Judith A. Kassis

The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs but pho mutants show only mild phenotypes compared with other PcG mutants. We characterize pho-like, a gene encoding a protein with high similarity to Pho. Pho-like binds to Pho-binding sites in vitro and pho-like, pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. We examined the distribution of five PcG proteins on polytene chromosomes from pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, does not cause misexpression of homeotic genes or reporter genes in imaginal disks.

Polycomb Group Response Elements in Drosophila and Vertebrates

Polycomb group genes (PcG) encode a group of about 16 proteins that were first identified in Drosophila as repressors of homeotic genes. PcG proteins are present in all metazoans and are best characterized as transcriptional repressors. In Drosophila, these proteins are known as epigenetic regulators because they remember, but do not establish, the patterned expression state of homeotic genes throughout development. PcG proteins, in general, are not DNA binding proteins, but act in protein complexes to repress transcription at specific target genes. How are PcG proteins recruited to the DNA? In Drosophila, there are specific regulatory DNA elements called Polycomb group response elements (PREs) that bring PcG protein complexes to the DNA. Drosophila PREs are made up of binding sites for a complex array of DNA binding proteins. Functional PRE assays in transgenes have shown that PREs act in the context of other regulatory DNA and PRE activity is highly dependent on genomic context. Drosophila PREs tend to regulate genes with a complex array of regulatory DNA in a cell or tissue-specific fashion and it is the interplay between regulatory DNA that dictates PRE function. In mammals, PcG proteins are more diverse and there are multiple ways to recruit PcG complexes, including RNA-mediated recruitment. In this review, we discuss evidence for PREs in vertebrates and explore similarities and differences between Drosophila and vertebrate PREs.

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.

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-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.

Spps, a Drosophila Sp1/KLF family member, binds to PREs and is required for PRE activity late in development.

The Polycomb group of proteins (PcG) is important for transcriptional repression and silencing in all higher eukaryotes. In Drosophila, PcG proteins are recruited to the DNA by Polycomb-group response elements (PREs), regulatory sequences whose activity depends on the binding of many different sequence-specific DNA-binding proteins. We previously showed that a binding site for the Sp1/KLF family of zinc-finger proteins is required for PRE activity. Here, we report that the Sp1/KLF family member Spps binds specifically to Ubx and engrailed PREs, and that Spps binds to polytene chromosomes in a pattern virtually identical to that of the PcG protein, Psc. A deletion of the Spps gene causes lethality late in development and a loss in pairing-sensitive silencing, an activity associated with PREs. Finally, the Spps mutation enhances the phenotype of pho mutants. We suggest that Spps may work with, or in parallel to, Pho to recruit PcG protein complexes to PREs.

Characterization of the polycomb group response elements of the Drosophila melanogaster invected Locus.

ABSTRACT The Polycomb group proteins (PcGs) play a vital role throughout development by maintaining precise gene expression patterns. In Drosophila melanogaster, PcG-mediated gene silencing is achieved through DNA elements called Polycomb response elements (PREs); however, the mechanism for establishing silencing and the requirements and composition of a working PRE are not fully understood. We have used the computer program jPREdictor to uncover PREs located within the invected (inv) locus. The functionalities of these predicted PREs were tested in two different assays: one analyzing their abilities to maintain expression of a β-galactosidase reporter gene and the other evaluating their abilities to establish pairing-sensitive silencing of the mini-white reporter in the vector pCaSpeR. We have identified two previously uncharacterized PREs at the inv gene and demonstrate that they produce similar results in the two assays. Our results indicate that clusters of protein binding sites do not accurately predict PREs and provide new insight into the DNA sequence requirements for the binding of the PcG protein Pho. Finally, our data show that PREs and regulatory DNA from different genes can function together to establish PcG-mediated silencing, highlighting the versatility of PREs despite discrepancies in the number and location of DNA binding sites.

Architectural and functional diversity of polycomb group response elements in Drosophila.

Polycomb group response elements (PREs) play an essential role in gene regulation by the Polycomb group (PcG) repressor proteins in Drosophila. PREs are required for the recruitment and maintenance of repression by the PcG proteins. PREs are made up of binding sites for multiple DNA-binding proteins, but it is still unclear what combination(s) of binding sites is required for PRE activity. Here we compare the binding sites and activities of two closely linked yet separable PREs of the Drosophila engrailed (en) gene, PRE1 and PRE2. Both PRE1 and PRE2 contain binding sites for multiple PRE–DNA-binding proteins, but the number, arrangement, and spacing of the sites differs between the two PREs. These differences have functional consequences. Both PRE1 and PRE2 mediate pairing-sensitive silencing of mini-white, a functional assay for PcG repression; however, PRE1 requires two binding sites for Pleiohomeotic (Pho), whereas PRE2 requires only one Pho-binding site for this activity. Furthermore, for full pairing-sensitive silencing activity, PRE1 requires an AT-rich region not found in PRE2. These two PREs behave differently in a PRE embryonic and larval reporter construct inserted at an identical location in the genome. Our data illustrate the diversity of architecture and function of PREs.

Combgap contributes to recruitment of Polycomb group proteins in Drosophila

Significance The ability of organisms to regulate gene expression spatially and temporally is a crucial aspect of development and differentiation. Polycomb group proteins (PcG) are a group of transcriptional repressors that mediate silencing of developmental genes in places where they should not be expressed. Mutations in PcG proteins have been implicated in cancer. We aim to understand mechanisms of PcG-mediated repression, and in this study we report the involvement of Combgap, a DNA-binding protein, in PcG recruitment. Combgap binds to GTGT motifs, which are present within the regulatory regions of PcG target genes. Genome-wide analyses suggest that Combgap may directly recruit Polyhomeotic, a PcG protein. Overall, our data help provide a mechanism for PcG recruitment to target genes. Polycomb group (PcG) proteins are responsible for maintaining the silenced transcriptional state of many developmentally regulated genes. PcG proteins are organized into multiprotein complexes that are recruited to DNA via cis-acting elements known as “Polycomb response elements” (PREs). In Drosophila, PREs consist of binding sites for many different DNA-binding proteins, some known and others unknown. Identification of these DNA-binding proteins is crucial to understanding the mechanism of PcG recruitment to PREs. We report here the identification of Combgap (Cg), a sequence-specific DNA-binding protein that is involved in recruitment of PcG proteins. Cg can bind directly to PREs via GTGT motifs and colocalizes with the PcG proteins Pleiohomeotic (Pho) and Polyhomeotic (Ph) at the majority of PREs in the genome. In addition, Cg colocalizes with Ph at a number of targets independent of Pho. Loss of Cg leads to decreased recruitment of Ph at only a subset of sites; some of these sites are binding sites for other Polycomb repressive complex 1 (PRC1) components, others are not. Our data suggest that Cg can recruit Ph in the absence of PRC1 and illustrate the diversity and redundancy of PcG protein recruitment mechanisms.

Polycomb group proteins bind an engrailed PRE in both the "ON" and "OFF" transcriptional states of engrailed.

Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the “OFF” and “ON” transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is “OFF”. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the “ON” transcriptional state. We tested these two models at the PcG target gene engrailed. engrailed exists in a gene complex with invected, which together have 4 well-characterized PREs. Our data show that these PREs are not transcribed in embryos or larvae. We also examined whether PcG proteins are bound to an engrailed PRE in cells where engrailed is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where engrailed is “ON” or “OFF”, we determined that components of three major PcG protein complexes are present at an engrailed PRE in both the “ON” and “OFF” transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of engrailed.