhou et al., 2001), a gene required for the formation of embryonic salivary glands (Myat and Andrew, 2000
; Weigel et al., 1989
). Although the sd gene has not been reported to be expressed in salivary glands, we found that like the fkh-Gal4 line, the sd-Gal4 line induced high levels of green fluorescent protein (GFP) from a UAS-GFP reporter construct in endoreduplicating salivary gland nuclei (Fig. 7A,B). Neither Gal4 line induced expression in imaginal ring cells or in the neighboring fat body, allowing for these cells to serve as internal controls. The results described below were virtually identical with both Gal4 drivers.
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To quantify the differences between the salivary gland nuclei from the various genotypes, the DNA signal ratios of salivary gland nuclei to non-expressing fat body nuclei were determined using the method described by Weiss et al. (Weiss et al., 1998). Our results reinforced the visual impressions that ectopic expression of either DMyb protein inhibited endoreduplication, but that the truncated
DMyb was a much more potent inhibitor than the full-length DMyb (see Fig. 7C for graphical representation and Fig. 7D-H for individual examples).
These conclusions were confirmed by in vivo labeling studies of S phase. In larvae raised at 24°C that were between the ages of 72 and 96 hours AED, BrdU incorporation could be detected in imaginal ring cells and the endoreplicating nuclei of salivary glands from wild-type controls and fkh-Gal4 or sd-Gal4/UAS-DMyb larvae (Fig. 8A,B and not shown). By contrast, no BrdU incorporation was observed in the salivary gland nuclei of fkh-Gal4 or sd-Gal4/UAS-DMyb larvae, even though it could still be detected in imaginal ring cells (Fig. 8C,D). Similar results were obtained when BrdU incorporation was examined in larvae between the ages of 96 and 120 hours AED (not shown).
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Ectopic expression of a G1/S regulator can override the DMyb induced inhibition of endoreduplication
To determine whether the inhibition of endoreplication in salivary glands could be overcome, a chromosome carrying the transgenes E2F and DP under the control of the Hsp70 promoter was mated into flies that also carried either sd-Gal4 or fkh-Gal4 and UAS-DMyb. E2F and DP encode the two subunits of the Drosophila E2F transcription factor, which promotes DNA replication in cells that are proliferating and in those that are endocycling (Duronio et al., 1995
; Dynlacht et al., 1994
; Ohtani and Nevins, 1994
; Royzman et al., 1997
). Daily heat shock treatments (30 minutes at 37°C) resulted in partial rescue of salivary glands, both with respect to the overall size of the glands and the size of individual nuclei (Fig. 7G), and BrdU incorporation could be detected in a number of salivary gland nuclei within a couple of hours after heat shock treatment (Fig. 8E). However, as the heat shock treatment induced expression of E2F in all cells, increased levels of BrdU incorporation were also observed in fat body nuclei.
As the ectopically induced DNA synthesis in fat body would presumably lead to excess endoreduplication, the extent of rescue calculated by the ratio of salivary gland nuclei to fat body nuclei is likely to be an underestimate (Fig. 7C). In addition, we suspected that as the GAL4/UAS system is known to be more efficient at higher temperatures (Greenspan, 1997; Morimura et al., 1996
), the heat shock treatments might be inducing higher levels of the
DMyb protein, which we would expect to further inhibit endoreduplication. To test these possibilities, sd-Gal/UAS-
DMyb or sd-Gal/UAS-
DMyb; HS-E2F/DP embryos were collected for 24 hours and then subjected to 30 minute heat-shock treatments every 12 hours. In accordance with our hypothesis that the heat-shock treatments were enhancing the inhibition of endoreduplication, the larvae had to be aged for an additional 24 hours (
144 hours instead of 120 hours) for the sd-Gal/UAS-
DMyb salivary glands to reach approximately the same size as in samples that had not been heat shocked (Fig. 7, compare I and F). The salivary glands dissected from the sd-Gal/UAS-
DMyb; HS-E2F/DP larvae were considerably larger than those dissected from the animals that did not carry the HS-E2F/DP transgenes (Fig. 7I,J), and the salivary gland nuclei were also enlarged, but so were the fat body nuclei, confirming the suspicion that the heat-shock treatments produced excess endoreduplication in the fat body.
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DISCUSSION |
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The similarities between the responses of both proliferating and endocycling cells to ectopic expression of DMyb and Cyclin E suggest the possibility that the effects of ectopic DMyb activity might be due to induction (either directly or indirectly) of high levels of Cyclin E, and preliminary results indicate that Cyclin E levels are increased in salivary glands expressing DMyb (C. A. F., unpublished). However, we also found that periodic expression of E2F/DP could overcome DMyb-induced inhibition of endoreduplication, whereas Follette and colleagues (Follette et al., 1998
) showed that E2F/DP expression could not override the replication block induced by Cyclin E. The finding that E2F induction can override the inhibition of DNA endoreduplication caused by ectopic DMyb activity, suggests that DMyb induced inhibition may be upstream of E2F or that E2F can circumvent the DMyb-induced block. In addition, it is unlikely that endogenous DMyb plays a role in regulating the levels of Cyclin E in endocycling larval cells, as Dm myb transcripts have not been detected in these cells and no deleterious effects on larval tissues have been observed in loss-of-function mutant alleles of Dm myb (Katzen and Bishop, 1996
; Katzen et al., 1998
). For these reasons, and because we do not detect any defects in salivary glands when we ectopically express another DMyb construct, which contains the DMyb DNA-binding domain fused to an engrailed repressor domain (C. A. F., unpublished), we do not believe that DMyb or
DMyb are acting to repress, rather than activate expression of target genes in salivary glands, a phenomena that has been observed with other transcriptional activators when they are overexpressed (e.g. Suppressor of Hairless) (Klein et al., 2000
). Therefore, the results from ectopic expression of DMyb reinforce our conclusions from studies of loss-of-function alleles, that one of the functions of Dm myb is to suppress endoreduplication and maintain genomic stability in proliferating diploid cells (Fung et al., 2002
; Katzen et al., 1998
).
