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.

The 35UZ transposon of Drosophila melanogaster reveals differences in maintenance of transcriptional control between embryonic and larval stages.

The D. melanogaster transposon P[35UZ] contains a lacZ reporter gene fused to 35 kb of Ubx upstream sequences which drive a Ubx-like expression in embryos and in metathoracic imaginal discs. Transposition of P[35UZ] followed by analysis of different lines in wild-type and mutant backgrounds allowed us to analyze the interplay between Ubx regulatory elements, including the Polycomb response element (PRE), located inside the transposon and cis-acting regulatory elements, located outside. We found that all lines show a Ubx-like beta-galactosidase expression pattern in the embryo, but proximity to strong imaginal enhancers can change this pattern drastically. These data illustrate how maintenance of gene expression depends on the chromosomal environment and on dynamic interactions between all developmentally regulated enhancers located close to a promoter under PcG control.

Needs and targets for the multi sex combs gene product in Drosophila melanogaster.

We have analyzed the requirements for the multi sex combs (mxc) gene during development to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila melanogaster. mxc is allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm (l(1)mbn). We show that the mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc has also a strong maternal effect. We find that hypomorphic mxc mutations enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. We also demonstrate that the mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. Our analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression previously reported for some PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development. We discuss these data in view of the numerous functions of PcG genes during development.

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.

The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis.

The polyhomeotic (ph) locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggested that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb, and the distal unit encodes a 6.4-kb embryonic mRNA. The proximal and distal transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon, and an internal ATG is used for a start codon. We show that the proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons. We find no evidence for differential binding of proximal and distal products to polytene chromosomes. Nevertheless, we show that mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated.

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.

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor.

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line development pathways, provoke the loss of cell affinities, but allow or promote growth.

Transposon-induced rearrangements in the duplicated locus ph of Drosophila melanogaster can create new chimeric genes functionally identical to the wild type.

Variation in the number of gene copies can play a major role in changing the coding capacities of eukaryotic genomes. Different mechanisms, such as unequal recombination or transposon-induced chromosome rearrangements, are believed to be responsible for these events. We have used the direct tandem duplication at the complex locus polyhomeotic (ph) of Drosophila melanogaster as a model system to study functional redundancy associated with chromosomal rearrangements, such as duplications or deletions. The locus covers 28.6 kb and comprises two independent units, ph proximal and ph distal, which are not only similar on the molecular level, but appear to be functionally redundant [Dura et al., Cell 51 (1987) 829-839; Deatrick et al., Gene 105 (1991) 185-195]. We present a molecular and phenotypic analysis of two hypomorphic ph mutants, ph2 and ph4, induced during hybrid dysgenesis. Each corresponds to an internal deletion in the ph locus that overlaps both transcription units. We show that the deletions are likely due to a P/M hybrid dysgenesis-induced rearrangement between proximal and distal ph, that created a single new chimerical ph gene. At least one of the breakpoints must be located in a 1247-bp region that is rich in single sequence, and 100% identical between proximal and distal ph. Junction points between units are in the protein-coding regions, but could not be exactly localized on the genomic sequence of either mutant, because of the precise molecular mechanism that caused the deletions. Protein products of the hybrid genes contain the same functional domains as either wild-type (wt) product.(ABSTRACT TRUNCATED AT 250 WORDS)

The complex genetic locus polyhomeotic in Drosophila melanogaster potentially encodes two homologous zinc-finger proteins.

Differential expression of the homeotic gene complexes, ANT-C and BX-C, of Drosophila melanogaster is partly controlled by trans-regulating factors located outside the two complexes. The complex genetic locus, polyhomeotic (ph), is one of these trans regulators required during development for correct expression of the homeotic selector genes. The ph locus comprises two genetically independent units whose functions are largely redundant. There are two duplicated sequences arranged as a tandem repeat in the ph region, defining two molecular ph units. Sequence analysis of the 28.6 kb of DNA comprising the locus shows varying degrees of sequence conservation between these two molecular units. Long open reading frames with a high degree of conservation have been localized in each tandem repeat. Putative protein products encoded by both the proximal and the distal unit contain several identical or practically identical protein domains: a zinc-finger-forming motif, an alpha-helix motif, a domain rich in serine and threonine residues and stretches of glutamine residues. The presence of these protein domains supports the hypothesis that ph encodes a transcription factor that may function as part of a protein complex. Possible molecular mechanisms leading to the particular structure of the locus are discussed.

Transcription of dispersed repeated sequences during Pleurodeles waltl oogenesis.

Several repeated DNA sequences were isolated from a partial genomic DNA library of the newt Pleurodeles waltl. These repeated DNA elements are dispersed over the 12 P. waltl bivalents, and some of them are transcribed in the oocyte. We describe the localization of label following in situ hybridization to nascent RNA attached to the lateral loops of lampbrush chromosomes. Variations in the number and the location of labelled loops were constantly found for several of the probes. The results are discussed in view of the "cotranscription model" of RNA synthesis on lampbrush chromosomes. We speculate on the possible origins of variation in transcription on lampbrush loops.

A complex genetic locus, polyhomeotic, is required for segmental specification and epidermal development in D. melanogaster.

Two mutagenic events are required to make null mutations of polyhomeotic (ph), which suggests that the locus is complex. Amorphic mutations (ph degrees) die in mid-embryogenesis and completely lack ventral thoracic and abdominal epidermal derivatives, whereas single-event mutations lead to transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes. After a chromosomal walk, the ph gene was localized using deficiencies and ph mutations that result from DNA rearrangements. Hybridization analyses show that there are two large, duplicated sequences in the ph region, and DNA lesions affecting either one of these repeats alter the function of the ph locus. We propose a model that may account for this unusual functional organization.