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Mutation of the Drosophila homologue of the Myb protooncogene causes genomic instability
Vertebrates have three related Myb genes. The c-Myb protooncogene is required for definitive hematopoiesis in mice and when mutated causes leukemias and lymphomas in birds and mammals. The A-Myb gene is required for spermatogenesis and mammary gland proliferation in mice. The ubiquitously expressed B-Myb gene is essential for early embryonic development in mice and is directly regulated by the p16/cyclin D/Rb family/E2F pathway along with many critical S-phase genes. Drosophila has a single Myb gene most closely related to B-Myb. We have isolated two late-larval lethal alleles of Drosophila Myb. Mutant imaginal discs show an increased number of cells arrested in M phase. Mutant mitotic cells display a variety of abnormalities including spindle defects and increased polyploidy and aneuploidy. Remarkably, some mutant cells have an aberrant S- to M-phase transition in which replicating chromosomes undergo premature histone phosphorylation and chromosomal condensation. These results suggest that the absence of Drosophila Myb causes a defect in S phase that may result in M-phase abnormalities. Consistent with a role for Drosophila Myb during S phase, we detected Dm-Myb protein in S-phase nuclei of wild-type mitotic cells as well as endocycling cells, which lack both an M phase and cyclin B expression. Moreover, we found that the Dm-Myb protein is concentrated in regions of S-phase nuclei that are actively undergoing DNA replication. Together these findings imply that Dm-Myb provides an essential nontranscriptional function during chromosomal replication.
The Myb gene family was discovered based on studies of the v-Myb oncogene of the avian myeloblastosis virus that causes leukemia in chickens (1). The normal c-Myb protooncogene is essential for hematopoiesis in mice (2) and when altered causes leukemias and lymphomas in mice and birds. Two additional Myb-related genes are present in vertebrates. The A-Myb gene is essential for spermatogenesis and for mammary gland proliferation in mice (3). Homozygous disruption of B-Myb in the laboratory mouse results in very early lethality at embryonic day 4.5–6.5 (4). These results are consistent with a requirement for B-Myb in cell proliferation that may be masked only briefly by maternal effect during early mouse development. The B-Myb gene is expressed widely throughout mouse development unlike A-Myb or c-Myb (5). The expression of B-Myb seems to correlate with cell division during embryogenesis and adulthood. When quiescent cultured fibroblasts enter the cell cycle, B-Myb mRNA and protein are induced in late G1 and S phases (6). The B-Myb promoter contains a binding site for the E2F transcription factor that negatively regulates gene expression during G0 and early G1 and the Rb family is required for this negative regulation (7–9). Other genes that are similarly regulated include cdc2, cyclin A, thymidylate synthetase, ribonucleotide reductase, and E2F-1 itself. Interestingly, constitutive expression of B-Myb has been reported to bypass p53-induced p21-mediated G1 arrest (10). Finally, the B-Myb protein itself is phosphorylated by CDK2/cyclinA, potentially serving as a signal for ubiquitination and proteolysis (11, 12). These results suggest that the regulation of B-Myb expression may be a critical step in cell cycle progression. However, the function of the B-Myb protein during the cell cycle has remained unclear.
The single Myb-related gene in Drosophila (Dm-Myb) seems to be more closely related to B-Myb than to A- or c-Myb. Indeed, phylogenetic analyses imply that A-Myb and c-Myb arose from a B-Myb/Dm-Myb-like ancestor by two rounds of gene duplication that included the acquisition of a central transcriptional activation domain (1, 13). The DNA-binding domain of all these Myb proteins consists of three tandem repeats of a helix–turn–helix domain that recognize a YAACNG motif (14, 15). The Dm-Myb protein binds to the same DNA sequences as the Myb proteins of vertebrates and of the cellular slime mold Dictyostelium discoideum (16, 17). However, no closely related Myb genes are present in the nematode Caenorhabditis elegans or in budding yeast (1). Therefore, genetic studies of Dm-Myb may shed light on the evolution of the vertebrate “three repeat” Myb gene family in general, and on the function of B-Myb in particular.
Dm-Myb is highly expressed in dividing cells throughout development, but no expression was detected by in situ hybridization in polytene larval tissues (18). Previous studies of two temperature-sensitive alleles of Dm-Myb have shown that this gene provides an essential function at multiple stages of development (18). Studies of pupal wing cells with these mutants have suggested that Dm-Myb is required during the G2/M transition of the cell cycle both for mitosis and for preventing endoreplication (19). To clarify further the role of Dm-Myb in cell cycle regulation and in development, we have now isolated and characterized two lethal alleles. Both of these mutants die as late third instar larvae with no detectable Dm-Myb protein.
