InteractiveFly: GeneBrief

pita: Biological Overview | References

The highly condensed chromosomes and chromosome breaks in mitotic cells of a Drosophila mutant, spotted-dick/pita, are the consequence of defects in DNA replication. Reduction of levels of Spotted-dick protein, by either RNAi or mutation, leads to the accumulation of cells that have DNA content intermediate to 2N and 4N in proliferating tissues and also compromises endoreduplication in larval salivary glands. The Spotted-dick Zinc-finger protein is present in the nuclei of cells committed to proliferation but necessary in cells undertaking S phase. Spotted-dick/Pita functions as a transcription factor. In cultured S2 cells, it is an activator of expression of some 30 genes that include the Orc4 gene, required for initiation of DNA replication. Chromatin immunoprecipitation indicates that it associates with the genes that it activates in S2 cells together with other sites that could represent genes activated in other tissues. Spdk as a probable downstream effector of DREF-mediated transcriptional regulation (Page, 2005).

Although proper transcriptional regulation of the cell cycle is vital to the developmental programmes of metazoans, relatively little is known about the mechanisms that coordinate these processes. A core of conserved transcriptional regulators control expression of genes required for the G1 to S transition in metazoans. CDKs bound by cyclin D or E can phosphorylate the retinoblastoma protein (RB) resulting in its dissociation from E2F1-DP heterodimers and the replacement of transcriptional corepressors with coactivators in this complex. Thus, upon S phase entry, the transcription of genes regulated by E2F can be 'switched on'. There are two known E2F genes in the Drosophila genome, E2F1 and E2F2. The product of the DP gene serves as a common binding partner for both. Functional antagonism between E2F1 and E2F2 has been demonstrated, although E2F2 appears not to be central to cell cycle control in normal somatic Drosophila cells. Among the S phase genes regulated by E2F1 is Orc1, which encodes the largest subunit of the origin recognition complex. The cell cycle regulatory genes cyclin E and string are also E2F1 targets (Page, 2005).

It has been suggested that E2F1 may perform an important homeostatic role in maintaining cell cycle period in the face of mild perturbations, rather than simply being necessary for the expression of its target genes. A rise in CDK activity (CDK1 or CDK2) was seen to inhibit transcription of the E2F1 gene in the cells of the wing imaginal disc. Thus, an increase in CDK2 accelerating progression through G1 would lead to a decrease in E2F1, a resulting decrease in string expression and a compensatory lengthening of G2 (Page, 2005).

In addition to the E2F family, another transcription factor, the Drosophila protein DREF, has been shown to regulate specific DNA replication genes. DREF binds to an 8 base pair palindromic 'DNA replication-related element' (DRE) present in the regulatory regions of the genes encoding PCNA and DNA pol. DREF is coexpressed in proliferating larval cells expressing PCNA and its ectopic expression induces DNA synthesis, as well as apoptosis and abnormal morphogenesis, in Drosophila eye imaginal discs. One-third of genes upregulated in a proliferating population of eye imaginal disc cells have been shown to have a DRE within 1000 bp of their upstream DNA. By contrast, only one of 23 genes upregulated in a differentiating population of eye disc cells had such a sequence in its regulatory regions. The DREF-mediated regulation of D-raf, e2f1 and Dm myb points further to its role in cellular proliferation. Moreover, the activation of cyclin A links DREF function to progression through both S phase and G2. Finally, the activation of the mitochondrial genes mtSSB provides a link between the transcriptional regulation of cellular proliferation and of organelle biogenesis (Page, 2005 and references therein).

Undoubtedly, further factors required for the transcriptional regulation of S phase remain to be discovered and the roles of known transcription factors are likely to be expanded to include aspects of S phase transcriptional regulation. Thus, for example, the proposed common regulatory factor for DNA replication and DREF genes is as yet unidentified, while Grainyhead exemplifies a specific transcription factor that is a positive regulator of mus209 (PCNA) transcription (Page, 2005).

