Mutation of the Drosophila homologue of the Myb protooncogene causes genomic instability

Abstract

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.

Figure 1

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.

Figure 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).

Figure 3

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.

Figure 4

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.

Figure 5

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).

Figure 6

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).

Figure 7

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.

Figure 8

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.

Figure 9

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).