polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs.

Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many developmental pathways. These include limb morphogenesis in Drosophila melanogaster, since PcG genes interact with identity and pattern specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. Our results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the level of engrailed expression in posterior ones. We suggest that PcG targets are not exclusively regulated by an on/off mechanism, but that the PcG also exerts negative transcriptional control on active genes.

engrailed and polyhomeotic interactions are required to maintain the A/P boundary of the Drosophila developing wing.

Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and wing morphogenesis. One of its regulatory targets in embryos was shown to be the Polycomb group gene, polyhomeotic. We show here that transheterozygous adult flies, mutant for both engrailed and polyhomeotic, show a gap in the fourth vein. In the corresponding larval imaginal discs, a polyhomeotic-lacZ enhancer trap is not normally activated in anterior cells adjacent to the anterior-posterior boundary. This intermediary region corresponds to the domain of low engrailed expression that appears in the anterior compartment, during L3. Several arguments show that engrailed is responsible for the induction of polyhomeotic in these cells. The role of polyhomeotic in this intermediary region is apparently to maintain the repression of hedgehog in the anterior cells abutting the anterior-posterior boundary, since these cells ectopically express hedgehog when polyhomeotic is not activated. This leads to ectopic expressions first of patched, then of cubitus interruptus and decapentaplegic in the posterior compartment, except for the dorsoventral border cells that are not affected. Thus posterior cells express a new set of genes that are normally characteristic of anterior cells, suggesting a change in the cell identity. Altogether, our data indicate that engrailed and polyhomeotic interactions are required to maintain the anterior-posterior boundary and the posterior cell fate, just prior to the evagination of the wing.

Molecular mechanism of polyhomeotic activation by Engrailed.

The Drosophila Engrailed homeoprotein has been shown to activate directly a Polycomb-group gene, polyhomeotic, during embryogenesis. The molecular mechanism involved in this activation has been studied. Two different types of Engrailed-binding fragments have been detected within the polyhomeotic locus. The P1 and D1 fragments contain several 'TTAATTGCAT' motifs, whereas the D2 fragment contains a long 'TAAT' stretch to which multiple copies of Engrailed bind cooperatively. Another homeodomain-containing protein, Extradenticle, establishes protein-protein interactions with Engrailed on the D2 fragment. We have shown by CAT assays that both types of Engrailed-binding sites (P1 or D1 and D2), as well as Extradenticle, are necessary to obtain activation by Engrailed. In vivo, we have also shown that normal polyhomeotic expression depends on extradenticle expression. Moreover, in the absence of Extradenticle, overexpression of Engrailed protein represses polyhomeotic expression.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

Selection and characterization of sequences with high affinity for the engrailed protein of Drosophila.

The engrailed gene helps to direct Drosophila melanogaster development by encoding a homeodomain-containing DNA binding protein. To identify genes whose transcription engrailed regulates, we developed a method to isolate genomic sequences to which engrailed protein binds with high affinity. Fragments of genomic DNA were fractionated on an engrailed protein affinity column, and fragments that were retained in the presence of 0.4-1.0 M KCl were isolated and cloned. The isolated fragments include regions of the engrailed and cubitus interruptus gene promoters, both of which are candidate targets of engrailed, and most fragments contain regions that engrailed protein protects from DNaseI digestion. Chromosomal deletions that remove some of the engrailed binding sites (located either at 64D, 96B or 99D) interact genetically with engrailed. Characterization of a transcript encoded in region 64D revealed its dependence on engrailed protein.

polyhomeotic appears to be a target of engrailed regulation in Drosophila.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.

Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster.

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.

Structure of the ecdysone-inducible P1 gene of Drosophila melanogaster.

The P1 gene codes for a major RNA, which accumulates specifically in the fat body cells at the late third larval stage of Drosophila melanogaster development under the positive control of the insect molting hormone 20-hydroxyecdysone. The primary structure of the P1 gene and the 5' upstream flanking region to position -776 relative to the transcription start was determined by sequence analysis of a cloned genomic DNA segment and two cDNAs containing sequences complementary to the 5' and 3' ends of the P1 transcript. The RNA coding region spans 3469 nucleotides and contains a 59-base-pair intron close to its 5' end, as predicted by computer analysis and established by S1 nuclease protection, primer extension and cDNA sequencing. The predicted P1 polypeptide contains 1030 amino acids, including a putative 16-amino acid signal peptide and two stretches of 12 and 11 aspartic and asparagine residues. Short stretches of nucleotide sequences similar to sequences located in the 5' regions of other genes expressed in the D. melanogaster fat body were found in the proximal promoter and transcribed region of the P1 gene.

Larval fat body-specific gene expression in D. melanogaster.

The Pl gene, together with the LSP-1 alpha, -1 beta, and -1 gamma, LSP-2, and P6 genes, is expressed exclusively in the larval fat body of D. melanogaster during the third instar. In vivo mapping of the cis-acting regulatory sequences of the P1 gene was carried out using hybrid constructs with three different reporter genes and a combination of transient and germline transformation assays. This revealed that regulatory elements involved in the setting up of the temporal and spatial specificities of transcription of the P1 gene are located in a short DNA region immediately upstream of the mRNA transcription start. This region includes an element that behaves as a fat-body transcriptional enhancer and element(s) required for ecdysone inducibility of transcription of the P1 gene.

Hormonal and developmental specificities of transcription lie within a 1.5 kb region 5' to the ecdysone inducible Drosophila P1 gene.

P-element-mediated transformation was used to investigate 5' cis-acting regulatory sequences flanking the P1 gene of Drosophila melanogaster, which is selectively expressed in the fat body of late third instar larvae under the positive control of ecdysone. A hybrid gene was constructed by fusing a 1.5 kb DNA fragment directly adjacent to and including the first 25 transcribed bases of the P1 gene to the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene itself linked at its 3' end to the SV40 t antigen splicing and polyadenylation sequences. Five transformed lines of D. melanogaster containing only one copy of the hybrid gene were established. In each of these lines the gpt sequence is transcribed with the same spatial, temporal and hormonal specificities as those of the P1 gene. This provides evidence that control elements essential for the ecdysone and developmentally regulated expression of P1 are located within a 1.5 kb region 5' to this gene.