Interactions of Polycomb and trithorax with cis regulatory regions of Ultrabithorax during the development of Drosophila melanogaster.

The activity of the Ultrabithorax gene is continuously required during imaginal development to maintain the morphogenetic identity of the third thoracic segment of Drosophila. The spatial pattern of Ultrabithorax gene expression depends on certain cis regulatory regions and several trans regulatory genes. Amongst the latter the Polycomb gene is necessary to maintain Ultrabithorax repressed in cells where it was not initially activated and the trithorax gene is required for maintaining the expression of the gene where initially active. We have studied genetic interactions between several Ultrabithorax mutations in coding and cis regulatory regions in combination with Polycomb and trithorax mutations. Our results suggest that Polycomb and trithorax gene products do not interact with Ultrabithorax protein products but interact (directly or indirectly) with specific and discrete cis regulatory regions such as those where anterobithorax and postbithorax but not bithorax mutations map. We discuss possible mechanisms of these interactions.     

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.     

Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.

The Polycomb group and trithorax group genes of Drosophila are required for maintaining the differential expression state of developmental regulators, such as the homeotic genes, in a stable and heritable manner throughout development. The Polycomb group genes have been suggested to act by regulating higher order chromatin and packaging repressed chromosomal domains in a heterochromatin-like structure. We have mapped, at high resolution, the distribution of Polycomb protein on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Our results further suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. We propose that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs and as trithorax group response elements.     

Association of trxG and PcG proteins with the bxd maintenance element depends on transcriptional activity

Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes can antagonize each other's function, whereas mutations of genes within each group have synergistic effects. Here, we show in Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and Asx require Trithorax in order to bind to their target genes. Together, our data argue that at the single-cell level, association of repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically.     

Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each approximately 400 bp) contain both TRX- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (approximately 90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group gene pleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators.     

A double-bromodomain protein, FSH-S, activates the homeotic gene ultrabithorax through a critical promoter-proximal region

More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic (fs(1)h) gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. We show that one of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. First, overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation. Second, FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner. Third, purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, we show that FSH-S is involved in phosphorylation of itself and other regulatory factors. We suggest that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers.     

The role of Polycomb-group response elements in regulation of engrailed transcription in Drosophila

Polycomb group proteins are required for long-term repression of many genes in Drosophila and all metazoans. In Drosophila, DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila engrailed promoter contains a multipartite PRE that can mediate mini-white silencing and act as a PRE in an Ubx-reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the engrailed gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in gene silencing, these two adjacent PRE fragments can facilitate the activation of the engrailed promoter by distant enhancers. The repressive action of the engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous engrailed gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.     

Enhancer Traps in the Drosophila Bithorax Complex Mark Parasegmental Domains

Eight P elements carrying a β-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure.     

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'''' Sites within a 1.6-Kb Region

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.     

Engrailed and polyhomeotic maintain posterior cell identity through cubitus-interruptus regulation

In Drosophila, the subdivision into compartments requires the expression of engrailed (en) and hedgehog (hh) in the posterior cells and of cubitus-interruptus (ci) in the anterior cells. Whereas posterior cells express hh, only anterior cells are competent to respond to the hh signal, because of the presence of ci expression in these cells. We show here that engrailed and polyhomeotic (ph), a member of the Polycomb Group (PcG) genes, act concomitantly to maintain the repression of ci in posterior compartments during development. Using chromatin immunoprecipitation (ChIP), we identified a 1 kb genomic fragment located 4 kb upstream of the ci coding region that is responsible for the regulation of ci. This genomic fragment is bound in vivo by both Polyhomeotic and Engrailed. In particular, we show that Engrailed is responsible for the establishment of ci repression early during embryonic development and is also required, along with Polyhomeotic, to maintain the repression of ci throughout development.