Since mutants showing S phase defects in Drosophila also lead to subsequent characteristic mitotic abnormalities, it was asked whether transcriptional activators of S phase genes might be identified within a screen for such mutants. This study shows that the spotted-dick (spdk) gene, originally identified in a screen of second chromosomal P-element insertion mutants showing such mitotic phenotypes (Ohkura, 1995), encodes a transcriptional regulator of the crucial S phase gene Orc4. Expression of the Orc4 gene is shown to be dependent upon Spdk, and Spdk binds to the genomic DNA in the region of Orc4. A possible role for Spdk as a downstream effector of DREF function is discussed (Page, 2005).

The spdk¹ mutant (line P^k14408) was first identified in a screen of a collection of second chromosomal P-element insertions (Török, 1993) for mutants with a mitotic phenotype. The highly condensed mitotic chromosomes seen in mutants resembled pieces of fruit in the British puddings after which they were named. spdk¹ mutant larvae developed melanotic tumours, had imaginal discs greatly reduced in size and failed to develop to the pupal stage. All of these aspects of the phenotype were reverted when the P-element was mobilised permitting plasmid rescue to be used as a means of mapping the insertion site of the P-element responsible for the mutation to within the coding region of the caspase gene dcp-1. Several other lines with P-elements inserted within this genomic region were identified of which two allelic mutants, spdk² (P⁰²¹²¹) and spdk³ (P⁰⁸⁸⁵⁹), were selected for further analysis (Page, 2005).

The P-element insertions resulted in downregulation of transcripts from both the dcp-1 gene and a longer transcription unit within which dcp-1 is a cryptic gene. Thus, to determine which gene corresponded to spdk, attempts were made to rescue spdk² mutants using the UAS-GAL4 bipartite system to drive expression of cDNAs for either dcp-1 or its surrounding gene. Whereas it was found that expression of dcp-1 cDNA failed to rescue the cell cycle phenotype, cDNA of the gene surrounding dcp-1 gave full rescue. Thus, the surrounding gene corresponds to spdk (Page, 2005).

The gene affected in spdk mutants corresponds to a gene that Laundrie (2003) subsequently called pita. Although the gene surrounds the dcp-1 caspase gene that is also downregulated by P-element insertions affecting spdk/pita, all of the mitotic and S phase phenotypes characteristic of spdk mutations can be rescued by expression of the spdk cDNA. Consistent with the presence of 10 C₂H₂ Zn fingers in the Spdk protein, it is required to regulate the expression of a number of genes in S2 cells including Orc4, a gene essential for DNA replication (Page, 2005).

The mitotic phenotype of spdk mutants bears a striking resemblance to the mitotic phenotype described for other Drosophila DNA replication mutants. Thus, Orc2, Orc5, mus209 and mcm4 all show delayed metaphases with a variety of abnormal figures similar to those found in spdk mutants (Pflumm, 2001). Since analysis of BrdU incorporation in combination with flow cytometry pointed to the perturbation of DNA replication in spdk mutants, this mitotic phenotype is likely to be secondary to the DNA replication phenotype. The increase in G2/M cells coupled with a normal mitotic index suggests that the replication defects result in a G2 delay and thus point to the presence of such a checkpoint in larval neuroblasts. Those cells that escape this checkpoint appear to be delayed in mitosis as a result of activation of a checkpoint related to the spindle integrity checkpoint, as indicated by the presence of BubR1 on metaphase chromosomes. It should be noted that the mitotic index rises in older spdk mutant third instar larvae, with some brains showing a mitotic index higher than 15%, presumably as G2/M checkpoint adaptation results in more cells delayed by this mitotic checkpoint. Thus, the raised mitotic index of some Drosophila DNA replication mutants is not inconsistent with the phenotype of spdk mutants (Page, 2005).

The failure to incorporate BrdU together with the increase in cells of DNA content intermediate to 2N and 4N, seen both in spdk mutant brains and following spdk RNAi in S2 cells, suggest that some cells can enter S phase but are very slow to, or become unable to, complete it presumably through reduced origin activity as a result of low levels of Orc4. The failure of larval salivary gland polytene chromosome endoreduplication in spdk mutants indicates that DNA replication also fails in endocycling tissues. In a study of pita mutant germline clones, Laundrie (2003) observed defects in the development of egg chambers (degeneration, abnormal or absent nurse cell nuclei followed by cell death). These can be explained by a failure in the division cycles of the germarium or a failure of endoreduplication cycles of nurse cells. Although the Spdk protein is found in nuclei of the proliferating or endoreduplicating cell types, its localisation suggests it is not directly involved in DNA replication as it is not found at replication punctae in either diploid S2 cells or the polyploid ovarian follicle cells. Thus, a role for Spdk/Pita in the regulation of transcription as originally suggested by Laundrie (2003) seems more probable. Indeed, the present data suggest that at least one of the genes activated by Spdk is absolutely required for passage through S phase (Page, 2005).