Drosophila Stocks.
The P element line (w1118, P{w[+mC] = EP}EP1071) was generated by P. Rorth (20) and used as a starting point in our screen. The green fluorescent protein (GFP) balancer line [FM7i, P{w[+mC] = ActGFP}JMR3/C(1)DX, f [1]] and a deficiency that uncovers 13F [Df(1)sd72b] were obtained from the Bloomington Drosophila stock center (Indiana University, Bloomington).
Immunoblotting.
Anti-Myb Abs were prepared by immunizing rabbits with a purified Escherichia coli-produced protein containing the three Myb repeats of Dm-Myb, followed by affinity purification with the same recombinant protein coupled to an Affigel-10 column (Bio-Rad) (S. Chao, B. Ganter, P. Fogarty, and J.S.L., unpublished data). The anterior halves of late third instar wild-type and mutant larvae were lysed and ground in 100 μl of 2X SDS/PAGE buffer (preheated to 65°C) containing standard protease inhibitors (Roche Molecular Biochemicals) and 5 mM EDTA. The samples were boiled for 5 min, cleared by centrifugation (at 10,000 × g for 5 min), electrophoresed in a 7.5% SDS-polyacrylamide gel, then electroblotted onto nitrocellulose and probed with rabbit anti-Dm-Myb Abs with the SuperSignal chemiluminescent kit (Pierce).
Pupariation Analysis.
Embryos from the MH107/FM7-GFP line were collected on standard corn meal-molasses medium for 2 h and then allowed to develop at 25°C. Care was taken to ensure that the bottles were not overcrowded with embryos. As larvae began to pupariate, they were scored daily for the presence of the GFP balancer chromosome and counted as mutant (lack of GFP) or wild type (presence of GFP).
Immunocytochemistry.
Late third instar larvae were bisected and the anterior halves were inverted in PBS and fixed in 4% formaldehyde in PBS for 30 min at room temperature. The tissue was washed in PBST (PBS and 0.1% Triton X-100) for 5 × 10 min, blocked with PBSTB (PBST and 1% BSA) for 40 min, incubated with primary Ab overnight at 4°C, washed in PBST for 5 × 10 min, reblocked with PBSTB for 40 min, and incubated with the appropriate fluorophore-conjugated secondary Ab (Molecular Probes) for 90 min at room temperature. The tissue was again washed for 5 × 10 min with PBST containing 2 mg/ml of DNase-free RNase A, and the imaginal discs, brains, and fat body were dissected and mounted in Vectashield containing propidium iodide (Vector Laboratories). Primary Abs included rabbit anti-phosphohistone H3 (PH3) (1:500; Upstate Biotechnology, Lake Placid, NY), rabbit anti-CID (1:1000; gift of S. Henikoff, Fred Hutchinson Cancer Research Center, Seattle) (21), mouse anti-β-tubulin (1:750; gift of B. Sullivan, Univ. of California, Santa Cruz), mouse anti-BrdUrd (1:20; no. RPN 202, Amersham Pharmacia Biotech), and affinity-purified rabbit anti-Myb (1:500). Images were obtained with a Nikon PCM confocal microscope.
BrdUrd/PH3 and BrdUrd/Myb Codetection.
Late third instar larvae were bisected and the anterior halves were inverted in Schneider's medium, incubated with 1 mg/ml of BrdUrd in Schneider's medium at 25°C for 20 min, and fixed for 30 min in 4% formaldehyde in PBS. The remaining steps were performed as described (22), except that the acid treatment was reduced to 16 min and an additional Ab was used after neutralization (either anti-PH3 or anti-Myb).
Flow Cytometry.
Wing imaginal discs were dissected and pooled from 15–20 late third instar wild-type or mutant larvae. Nuclei were prepared as described, treated with RNase, stained with propidium iodide, and analyzed for DNA content (23, 24).
Karyotypic Analysis.
Chromosomal squashes were performed on wild-type and mutant late third instar larval brains with hypotonic treatment as described (22), mounted in Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories), and viewed with a Nikon ES800 microscope and a SPOT digital camera. The images presented were converted to gray scale and black/white-inverted with photoshop (Adobe Systems, Mountain View, CA).
Generation of Dm-Myb Mutants.
