Dynamic Competition of Polycomb and Trithorax in Transcriptional Programming.

Predicting regulatory potential from primary DNA sequences or transcription factor binding patterns is not possible. However, the annotation of the genome by chromatin proteins, histone modifications, and differential compaction is largely sufficient to reveal the locations of genes and their differential activity states. The Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are the central players in this cell type-specific chromatin organization. PcG function was originally viewed as being solely repressive and irreversible, as observed at the homeotic loci in flies and mammals. However, it is now clear that modular and reversible PcG function is essential at most developmental genes. Focusing mainly on recent advances, we review evidence for how PcG and TrxG patterns change dynamically during cell type transitions. The ability to implement cell type-specific transcriptional programming with exquisite fidelity is essential for normal development.

The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation.

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal discs; in contrast, small IDE-reporter transgenes are expressed mainly in the anterior compartment. We found that En binds to the IDEs and suggest that it may directly repress IDE function and modulate En expression levels. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from an endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data suggest that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic memory set by the embryonic enhancers. This epigenetic regulation is similar to that of the Ultrabithorax IDEs and we suggest that the activity of late-acting enhancers in other genes may be similarly regulated.

Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila.

The Polycomb group (PcG) proteins are fundamental epigenetic regulators that control the repressive state of target genes in multicellular organisms. One of the open questions is defining the mechanisms of PcG recruitment to chromatin. In Drosophila, the crucial role in PcG recruitment is thought to belong to DNA-binding proteins associated with Polycomb response elements (PREs). However, current data suggests that not all PRE-binding factors have been identified. Here, we report the identification of the transcription factor Crooked legs (Crol) as a novel PcG recruiter. Crol is a C2H2-type Zinc Finger protein that directly binds to poly(G)-rich DNA sequences. Mutation of Crol binding sites as well as crol CRISPR/Cas9 knockout diminish the repressive activity of PREs in transgenes. Like other PRE-DNA binding proteins, Crol co-localizes with PcG proteins inside and outside of H3K27me3 domains. Crol knockout impairs the recruitment of the PRC1 subunit Polyhomeotic and the PRE-binding protein Combgap at a subset of sites. The decreased binding of PcG proteins is accompanied by dysregulated transcription of target genes. Overall, our study identified Crol as a new important player in PcG recruitment and epigenetic regulation.

Epigenetic Regulation of Tick Biology and Vectorial Capacity.

Ticks exist across diverse environments and transmit numerous pathogens. Due to their long and unique life cycles, these arthropods likely evolved robust epigenetic mechanisms that provide sustainable responses and buffers against extreme environmental conditions. Herein, we highlight how the study of the epigenetic basis of tick biology and vectorial capacity will enrich our knowledge of tick-borne infections.

Global changes of H3K27me3 domains and Polycomb group protein distribution in the absence of recruiters Spps or Pho.

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes in animals, but how these proteins are recruited to specific regions of the genome is still poorly understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) that include combinations of sites for sequence specific DNA binding "PcG recruiters," including Pho, Cg, and Spps. To understand their roles in PcG recruitment, we compared Pho-, Cg-, and Spps-binding sites against H3K27me3 and key PcG proteins by ChIP-seq in wild-type and mutant third instar larvae. H3K27me3 in canonical Polycomb domains is decreased after the reduction of any recruiter. Reduction of Spps and Pho, but not Cg, causes the redistribution of H3K27me3 to heterochromatin. Regions with dramatically depleted H3K27me3 after Spps knockout are usually accompanied by decreased Pho binding, suggesting their cooperative binding. PcG recruiters, the PRC2 component E(z), and the PRC1 components Psc and Ph cobind thousands of active genes outside of H3K27me3 domains. This study demonstrates the importance of distinct PcG recruiters for the establishment of unique Polycomb domains. Different PcG recruiters can act both cooperatively and independently at specific PcG target genes, highlighting the complexity and diversity of PcG recruitment mechanisms.

Formation of a Polycomb-Domain in the Absence of Strong Polycomb Response Elements.

Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. Our data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain.

Combgap contributes to recruitment of Polycomb group proteins in Drosophila.

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.

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.

Wapl antagonizes cohesin binding and promotes Polycomb-group silencing in Drosophila.

Wapl protein regulates binding of the cohesin complex to chromosomes during interphase and helps remove cohesin from chromosomes at mitosis. We isolated a dominant mutation in wapl (wapl(AG)) in a screen for mutations that counteract silencing mediated by an engrailed Polycomb-group response element. wapl(AG) hemizygotes die as pharate adults and have an extra sex combs phenotype characteristic of males with mutations in Polycomb-group (PcG) genes. The wapl gene encodes two proteins, a long form and a short form. wapl(AG) introduces a stop codon at amino acid 271 of the long form and produces a truncated protein. The expression of a transgene encoding the truncated Wapl-AG protein causes an extra-sex-comb phenotype similar to that seen in the wapl(AG) mutant. Mutations in the cohesin-associated genes Nipped-B and pds5 suppress and enhance wapl(AG) phenotypes, respectively. A Pds5-Wapl complex (releasin) removes cohesin from DNA, while Nipped-B loads cohesin. This suggests that Wapl-AG might exert its effects through changes in cohesin binding. Consistent with this model, Wapl-AG was found to increase the stability of cohesin binding to polytene chromosomes. Our data suggest that increasing cohesin stability interferes with PcG silencing at genes that are co-regulated by cohesin and PcG proteins.

