Co-regulation of invected and engrailed by a complex array of regulatory sequences in Drosophila.

invected (inv) and engrailed (en) form a gene complex that extends about 115 kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. Our dissection of inv/en regulatory DNA shows that most enhancers are spread throughout a 62 kb region. We used two types of constructs to analyze the function of this DNA: P-element based reporter constructs with small pieces of DNA fused to the en promoter driving lacZ expression and large constructs with HA-tagged en and inv inserted in the genome with the phiC31 system. In addition, we generated deletions of inv and en DNA in situ and assayed their effects on inv/en expression. Our results support and extend our knowledge of inv/en regulation. First, inv and en share regulatory DNA, most of which is flanking the en transcription unit. In support of this, a 79-kb HA-en transgene can rescue inv en double mutants to viable, fertile adults. In contrast, an 84-kb HA-inv transgene lacks most of the enhancers for inv/en expression. Second, there are multiple enhancers for inv/en stripes in embryos; some of these may be redundant but others play discrete roles at different stages of embryonic development. Finally, no small reporter construct gave expression in the posterior compartment of imaginal discs, a hallmark of inv/en expression. Robust expression of HA-en in the posterior compartment of imaginal discs is evident from the 79-kb HA-en transgene, while a 45-kb HA-en transgene gives weaker, variable imaginal disc expression. We suggest that the activity of the imaginal disc enhancer(s) is dependent on the chromatin structure of the inv/en domain.

Enhancer-promoter communication at the Drosophila engrailed locus.

Enhancers are often located many tens of kilobases away from the promoter they regulate, sometimes residing closer to the promoter of a neighboring gene. How do they know which gene to activate? We have used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. Here we show that engrailed enhancers can act over large distances, even skipping over other transcription units, choosing the engrailed promoter over those of neighboring genes. This specificity is achieved in at least three ways. First, early acting engrailed stripe enhancers exhibit promoter specificity. Second, a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). Our data suggest that there are two partially redundant promoter-tethering elements. Third, the long-distance action of engrailed enhancers requires a combination of the engrailed promoter and sequences within or closely linked to the promoter proximal Polycomb-group response elements. These data show that multiple mechanisms ensure proper enhancer-promoter communication at the Drosophila engrailed locus.

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.

An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene.

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.