With a P element mobilization screen, we have generated previously uncharacterized null alleles of Dm-Myb (MH30 and MH107; Fig. Fig.1).1). Both mutants die as late third instar larvae/prepupae and exhibit a variety of morphological defects, including small brains and imaginal discs, reduced fat body, and occasional melanotic tumors. The mutants also show a dramatic delay in development, taking about twice as long to reach the late third instar larval stage. This period of lethality and the delay in imaginal disc development are reminiscent of a class of recessive mutants that affect the mitotic cell cycle and survive into late larval stages as a result of embryonic mRNA deposition by heterozygous mothers (25). MH107 lacks the 5′ upstream sequences and 5′ untranslated region of Dm-Myb, whereas MH30 lacks the entire ORF. No Dm-Myb protein was detectable in third instar larvae of either mutant (Fig. (Fig.11 and data not shown). Similar phenotypes were observed when these two new alleles of Dm-Myb were each made heterozygous with a chromosomal deficiency that uncovers this region, again suggesting that the new alleles are functional nulls. Although both mutants appear to have a similar phenotype, MH107 was used in the remaining studies described here because MH30 lacks an additional gene 3′ of Dm-Myb.
Isolation and characterization of Dm-Myb mutants. A viable P element at 13F near the Dm-Myb gene [EP(X)1071] was mobilized by standard techniques (Upper Left). Approximately 2,000 lines were generated, 120 of these contained X-linked lethal mutations, and 2 of these were rescued by an autosomal P element containing the [Myb+, AlkB+] construct but not by an autosomal P element containing the [Myb−, AlkB−] construct. These two mutants (MH107 and MH30) were further characterized by Southern blotting, plasmid rescue, and DNA sequencing. In both cases, imprecise excision resulted in deletion of a 3′ portion of the original P element along with adjacent cellular DNA as indicated by the dotted lines. MH107 contains a deletion of the entire AlkB gene as well the 5′ untranslated region of Dm-Myb. MH30 contains a deletion of AlkB, the entire Dm-Myb ORF, and an adjacent 3′ gene. No Dm-Myb protein was detected in mutant third instar larvae by immunoblotting (Upper Right). Although the phenotypes of MH107 and MH30 were quite similar, we have focused on MH107 because MH30 has deleted another adjacent gene. AlkB itself is not an essential gene for growth in the laboratory, because an autosomal P element containing Dm-Myb but not AlkB [Myb+, AlkB−] completely rescued MH107. Conversely, an autosomal P element line containing AlkB but not Dm-Myb [Myb−, AlkB+] failed to rescue MH107 and exhibited the same mutant phenotypes as MH107, suggesting that AlkB does not contribute to the MH107 phenotype. Pupariation was greatly reduced and delayed in the MH107 line (Lower Left). Mutant larvae and pupae were readily identified by the absence of an FM7-actin-GFP balancer chromosome (Lower Right, Upper). The few GFP-negative Dm-Myb mutant pupae that were identified displayed extensive autolysis (Lower Right, Lower). WT, wild type.
Loss of Dm-Myb Causes Abnormal Mitoses but Not an Accumulation of Cells with G2 DNA Content.
A previous analysis of two temperature-sensitive alleles of Dm-Myb suggested that this gene was required for progression from G2 into M phase (19). To directly test whether a G2/M delay or arrest occurred in the absence of Dm-Myb protein, we used flow cytometry to determine the DNA content of imaginal wing disc cells from late third instar wild-type and MH107 Dm-Myb mutant larvae. Wing imaginal discs contain a largely unsynchronized population of cells that cycle in small clusters, increasingly accumulating in G2 as third instar larval development proceeds (23, 26). Therefore, if the primary defect in the Dm-Myb mutant occurred during G2/M progression as reported, one would expect to see an increase in the fraction of mutant cells with G2 DNA content. Instead, we observed a decrease in this fraction (Fig. (Fig.2).2).
Fewer cells have G2 DNA content but more cells have undergone chromosome condensation in Dm-Myb mutant imaginal discs. Imaginal wing discs from wild-type (WT) or Dm-Myb mutant (MH107) third instar larvae were isolated and analyzed for nuclear DNA content by flow cytometry (Left), stained for total DNA with propidium iodide (PI) (Center), and for condensed chromosomes with anti-PH3 Ab (Phospho-H3) (Right).