Microarray analysis suggests that a relatively small number of genes are regulated by Spdk in S2 cells. Of these, 33 are downregulated upon reduction of Spdk levels and 10 are upregulated. ChIP suggests that, in contrast to the upregulated genes, most of those downregulated (6-9 of 13 represented) are likely to have Spdk binding sites in their flanking chromatin. This suggests that most of those genes significantly downregulated by reduction of Spdk are directly regulated by Spdk through its binding to regulatory regions to presumably activate transcription. However, the majority of significantly upregulated genes are likely not to be direct targets of Spdk-mediated repression. Despite the apparently small number of Spdk target genes in S2 cells, both precipitation of chromatin with anti-Spdk serum and the immunolocalisation of Spdk on polytene chromosomes of larval salivary glands point to its having a large number of genomic binding sites. It is possible that the majority of Spdk binding sites have relatively little or no effect on the transcription of nearby genes and so only subtle changes in the transcription of such genes would result from Spdk depletion. Alternatively, these binding sites may only be important in specific developmental or physiological contexts. If this were the case, expression studies in a range of tissue types, rather than just in S2 cells, may reveal regulation of a larger number of genes by Spdk (Page, 2005).

Of the known putative target genes identified to be activated by Spdk, perhaps the most interesting are Orc4, TFIIEα and mRpL1. Not only has Spdk been shown to be likely to bind to chromatin in the region of these genes, but their expression was also greatly reduced following downregulation of spdk by mutation or RNAi. This suggests that Spdk may act as a factor that promotes cellular proliferation, by activating expression of Orc4, while also activating cellular and mitochondrial growth, by activation of TFIIEα and mRpL1, respectively. By analogy with the cyclin D-Cdk4-mediated regulation of mRpL12, the Spdk-mediated activation of mRpL1 transcription may be important in the activation of cellular growth as well as mitochondrial biogenesis. Given that Orc4 mRNA levels are extremely low in spdk mutants, and that expression of Orc4 in spdk dsRNA-treated DMEL cells can rescue their cell cycle defects, it seems likely that the cell cycle phenotype of spdk mutants is caused by a lack of Orc4 protein. However, it cannot be ruled out that other proteins required for DNA replication are also depleted in these mutants (Page, 2005).

To date, DREF and E2F1 are the principal Drosophila transcription factors known to be important in regulating G1 and S phases. Examination of the genomic DNA flanking spdk revealed one perfect match for the Drosophila DNA replication-related element (DRE) 54-61 base pairs upstream from the putative transcription start site. Since Spdk is expressed in proliferating cells, it seems likely that transcriptional regulation by DREF alone, or in combination with autoregulation by Spdk (as suggested by its own precipitation by anti-Spdk ChIP), could explain the expression pattern of spdk. This would place Spdk as a probable downstream effector of DREF-mediated transcriptional regulation. Given that DREF has been implicated in the activation of cell proliferation and its coordination with mitochondrial growth, the identified targets of Spdk fit well with this role. Intriguingly, it was shown that Spdk may also bind to the regulatory regions of dref. This may enable mutual regulation of Spdk and DREF, although this is highly speculative. It will be of future interest to see how the expression of other genes activated by Spdk might contribute to the coordination of cell proliferation with S phase progression (Page, 2005).

Since many of the factors involved in the transcriptional regulation of the cell cycle are conserved, it seems likely that Spdk might have its counterpart in human cells as, for example, does DREF. Anopheles gambiae has a clear orthologue of spdk, XP319587, which shares 45% identity with residues 225-506 of Spdk with homology extending beyond this most conserved region. A similar region of Spdk shares 41% identity with a region of Human Zinc-Finger Protein 155. However, given that the gene that encodes this protein is a member of a cluster of paralogous genes (Shannon, 2003), future work will be necessary to downregulate each of these genes to identify a functional orthologue of Spdk (Page, 2005).

Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila

Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, it was found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C₂H₂ zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1⁺ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis (Laundrie, 2003; full text of article).

All four P-element mutants showed abnormal oogenesis in GLCs, and the strength of the phenotype varied depending on the allele. Nuclear ß-galactosidase (ß-gal) was used to visualize the breakdown of nurse cell nuclei in late oogenesis. In wild-type nurse cells, nuclear ß-gal diffused into the cytoplasm beginning in stage 10, before nurse cell dumping occurred. The PZ01862 and PZ02132 GLCs have a variable dumpless phenotype with persisting ß-gal-positive nurse cell nuclei and a similar moderate phenotype was seen in k05606 GLCs. However, the ovarian phenotype of PZ08859 was significantly stronger, with ~95% of late egg chambers displaying a strong dumpless phenotype. In addition, egg chambers frequently displayed abnormalities at earlier stages, including reduced size, an unusually thick follicle cell layer, and abnormal nurse cell nuclear morphology (Laundrie, 2003).

The EMS alleles displayed a range of GLC phenotypes, with some similarities but also distinct differences compared to the P-element alleles. The pita¹ allele had a moderate dumpless phenotype, with 80% dumpless stage 14 egg chambers. However, pita¹ GLCs had notably fewer stage 14 egg chambers, suggesting that many of the pita¹ egg chambers degenerated before reaching late oogenesis. pita³ had a stronger phenotype than pita¹ or PZ08859, with many abnormal early egg chambers. Furthermore, early egg chambers (stages 6-9) from both the pita¹ and pita³ alleles often displayed a 'bowling pin' shape, lacking nurse cell nuclei. This phenotype was not seen in any of the P-element alleles. The GLC phenotype of the pita² allele was much weaker than the other alleles and although the flies were largely infertile, the majority of egg chambers appeared wild type. Thus, the EMS alleles showed variability in phenotypes, with dumpless egg chambers and abnormal nurse cell nuclear morphology like the P-element alleles. However, the stronger EMS alleles also showed significant levels of premature nurse cell death. This premature nurse cell death was not observed in the dcp-1/pita double mutants, suggesting that dcp-1⁺ activity was required for the mid-oogenesis cell death seen in pita mutants (Laundrie, 2003).

The P-element and EMS alleles showed altered nurse cell nuclear morphology. To investigate the nuclear organization further, egg chambers stained with propidium iodide were examined. Early stage wild-type nurse cell chromosomes are polytene and appear as discrete 'blobs' until stage 5, after which the chromosomes disperse, giving the nuclei a diffuse appearance. However, PZ08859 and pita¹ GLCs showed persistent individualized chromosome blobs through late stages. Thus, pita may play a role in chromosome dispersal that normally occurs during stage 5 (Laundrie, 2003).

To further confirm that the observed phenotypes were due to pita, rescue experiments of the P-element and EMS alleles were performed. The pita cDNA was expressed under the control of a heat-shock-inducible promoter (HS-pita) and 1-hr heat shocks were performed daily during larval and pupal development. Homozygous PZ02132, PZ08859, pita¹, or pita³ flies carrying the HS-pita transgene survived to adulthood and appeared normal. In contrast, the lethality of the P-element alleles could not be rescued by expression of dcp-1, using HS-dcp-1, UASp-truncated-dcp-1, or pCaSpeR-4.4dcp-1. Thus, expression of pita but not of dcp-1 could rescue the larval lethality, as well as the imaginal disc and melanotic tumor phenotypes of the different alleles (Laundrie, 2003).