The transgenic experiments reported here demonstrate that DMyb is a much more potent inhibitor of endoreduplication than DMyb. The C-terminal region of the vertebrate A-Myb and c-Myb proteins has been shown to contain negative regulatory domains that downregulate the DNA-binding and transcriptional activation abilities of the proteins; the equivalent portion of the B-Myb protein contains both negative and positive regulatory sequences (reviewed by Gonda et al., 1996
; Oh and Reddy, 1999
; Saville and Watson, 1998
). Our findings indicate that the C-terminal sequences that were deleted in the DMyb protein (by analogy to c-Myb) act to strongly downregulate DMyb activity in salivary glands. By contrast,
DMyb appeared to be only slightly more active than DMyb at promoting proliferation in imaginal disc cells, even those in the ZNC, which should be specifically arrested in either G1 or G2. This finding is in agreement with a growing body of evidence from studies with the vertebrate Myb proteins, that their ability to activate transcription is strongly dependent on the presence and/or abundance of other cellular factors (Ness, 1999
). Therefore, one rationale for the difference between the behavior of the DMyb proteins in imaginal discs and salivary glands is that imaginal disc cells may contain an activating factor that is absent in salivary glands, that interacts with full-length DMyb to relieve the repression of its transcriptional activating potential that is mediated via the C-terminal domain. Another possibility is that salivary gland cells contain a factor that specifically interacts with full-length DMyb to repress its activity, but this seems less likely as endogenous Dm myb expression has not been detected in these cells.
E2F/DP and DREF (DNA replication-related element binding factor) are transcription factors that have been shown to be crucial for cell cycle regulation in Drosophila. These factors promote, and are required for DNA replication in both mitotic and endocycling cells (Duronio et al., 1995; Hirose et al., 1999
; Royzman et al., 1997
). We have now demonstrated that like these factors, DMyb promotes DNA replication in mitotic cells. However, the situation differs in endocycling cells. Previously reported results have shown that DMyb is not required for DNA replication in endocycling cells (see above) (Katzen and Bishop, 1996
; Katzen et al., 1998
), and the data presented here demonstrate that DMyb can actively inhibit endoreduplication. The ability of DMyb to have directly opposing effects on DNA replication, depending upon cell cycle context, makes DMyb unique among the transcription factors in Drosophila that have been implicated in cell cycle regulation (see Fig. 9). Further investigation should elucidate how these transcription factors interact to coordinate cell cycle progression.
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There is a substantial amount of data indicating that vertebrate Myb genes function to promote the G1/S transition. By contrast, loss-of-function mutations in Drosophila, cause either a block at the G2/M transition followed by endoreduplication or mitotic defects, which have implicated Dm myb in several aspects of cell cycle regulation, but not directly in the initiation of S phase. These discrepancies have prompted the question of whether the functions of the insect and vertebrate Myb genes are really equivalent? However, mitotic defects (chromosome breakage and cells arrested in metaphase) have recently been observed with mutations in several other genes that are known to be required for DNA replication, including MCM4 (dpa FlyBase) PCNA (mus209 FlyBase) and three genes encoding proteins crucial for assembly of the pre-initiation complex: Orc2, Orc5 and dup (also known as cdt1) (Pflumm and Botchan, 2001; Whittaker et al., 2000
). These findings indicate that the mitotic defects observed in Dm myb mutants could be secondary consequences of replication defects, a viewpoint supported by Manak and colleagues in a recent paper (Manak et al., 2002
). By contrast, the findings that DMyb is an activator of cyclin B expression in the imaginal eye disc (Okada et al., 2002
) and that DMyb activity can induce mitosis in cells within the ZNC that are normally blocked in G2 (see Fig. 5), provide support for our earlier conclusions that DMyb has a direct involvement in promoting mitosis. Additionally, three experimental observations provide strong circumstantial evidence that Dm myb function is not an absolute requirement for DNA replication per se: Dm myb expression is not detected in larval endoreplicating tissues; endoreduplication in larval tissues appears to occur normally in loss-of-function mutant alleles of Dm myb; and de novo endoreduplication is observed in mutant wing cells during pupal development (Katzen and Bishop, 1996
; Katzen et al., 1998
).
We have presented evidence that, in addition to inducing increased levels of mitosis, Dm myb, like its vertebrate counterparts, can promote the G1/S transition. Our studies also demonstrate that the C termini of the vertebrate and Drosophila Myb proteins share the function of downregulating their activities. Finally, the finding that ectopic DMyb can actively inhibit endoreduplication reinforces our conclusions from previous analyses of loss-of-function alleles, that Dm myb normally acts in proliferating cells to maintain diploidy by suppressing reinitiation of S phase prior to mitosis. Our demonstration that at least one aspect of myb function is conserved between the Drosophila and vertebrate Myb proteins, raises the issue of whether one or more of the vertebrate Myb proteins may also act to inhibit endoreduplication and/or to promote mitosis. In conclusion, our studies demonstrate that DMyb functions in multiple aspects of the cell division cycle to promote proliferation and maintain the integrity of the genome.
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ACKNOWLEDGMENTS |
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