The inversion of the G1:G2 ratio of DNA content in mutant vs. wild-type imaginal wing disk cells may be the result of a delay in G1/S. It is also possible that the observed decrease in mutant cells with a G2 DNA content is the result of a developmental delay despite the nearly wild-type size of the discs in late third instar mutant larvae. However, this seems unlikely for two reasons. First, wandering mutant larvae like those used for DNA content analysis begin to pupariate within a day, implying that they are indeed late third instar. Second, never in mid to late third instar wild-type larvae does the fraction of cells with G2 DNA content exceed the fraction of cells with G1 DNA content (23).
Because only a very small fraction of cells with G2/M DNA content is generally in M phase (1–2%), we directly enumerated mitotic cells in wild-type and mutant imaginal discs with Abs directed against condensed chromatin (PH3). We found a 2–3-fold increase in the percentage of cells with condensed chromosomes in mutant imaginal discs (Fig. (Fig.2),2), rather than a decrease as would be expected for cells with a primary defect in the G2/M transition. Intriguingly, many of the mutant nuclei appeared to contain hypercondensed chromosomes as judged by the increased intensity of their PH3 staining. Not surprisingly, a substantial increase in apoptosis was also seen in the imaginal discs of the Dm-Myb mutant (data not shown).
Because of the increased number of mitoses in the Dm-Myb mutant, we wished to investigate whether M phase proceeded normally. We therefore examined third instar wild-type and mutant larval brains and imaginal discs at high power with Abs directed against γ-tubulin and PH3 (Fig. (Fig.3).3). Wild-type cells with highly condensed chromosomes contained two symmetrically arrayed centrosomes at the spindle poles, whereas mutant cells with highly condensed chromosomes often contained only one or no centrosomes. Many of the PH3-positive mutant cells contained small, sausage-shaped masses of DNA, reminiscent of the hypercondensed chromosomes seen in mutants of morula (27).
Hyperconsdensed chromatin in Dm-Myb mutant imaginal discs. Imaginal disc cells were stained with anti-γ-tubulin (red) to visualize centrosomes and with anti-PH3 (green) to visualize condensed chromosomes. Note that wild-type (WT) nuclei display distinct metaphase plates with symmetric bipolar centrosomes. In contrast, the mutant (MH107) nuclei often contain one or no centrosomes as verified by serial optical sections and disarrayed sausage-like blobs of condensed chromosomes.
To determine whether centromeres were properly assembled in mutant cells, we costained cells with anti-CID (CENP-A) and anti-β-tubulin Abs (Fig. (Fig.4).4). Brightly staining centromeres were clearly visible in both wild-type and mutant chromosomes. However, the mutant cells displayed a variety of abnormalities, including monopolar spindle attachment and massive polyploidy. To examine chromosomal segregation we costained cells with anti-PH3 and anti-β-tubulin (Fig. (Fig.5).5). We again observed a variety of mitotic abnormalities in mutant cells, including monopolar and multipolar spindles. Occasionally, we saw condensed chromatin untethered to the spindle during metaphase. This configuration could represent either a failure of congression at the metaphase plate or precocious segregation. These data are certainly consistent with defects in chromosomal segregation and/or cytokinesis. For example, if chromatids failed to segregate and the cell underwent an additional mitotic cycle without cytokinesis, alterations in centrosome number and ploidy could result.
Abnormal mitoses in Dm-Myb mutant cells. Imaginal disc cells were stained with anti-β-tubulin (green) to visualize mitotic spindles, anti-CID (red) to visualize centromeric heterochromatin, and propidium idodide (blue) to visualize DNA. Note the presence of a monopolar spindle (Center) and polyploidy (Right) in the mutant (MH107) nuclei. WT, wild type.
Abnormal spindles and chromosome segregation in Dm-Myb mutant cells. Imaginal disk cells were stained with anti-β-tubulin (green) to visualize mitotic spindles, anti-PH3 (red) to visualize condensed chromosomes, and propidium idodide (blue) to visualize DNA. Note the cell with a mass of condensed chromatin separated from the metaphase plate (left arrow) and the cell with a multipolar mitotic spindle and multiple metaphase plates (right arrow).
Loss of Dm-Myb Causes Aneuploidy and Polyploidy.
To directly visualize mitotic chromosomes in cells without Dm-Myb, we performed brain squashes of late third instar mutant larvae (Fig. (Fig.6).6). We observed a substantial increase in both aneuploid and polyploid karyotypes from Dm-Myb mutant brains. These phenotypes are consistent with the observed segregation and mitotic spindle defects (Fig. (Fig.5).5). We found that ≈5% of all mutant mitoses are aneuploid, whereas 1–2% are polyploid. We also observed that the percentage of mitotic nuclei in anaphase drops from 9% in the wild type to 4% in the mutant. These data are consistent with a delay or arrest in M phase before anaphase in a substantial fraction of the cells lacking Dm-Myb.
