InteractiveFly: GeneBrief

pita: Biological Overview | References

Gene name - pita

Synonyms - spotted dick

Cytological map position - 59E3-59E3

Function - transcription factor

Keywords - cell cycle, transcriptional regulator of orc4

Symbol - pita

FlyBase ID: FBgn0034878

Genetic map position - 2R:19,436,488..19,442,241 [-]

Classification - C2H2 zinc finger

Cellular location - nuclear

NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Zolotarev, N., Fedotova, A., Kyrchanova, O., Bonchuk, A., Penin, A.A., Lando, A.S., Eliseeva, I.A., Kulakovskiy, I.V., Maksimenko, O. and Georgiev, P. (2016). Architectural proteins Pita, Zw5,and ZIPIC contain homodimerization domain and support specific long-range interactions in Drosophila. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27137890
According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer-promoter interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. This study shows that the ZAD domains of ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene promoter. However, no functional interaction between binding sites for different proteins was found, suggesting that such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.


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 Pk14408) 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).

Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax Complex

Boundaries in the Bithorax Complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two critical functions: they must block crosstalk between adjacent regulatory domains, and at the same time facilitate boundary bypass. The C2H2 zinc finger Pita protein binds to several BX-C boundaries including Fab-7 and Mcp. To study Pita functions, a boundary replacement strategy was used by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6<-->ab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7 a novel sensitized background was used to show that the two Pita sites contribute its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7; it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function (Kyrchanova, 2017).

Previous studies on the Pita (also known as Spotted Dick) protein suggested that it is a transcriptional activator and showed that the replication defects in pita mutants and in RNAi knockdowns were due to a reduction in the expression of the replication origin protein Orc4. The experiments presented in this study, together with previous studies (Maksimenko, 2015), indicate that pita has an additional, if not an entirely different, function, which is chromosome architecture. This paper details the evidence in favor of this conclusion, and also discuss the implications of findings for boundary function in the context of BX-C (Kyrchanova, 2017).

Boundary replacement experiments provide compelling evidence that the zinc-finger protein Pita functions just like other insulator/architectural proteins. When placed in the context of Fab-7, multimerized Pita-binding sites block crosstalk between iab-6 and iab-7, but are not permissive for the regulatory interactions between iab-6 and the Abd-B gene. In this respect, the functioning of the multimerized Pita-binding sites is similar to that observed when multimerized sites for 'canonical' boundary factors, dCTCF and Su(Hw), are substituted for the Fab-7 boundary. In the context of Fab-7, they also block crosstalk between iab-6 and iab-7, but do not support bypass (Kyrchanova, 2017).

The boundary functions of the Pita protein are also supported by experiments testing its activity in a native context. For Fab-7, there are two Pita-binding sites in the HS2 hypersensitive region. Since previous studies have shown that HS1 is sufficient for full boundary activity, when the iab-7 PRE (HS3) is present, it is clear that Pita function is redundant. This was confirmed by introducing mutations in the two Pita-binding sites in a Fab-7 boundary, HS1+2+3, that lacks the '*' nuclease-hypersensitive site, but contains the iab-7 PRE. However, a different result was obtained in the context of a sensitized replacement, HS1+2, in which the iab-7 PRE (HS3) is deleted. In this sensitized background, the two Pita-binding sites in HS2 are essential for boundary activity (Kyrchanova, 2017).

Interestingly, the sensitized HS1+2 Fab-7 replacement has unprecedented properties. Unlike previously described Fab-7 mutations, which are dominant, the boundary defects of HS1+HS2 can be fully complemented by a wild-type boundary in trans. Additionally, as a homozygote, it has differential effects on the specification of dorsal and ventral tissues. The A6 (PS11) sternite is missing in HS1+2 males. This gain-of-function transformation indicates that boundary activity is disrupted in the cells that give rise to this ventral cuticular structure. By contrast, the A6 tergite is not only nearly normal in size, but is also properly specified. This finding means that boundary activity is largely retained in the PS11 cells that give rise to dorsal cuticle structures (Kyrchanova, 2017).

It is also worth noting that HS1+2 is very different from mutations that delete the iab-7 PRE (HS3) but retain the entire Fab-7 boundary. First, the vast majority of homozygous iab-7 PRE (HS3) deletion males are indistinguishable from wild type, arguing that the HS3 deletion retains full boundary function. Second, in a few of the males (~2.5%), small sections of the dorsal A6 tergite are missing. This phenotype is most readily explained by a loss of PRE silencing, and consequent gain-of-function transformation, in a subset of the cells that give rise to the dorsal cuticle. As the HS1+2 replacement differs from all of the iab-7 PRE (HS3) deletions isolated previously in that it lacks 'HS*', it would appear that this part of the boundary contains binding motifs for factors that are important for boundary function specifically in ventral tissues (Kyrchanova, 2017).

This would not be the only Fab-7 boundary factor that has 'developmentally' restricted activity. The two large complexes known to be important for Fab-7 HS1 boundary function, Elba and LBC, are active at different stages of development; the former in early embryos and the latter from mid-embryogenesis onwards. The fact that there is likely to be yet another boundary factor whose activity is developmentally restricted, fits with the idea that boundary function in flies can be subject to stage- and/or tissue-specific regulation (Magbanua, 2015; Kyrchanova, 2017 and references therein).