The HS-pita transgene was also sufficient to rescue the ovary phenotype of the mutants. Homozygous PZ02132 females that reached adulthood following larval and pupal expression of HS-pita were initially fertile and showed normal oogenesis. However, aged flies that were no longer subjected to expression of HS-pita showed egg chambers with follicle cell defects. Flies >3 days post-heat shock showed an accumulation of abnormal egg chambers, with relatively normal nurse cells but very few surrounding follicle cells. Similarly, homozygous PZ08859 flies rescued with HS-pita showed normal oogenesis in young flies and had egg chambers that lacked follicle cells in older flies. However, rescued PZ08859 flies were sickly and infertile. Thus, the pita transgene rescued the viability and ovary phenotype of the dcp-1/pita mutants but defects arose several days post-heat shock, suggesting that continued expression of pita was necessary for normal oogenesis (Laundrie, 2003).

To determine whether the abnormal egg chambers were caused by the loss of dcp-1 or pita, the ovaries of rescued pita¹ and pita³ flies were examined. As seen with the PZ02132 allele, pita¹ and pita³ flies were initially fertile, but became sterile a few days post-heat shock, suggesting that fertility was dependent on pita expression. However, flies that were aged beyond 3 days showed egg chambers with degenerating germline and follicle cells, rather than the selective follicle cell death seen in the double mutants. These results suggest that pita function is required for the survival of follicle cells. The germline cannot normally survive when follicle cells are defective or dying, and as such the germline cells also died in the pita mutant. However, the germline survived in the pita/dcp-1 double mutants, suggesting that dcp-1 function is required for the death of the germline at the mid-oogenesis checkpoint (Laundrie, 2003).

Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis

This study demonstrates the characteristic morphological features of Drosophila egg chambers lacking both dcp-1 and pita functions in the germline cells. Dcp-1 is an effector caspase and it has been previously shown to play an important role during Drosophila oogenesis. The completion of sequencing and annotation of the Drosophila genome has revealed that the dcp-1 gene is nested within an intron of another distinct gene, called pita, a member of the C2H2 zinc finger protein family that regulates transcriptional initiation. The dcp-1^-/-/pita^-/- nurse cells exhibit euchromatic nuclei (delay of apoptosis) during the late stages of oogenesis, as revealed by conventional light and electron microscopy. The phalloidin-FITC staining discloses significant defects in actin cytoskeleton arrangement. The actin bundles fail to organize properly and the distribution of actin filaments in the ring canals is changed compared to the wild type. The oocyte and the chorion structures have been also modified. The oocyte nucleus is out of position and the chorion appears to contain irregular foldings, while the respiratory filaments obtain an altered morphology. The dcp-1^-/-/pita^-/- egg chambers do not exhibit the rare events of spontaneously induced apoptosis, observed for the wild type flies, during mid-oogenesis. Interestingly, the mutated egg chambers are protected by staurosporine-induced apoptosis in a percentage of 40%, strongly suggesting the essential role of dcp-1 and/or pita during mid-oogenesis (Nezis, 2005).

Search PubMed for articles about Drosophila Pita

Laundrie, B., et al. (2003). Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila. Genetics 165: 1881-1888. PubMed citation: 14704173

Nezis, I. P., et al. (2005). Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis. Cell Motil. Cytoskeleton 60(1): 14-23. PubMed citation: 15547953

Ohkura, H., Török, T., Tick, G., Alvarado, M., Kiss, I. and Glover, D. (1995). Screening for mitotic mutants on the second chromosome. Eur. Dros. Res. Conf. 14: 254

Page, A. R., et al. (2005). Spotted-dick, a zinc-finger protein of Drosophila required for expression of Orc4 and S phase. EMBO J. 24(24): 4304-15. PubMed citation: 16369566

Pflumm, M. F. and Botchan, M. R. (2001). Orc mutants arrest in metaphase with abnormally condensed chromosomes. Development 128: 1697-1707. PubMed citation: 11290306

Shannon, M., Hamilton, A. T., Gordon, L., Branscomb, E. and Stubbs, L. (2003). Differential expansion of zinc-finger transcription factor loci in homologous human and mouse gene clusters. Genome Res 13: 1097-1110. PubMed citation: 12743021

Török, T., Tick, G., Alvarado, M. and Kiss, I. (1993). P-lacW insertional mutagenesis on the second chromosome of Drosophila melanogaster: isolation of lethals with different overgrowth phenotypes. Genetics 135: 71-80. PubMed citation: 8224829

date revised: 7 July 2008

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