Aneuploidy and polyploidy in Dm-Myb mutant larval brains. Chromosome squashes were prepared from MH107 late third instar larval brains. A variety of abnormalities were seen, including two large autosomes instead of four (Upper Left), two darkly staining Y chromosomes and four dot-like chromosome IVs (Upper Right), euploidy with hypercondensation (Lower Left), and massive polyploidy (Lower Right).
Loss of Dm-Myb Causes Abnormal Overlap of S- and M-Phase Markers.
We observed approximately the same proportion of cells in S phase in mutant and wild-type imaginal discs by flow cytometry of isolated nuclei (Fig. (Fig.5)5) as well as by microscopy to visualize BrdUrd incorporation (data not shown). However, in mutant brains stained for both BrdUrd and condensed chromatin after a brief period of BrdUrd incorporation, we observed an intriguing overlap of signal in some mutant nuclei that we have never observed in the wild type (Fig. (Fig.7).7). These results suggest that the relationship between S phase and M phase is abnormal in Dm-Myb mutants. Because we labeled with BrdUrd for only 20 min, this establishes the maximum amount of time these cells might be in G2 and suggests that the G2 phase is very short, if not completely absent. This result is consistent with the significant decrease in the fraction of nuclei with a G2 content of DNA as determined by flow cytometry of late third instar mutant wing discs relative to wild type (Fig. (Fig.2).2).
Abnormal overlap of S and M characteristics in Dm-Myb mutant cells. Mutant larval brains were incubated with BrdUrd for 20 min and then immunostained for DNA synthesis (BrdUrd) and chromosome condensation (PH3). Note the abnormally double-labeled cells (leftward arrowheads). Such cells were never observed in wild-type brains. Representative cells that labeled normally with BrdUrd alone (upward arrowhead) or with PH3 alone (downward arrowhead) are also indicated.
Dm-Myb Protein Localizes to Replicating DNA in Mitotic and Endocycling Cells.
The unusual overlap of S- and M-phase characteristics in Dm-Myb mutant cells in mitotic tissues suggests that a primary function of Dm-Myb protein might occur in S phase. We therefore looked for Dm-Myb protein in wild-type larval fat body, an endocycling tissue in which cells undergo alternating rounds of S and G phases without intervening mitoses (28). We used BrdUrd incorporation followed by fixation and staining with anti-BrdUrd and anti-Myb Abs to visualize Myb distribution in S-phase nuclei (Fig. (Fig.8).8). Remarkably, the few nuclei that stained strongly with anti-Myb were also undergoing active DNA replication. Furthermore, the Dm-Myb protein appeared to be most concentrated in subnuclear structures that contained recently replicated DNA. Previous studies reported that Dm-Myb RNA expression was not detected in polyploid larval tissues by in situ hybridization (18). However, this may be a result of the relatively small number of cells that express Dm-Myb and/or the transient nature of this expression during the endocycle. A similar colocalization of Dm-Myb protein and recently replicated DNA was observed in larval brain, a mitotic tissue (Fig. (Fig.9).9). These results imply a functional role for Dm-Myb protein during DNA replication in both endocycling and mitotic tissues. Consistent with a role for Dm-Myb in endocycling cells, mutant third instar larvae appear thin and translucent because of a decreased mass of fat body that normally fills the subcuticular space of the animal.
Dm-Myb protein localizes to replicating DNA in endocycling cells. Wild-type larval fat body was incubated with BrdUrd for 60 min and then stained for total DNA with propidium iodide (Upper Left; blue in Lower Right), for BrdUrd incorporation (Upper Right; red in Lower Right), and Dm-Myb protein (Lower Left; green in Lower Right). Note that the single hypocondensed nucleus in the field displays both BrdUrd incorporation and strong Dm-Myb staining.
Dm-Myb protein localizes to replicating DNA in mitotically cycling cells. Wild-type larval brain was incubated with BrdUrd for 30 min and then stained for total DNA with propidium iodide (Upper Left; blue in Lower Right), for BrdUrd incorporation (Upper Right; red in Lower Right), and Dm-Myb protein (Lower Left; green in Lower Right). Note that the two nuclei with strong Dm-Myb staining also stain for BrdUrd incorporation in a similar pattern (arrows).
