4.1 SUMMARY AND GENERAL DISCUSSION
In this study a cDNA for an additional member of the lipid phosphate phosphohydrolase family in Drosophila melanogaster was clonded. This encodes a 350 amino acid protein, which was called Tunen and shares 34% amino acid homology with Wunen the first Drosophila LPP identified (Zhang et al., 1997). The most conserved region between these two LPPs is around the proposed phosphatase domain. These two proteins show the greatest diversity at the amino and carboxy termini, with Tunen having a much longer N-terminus and shorter carboxy terminus than Wunen. Since the amino and carboxy termini are predicted to be cytoplasmic, this diversity suggests these regions may play a role in controlling the specific activity o f these proteins via interaction with isoform specific intracellular proteins. When compared to the mammalian homologs, Tunen, like Wunen does not obviously fit into the isoform classes identified to date. The LPP-3 isoforms identified in rat and human are longer at the amino terminus than the LPP2 and LPPl isoforms but the amino acid homology with Tunen in this region is negligible.
In situ hybridisation analysis of developing embryos showed that Tunen transcript is
expressed in a very similar pattern to Wunen. This common expression pattern suggested Tunen might also play a role in guiding germ cells from the gut to the gonadal mesoderm. This prediction was confirmed by creating a Tunen transcript null,
tunen In tunen embryos germ cell migration was disturbed when compared to a
wild type control. However this germ cell migration phenotype was not so severe as that shown by the Wunen null wun ^^^^^\Tunen flies are homozygous viable and though germ cell migration is somewhat perturbed many germ cells still find their way to the gonads. It has been shown by germ cell transplantation experiments that only a few germ cells are actually required for functional gonads to form (Marsh and Wieschaus, 1977) It was also shown that Tunen guides germ cell by repulsion by overexpressing it in the mesoderm in a manner analogous to the experiment carried out by Zhang et al for Wunen (Zhang et a l, 1997).
The initial data acquired from this work suggested that two LPPs exist in Drosophila
cells from the gut to the mesoderm by repulsion. The differences in severity of phenotypes suggested that Wunen played a more significant role than Tunen in germ cell guidance. However, analysis of the genomic sequence of tunen and wunen revealed that both proteins are encoded by the same locus, which I have called wuntun and are divergently transcribed. The intergenic sequence in the wuntun locus between the transcript start sites is approximately 5kb. Analysis o f this intergenic sequence using promoter prediction and transcription factor binding sites programmes identified a number of motifs that suggest promoter activity in this region. These data raise a number o f interesting questions. First, are these proteins transcriptionaly regulated? Do they function independently or synergistically in germ cell guidance? Have these two transcripts arisen from one ancestral gene as a consequence of an early evolutionary event? What does this genomic organisation imply with regards to the data available on
the wuntun locus so far?
Divergently transcribed genes have frequently been found among members o f gene families that are organised in clusters or groups and various modes of transcriptional regulation are employed. In Drosophila, salivary glue protein genes sgs-1 and sgs-S are divergently transcribed, separated by 475 nucleotides and a cw-acting element located in the intergenic sequence is required for correct expression of both genes. (Hofrnann et al., 1991). Msr84Da and Ms?84Db are members of the Mst{3)CGP gene family involved in the formation of the Drosophila sperm tail. These two genes are divergently transcribed, structurally similar and show a common expression pattern (Gigliotti et al.,
1997). These features are reminiscent of tunen and wunen, however the intergenic region between Ms'/84Da and Mst%ADh is only 194 nucleotides and transcription of these genes is not co-regulated. Expression of chicken beta- and epsilon-globin genes in erythroid cells is controlled by a shared enhancer located in the 3.1kb intergenic sequence (Nickol and Felsenfeld, 1988). It is not obvious from the data available on the WunTun locus whether these genes are transcriptionaly coregulated or not. However some of the data acquired in this work suggests this may be so.
It was demonstrated by in situ analysis that wun which was previously thought to be null only for Wunen transcript is actually a null for both Tunen and Wunen. Wun
is a consequence of a P-element insertion in a zinc-finger transcription factor GFI-1, approximately 2kb downstream of the wunen transcription unit. This insertion
results in the truncation of the product of this gene and downregulation of wunen
expression (Zhang et al., 1997). These data, which shows that this insertion also downregulates the expression of tunen suggest the P-element insertion in some way interferes with the transcription machinery for both tunen and wunen. How might this occur?