One of the paradoxes posed by the BX-C boundaries is that six of the nine regulatory domains in the complex are separated from their homeotic target genes by one or more boundaries. Consequently, these boundaries must, on the one hand, block regulatory interactions between adjacent domains and, on the other, facilitate boundary bypass. One of models to explain these two contradictory activities is that BX-C boundaries have unique properties, i.e. they are designed to block interactions between enhancers/silencers, but not between enhancers/silencers and promoters. This model gained currency from replacement experiments, which showed that the BX-C boundary Fab-8 can substitute for Fab-7, while two heterologous boundaries cannot. A prediction of the model is that other BX-C boundaries could also substitute for Fab-7. However, contrary to this prediction, the current experiments indicate that Mcp340 boundary behaves like the heterologous fly boundaries -- it blocks both crosstalk and bypass (Kyrchanova, 2017).

Analysis of the effects of mutations in the Pita and dCTCF sites of the Mcp340 boundary suggest that there is a complicated relationship between blocking crosstalk and blocking or enabling bypass. Although mutations in the Pita and dCTCF disrupt the functioning of Mcp340 replacement, the actual consequences of each mutation are quite distinct. In the case of the Pita mutation, loss of Pita binding was found to lead to a substantial reduction in the binding of dCTCF. This means that for this particular boundary, Pita association is required to recruit dCTCF. The requirement is not, however, reciprocal: deleting the Mcp dCTCF site has no effect on Pita association (Kyrchanova, 2017).

Correlated with the differential effects on protein binding, the M340ΔPita and M340ΔCTCF mutants have quite different phenotypes. The phenotype (mixed gain and loss of function) of the former resembles a classic Fab-7 boundary deletion in which the iab-7 PRE (HS3) is retained. In contrast, the phenotype of the latter is a mixture of gain and loss of function, together with cuticle that has morphological features identical to that in A6 (PS11) of wild-type flies. The presence of cuticle that has the proper PS11 identity argues that M340ΔCTCF retains residual boundary function that, in a subset of cells, is sufficient to not only block crosstalk between iab-6 and iab-7, but is also able to facilitate iab-6 bypass (Kyrchanova, 2017).

A simple interpretation of this finding is that the Pita protein differs from dCTCF in that it blocks crosstalk but can facilitate bypass. However, this simple model is not supported by other findings. First, as noted above, just like multimerized dCTCF sites, multimerized Pita sites block both crosstalk and bypass when substituted for Fab-7. Second, the two Pita sites in Fab-7 are not in themselves sufficient for blocking crosstalk. Third, the Fab-8 boundary, which has two dCTCF sites, but no sites for Pita, has both blocking and bypass activity when substituted for Fab-7. Moreover, these dCTCF sites appear to contribute to the bypass activity of the Fab-8 replacement. Thus, a more likely hypothesis is that there are other, as yet unidentified, factors that are bound to Mcp340 and contribute to the blocking and (newly acquired) bypass activities of the M340ΔCTCF mutant, in addition to the Pita protein. Taken together with the finding that reversing Fab-8 eliminates bypass activity (Kyrchanova, 2016), the current experiments with Mcp suggest that there may not be a common mechanism for generating both blocking and bypass activity. Rather, each BX-C boundary would appear to deploy distinct mechanisms that are adapted for their specific context within the complex (Kyrchanova, 2017).

Based on RNAi knockdown experiments in S2 cells, it has been suggested that Pita is a transcriptional activator and that it could play a crucial role in coordinating S phase progression. This idea was supported by experiments showing that the replication defects induced by Pita depletion are caused by a reduction in Orc4 expression. However, only 32 genes are downregulated (and 10 upregulated) after Pita RNAi, and most appear to have nothing to do with replication. Furthermore, as there are several thousand Pita sites in the genome, the number of affected genes is surprisingly low. In this light, an obvious question is whether blocking, instead of transcriptional activation, might account for the effects of Pita depletion on the Orc4 transcription? Although this study did not investigate how Pita functions in the S2 cells, there are reasons to think that this is a distinct possibility. ChIP experiments have shown that Pita binds to a region upstream of the Orc4 gene in S2 cells. ModEncode ChIP experiments indicate that there is a large PcG silenced domain just beyond this Pita site. Thus, an alternative possibility is that Orc4 expression is reduced when Pita is depleted, because the gene is silenced by the PcG spreading. Several of the other Pita transcriptional targets are also close to the PcG domains, and could be silenced in a similar manner (Kyrchanova, 2017).

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

Kyrchanova, O., Zolotarev, N., Mogila, V., Maksimenko, O., Schedl, P. and Georgiev, P. (2017). Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax Complex. Development [Epub ahead of print]. PubMed ID: 28619827

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 ID: 14704173

Magbanua, J. P., Runneburger, E., Russell, S. and White, R. (2015). A variably occupied CTCF binding site in the ultrabithorax gene in the Drosophila bithorax complex. Mol Cell Biol 35(1): 318-330. PubMed ID: 25368383

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Biological Overview

date revised: 22 December 2017

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