Our results demonstrate that mitotic defects including spindle and ploidy abnormalities are a prominent feature in our Dm-Myb mutants. However, these phenotypes may be the consequence of a primary defect in S rather than G2 or M phase. Several observations support this hypothesis: the mitotic defects are quite variable; flow cytometric analysis of wing imaginal discs shows a decreased fraction of nuclei with G2 DNA content rather than an increase as would be expected with a G2/M phase delay or arrest; mutant cells sometimes display both S- and M-phase characteristics simultaneously; and the Dm-Myb protein itself colocalizes with recently replicated DNA both in mitotically cycling cells and in endocycling cells that lack an M phase.
The difference between our results and the previous report of a G2/M progression defect in a ts mutant of Dm-Myb may be because of the difference in alleles, the stage of development examined, or the methods used. The previous report studied hypomorphic alleles in pupal wing cells, whereas we studied apparent null alleles in imaginal wing discs. The main argument for a G2/M progression defect in the previous report was the ability of ectopically expressed mitotic regulators (cdc2 and stg) to induce additional mitoses in mutant pupal wings (19). However, ectopic expression of stg has been shown to induce additional mitoses in some wild-type tissues, including imaginal wing discs (29, 30). In contrast, we used flow cytometry to directly measure the DNA content of wing disc nuclei. We observed a decrease in the fraction of cells with a G2 DNA content in mutant vs. wild-type imaginal wing discs rather than an increase as would be predicted for a block in G2/M progression. Moreover, the increased mitotic index in mutant vs. wild-type imaginal discs also argues against a delay in G2 as does the unusual overlap of S and M characteristics in some mutant cells. In this regard, we note that M-phase arrest has recently been observed in mutants of several genes directly involved in DNA synthesis (ORC2, ORC5, PCNA, MCM4, and dup/Cdt1) (31–33). In addition, M-phase defects were observed after injection of the DNA polymerase inhibitor aphidicolin into wild-type embryos (34).
While this manuscript was in review, two new analyses of temperature-sensitive mutants of Dm-Myb appeared in the literature. One study demonstrated that in ts mutant animals, abdominal histioblasts progressed more slowly through the cell cycle with marked centrosome amplification and genomic instability (35). These results are generally consistent with those reported here. However, we observed a greater variety of centrosome/spindle abnormalities in mutant imaginal disc and larval brain cells, including anastral, monopolar, and multipolar spindles. These differences may be the results of the nature of the alleles (hypomorph vs. null) and/or the marked differences in cell cycle regulation in wild-type abdominal histioblasts vs. imaginal disk cells, the former being resistant to ectopic stg (30). Furthermore, our additional data support an essential role for Dm-Myb in S phase that may cause the observed variety of M-phase defects (see next section).
A second recent study proposed that Dm-Myb regulates G2/M progression in the eye imaginal disc by transcriptionally activating the cyclin B gene (36). The data presented here do not directly address this question. However, we do not believe that the primary function of Dm-Myb is to facilitate the transition from G2 into M phase. Rather, our observation of mutant but not wild-type cells that stain with both anti-BrdUrd and anti-PH3 after a short period of BrdUrd incorporation suggests that Dm-Myb may be required for the proper completion of S phase and/or for prevention of premature chromosome condensation. Furthermore, the colocalization of Dm-Myb protein with newly replicated DNA during S phase both in mitotically cycling and in endocycling cells that lack cyclin B expression argues for a nonmitotic, nontranscriptional function for Dm-Myb. Intriguingly, Myb domains are found in a number of proteins that directly regulate covalent histone modifications (ADA2, N-CoR), chromatin structure (SWI3, I-SWI), and telomere metabolism (RAP1, TAZ1, TRF1, and TRF2) (1). Therefore, we hypothesize that Dm-Myb might function in S phase to establish proper chromosomal structure, either by directing covalent modification of histones and chromatin-associated proteins or by the loading/unloading of these proteins.
We thank Jeff Axelrod, Mike Botchan, Lolli Beall, Brian Calvi, Abby Dernburg, Margarete Heck and her laboratory, Peter Jackson, Michelle Pflumm, Tin Tin Su, Bill Sullivan, Dave Tree, Terri Orr-Weaver, and the members of our laboratory for helpful advice and discussions. This work was supported by grants from the U.S. Public Health Service (to J.S.L.).
| GFP | green fluorescent protein |
| PH3 | phosphohistone H3 |
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