The intergenic region between tunen and wunen is quite large and it is possible that independent promoters and a common enhancer reside in this intergenic region, and regulates both these genes. Enhancer proteins bind activator and repressor proteins and have been identified in locations from 100 to 3,000base pairs upstream or down stream of eukaryotic genes. It is assumed that the activator proteins recruit basal transcriptional machinery such as TATA-binding proteins and RNA polymerase II holoenzyme to the promoter (Ptashne and Gann, 1997). In this way activators and co-activators contact components of the basal machinery. The recruitment model works well when activators bind close to the promoter. However in the case o f yeast the upstream activation sequence (UAS) can be up to one kilo base or so upstream o f the promoter. Evidence from several organisms indicates that the chromatin between activators and promoters can bend and loop to accommodate protein-protein interactions over these distance (Ptashne, 1986; Ptashne, 1988).
It has been proposed by Dorsett (Dorsett, 1999) that facilitator factors such as Chip (Morcillo et al., 1997) and Nipped B (Rollins et al., 1999) in Drosophila function between remote enhancers and promoters to bring them physically together through their interaction with homeodomain proteins. There is also evidence that enhancers can simultaneously activate two promoters and some promoters actually compete with each other for activation (Ohtsuki et al., 1998). So wunen and tunen may share enhancers which activate individual promoters and might use looping to bind promoter elements. The P-element insertion in wun could interfere with this architecture thus preventing transcription of both genes. The Tunen null, tunen on the other hand is the result of a deletion in the 5' UTR and Tunen coding sequence thus does not affect Wunen expression.
Genomic organisation, shared expression pattern, similar function and possible co regulation o f transcript expression also suggests that these two proteins may work together to guide germ cell migration. The in situ hybridisation data shows that the transcripts for both these proteins are expressed in the same tissues at the same time. In
tunen flies that germ cell migration is disrupted, however, in these embryos some
germ cells still populated the gonads and as a result the flies are fertile. In the tunen
embryos Wunen transcript is still expressed. This strongly suggests that Wunen can compensate for the lack o f Tunen but does not guide germ cell migration as efficiently as when Tunen is also present. This theory is further supported by the my finding that
wun which shows a much stronger germ cell migration phenotype than tunen is actually null for both Wunen and Tunen transcripts and not just wunen as previously thought. To date a Wunen only null is not available but the observations made so far would suggest this would show a similar weak phenotype to tunen How these proteins act together to guide germ cell migration is not clear.
It is possible that these two LPP isoforms which appear to co-regulate germ cell migration work together by forming a heterodimer or a larger complex at the plasma membrane. Receptor dimérisation is a common theme used by growth factor receptors as a means of controlling receptor activation. Several growth factors induce receptor dimérisation by virtue o f their dimeric nature e.g. PDGF (Bishayee et a l, 1989). Monomeric growth factors such as EGF contain two binding sites for their receptors and can cross-link two neighbouring EGF receptors (Lemmon et al., 1997). FGFs bind to their receptors monovalently but utilise accessory molecules to facilitate the formation of multimeric ligand-receptor complexes (Spivak-Kroizman et al., 1994). Receptor dimérisation in these instances is essential for stimulation o f the intrinsic catalytic activity and for autophosphorylation of growth factor receptors.
Growth factor receptors can undergo both homo and heterodimerisation (Lemmon and Schlessinger, 1994). Ligand induced receptor oligomerisation has been established as a universal mechanism for activation o f hormone and growth factor receptors, lymphokine receptors, T-cell receptors and B-cell receptors as well as many other families of receptors. More recently Dekker (Dekker et al., 1997) et al described an integral membrane enzyme from E. coli, outer membrane phospholipase A (OMPLA),
whose activity is regulated by reversible dimérisation. Dimersisation o f these enzyme results in productive active sites, with two substrate binding pockets created at the interface between two monomers.
Data on LPP purified from rat liver membranes suggests that LPPs could form oligomers (Siess and Hofstetter, 1996). Using both chromatographic and immunological methods for the purification of LPP a molecular mass of 186 kDa was estimated for the native enzyme. Since the denatured protein migrated at 31 kDA on an SDS gel it was suggested that a heterodimer of LPP is formed. Four conserved cysteine residues located in the predicted extracellular loops of all LPP isoforms identified further, suggests that such dimérisation could occur through disulphide bond formation. The schematic below (Figure 41) shows the localisation of these residues on Tunen and Wunen transmembrane proteins and the potential disulphide bonds that could be formed.