The DNA-binding polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene.

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.     

A yolk protein mutant leads to defects in the secretion machinery of Drosophila melanogaster.

The three yolk proteins of Drosophila melanogaster are synthesized in the fat body and ovarian follicle cells. A mutation in yolk protein 3, YP3S1, has been described in which the leader sequence is not cleaved from the protein. We describe here ultrastructural and molecular studies on the YP3S1 mutant and show that the mutant protein enters the secretory pathway and forms precipitates, often as electron dense material in excessive elaborations of the plasma membrane. Females homozygous for YP3S1 lay fewer eggs than wild type flies and these embryos are less viable. The abnormal ultrastructure of the yolk spheres observed suggests that whilst YP3 is not completely essential for viability, it is required for normal yolk sphere morphogenesis.     

The zygotic mutant tailless affects the anterior and posterior ectodermal regions of the Drosophila embryo

The recessive zygotic lethal mutation tailless maps to region 100A5,6-B1,2 at the tip of the right arm of chromosome 3, and results in shortened pharyngeal ridges in the head skeleton of the mature embryo and the elimination of the eighth abdominal segment and telson. Although they have a normal body length, tailless embryos have a smaller number of abdominal segments, some of which are larger than normal. The mutant phenotype is seen as early as 8 hr postfertilization, when tailless embryos are observed to have fewer tracheal pits than wildtype. At 9 hr, tailless embryos appear to be missing segments A8, A9, and A10 and have an abnormal clypeolabrum, optic lobes, and procephalic lobe. Segments A4, A5, A6, and A7 appear larger in tailless embryos than wildtype at this stage. The tailless mutation, although affecting anterior and posterior ectodermal structures in the mature embryo, does not affect the formation of pole cells, the posterior midgut, or the proctodeum, which arise from the most posterior region of the embryo. The mutation does result, however, in the failure of Malpighian tubule formation. Consistent with its effect on ectodermal segments, tailless leads to a reduction in the number of segmented, paired ganglia in the ventral nerve cord as well as to an abrupt alteration in the posterior region of the tracheal system. The role the tailless gene may play in the formation of the most anterior and posterior regions of the embryo's ectodermal body plan is discussed.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.     

Exposure to static electric fields leads to changes in biogenic amine levels in the brains of Drosophila

Natural and anthropogenic static electric fields are commonly found in the environment and can have both beneficial and harmful effects on many animals. Here, we asked how the fruitfly responds to these fields and what the consequences of exposure are on the levels of biogenic amines in the brain. When given a choice in a Y-tube bioassay Drosophila avoided electric fields, and the greater the field strength the more likely Drosophila were to avoid it. By comparing wild-type flies, flies with wings surgically removed and vestigial winged flies we found that the presence of intact wings was necessary to produce avoidance behaviour. We also show that Coulomb forces produced by electric fields physically lift excised wings, with the smaller wings of males being raised by lower field strengths than larger female wings. An analysis of neurochemical changes in the brains showed that a suite of changes in biogenic amine levels occurs following chronic exposure. Taken together we conclude that physical movements of the wings are used by Drosophila in generating avoidance behaviour and are accompanied by changes in the levels of amines in the brain, which in turn impact on behaviour.     

Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.     

The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing

Polycomb group (PcG) and Trithorax (TRX) complexes assemble at Polycomb response elements (PREs) and maintain respectively the repressed and active state of homeotic genes. Although PcG and TRX complexes are distinct, their binding to some PRE fragments in vitro depends on GAGA motifs. GAGA factor immunoprecipitates with both complexes. In presence of a PRE, TRX stimulates expression and prevents the return of repression at later stages. When TRX levels are reduced, repression is re-established in inappropriate regions of imaginal discs, suggesting that TRX insufficiency impairs the epigenetic memory of the active state. Targeting a GAL-TRX fusion shows that TRX is a coactivator that stimulates expression of an active gene but cannot initiate expression by itself. Targeting a histone acetylase to a PRE does not affect embryonic silencing but causes a loss of memory in imaginal discs, suggesting that deacetylation is required to establish the memory of the repressed state.     

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.