Polycomb protein binding and looping in the ON transcriptional state.

Polycomb group (PcG) proteins mediate epigenetic silencing of important developmental genes by modifying histones and compacting chromatin through two major protein complexes, PRC1 and PRC2. These complexes are recruited to DNA by CpG islands (CGIs) in mammals and Polycomb response elements (PREs) in Drosophila. When PcG target genes are turned OFF, PcG proteins bind to PREs or CGIs, and PREs serve as anchors that loop together and stabilize gene silencing. Here, we address which PcG proteins bind to PREs and whether PREs mediate looping when their targets are in the ON transcriptional state. While the binding of most PcG proteins decreases at PREs in the ON state, one PRC1 component, Ph, remains bound. Further, PREs can loop to each other and with presumptive enhancers in the ON state and, like CGIs, may act as tethering elements between promoters and enhancers. Overall, our data suggest that PREs are important looping elements for developmental loci in both the ON and OFF states.

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.

The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation.

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. Our data show that the En protein binds to en IDEs, and we suggest that En directly represses IDE function. We identified two en IDEs, 'O' and 'S'. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin.

Context-dependent role of Pho binding sites in Polycomb complex recruitment in Drosophila.

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, "PcG recruiters," including Pho, Spps, Cg, and GAF. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here, we directly addressed the importance of Pho binding sites in 2 engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, 2 PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho. This supports the view that multiple mechanisms contribute to PcG recruitment in Drosophila.

Polycomb protein binding and looping mediated by Polycomb Response Elements in the ON transcriptional state.

Polycomb group proteins (PcG) mediate epigenetic silencing of important developmental genes and other targets. In Drosophila, canonical PcG-target genes contain Polycomb Response Elements (PREs) that recruit PcG protein complexes including PRC2 that trimethylates H3K27 forming large H3K27me3 domains. In the OFF transcriptional state, PREs loop with each other and this looping strengthens silencing. Here we address the question of what PcG proteins bind to PREs when canonical PcG target genes are expressed, and whether PREs loop when these genes are ON. Our data show that the answer to this question is PRE-specific but general conclusions can be made. First, within a PcG-target gene, some regulatory DNA can remain covered with H3K27me3 and PcG proteins remain bound to PREs in these regions. Second, when PREs are within H3K27ac domains, PcG-binding decreases, however, this depends on the protein and PRE. The DNA binding protein GAF, and the PcG protein Ph remain at PREs even when other PcG proteins are greatly depleted. In the ON state, PREs can still loop with each other, but also form loops with presumptive enhancers. These data support the model that, in addition to their role in PcG silencing, PREs can act as "promoter-tethering elements" mediating interactions between promoter proximal PREs and distant enhancers.

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.

Decreasing Wapl dosage partially corrects embryonic growth and brain transcriptome phenotypes in Nipbl+/- embryos.

Cohesin rings interact with DNA and modulate the expression of thousands of genes. NIPBL loads cohesin onto chromosomes, and WAPL takes it off. Haploinsufficiency for NIPBL causes a developmental disorder, Cornelia de Lange syndrome (CdLS), that is modeled by Nipbl+/- mice. Mutations in WAPL have not been shown to cause disease or gene expression changes in mammals. Here, we show dysregulation of >1000 genes in WaplΔ/+ embryonic mouse brain. The patterns of dysregulation are highly similar in Wapl and Nipbl heterozygotes, suggesting that Wapl mutations may also cause human disease. Since WAPL and NIPBL have opposite effects on cohesin's association with DNA, we asked whether decreasing Wapl dosage could correct phenotypes seen in Nipbl+/- mice. Gene expression and embryonic growth are partially corrected, but perinatal lethality is not. Our data are consistent with the view that cohesin dynamics play a key role in regulating gene expression.

Polycomb response elements and targeting of Polycomb group proteins in Drosophila.

Polycomb group (PcG) proteins are conserved regulatory proteins that repress transcription of particular target genes in animals and plants. Studies over the past decade have established that most PcG proteins are not classic DNA binding factors but that they exist in multisubunit protein complexes that bind to and modify chromatin. Nevertheless, PcG repression of target genes in Drosophila requires specific cis-regulatory sequences, called Polycomb response elements (PREs), and chromatin immunoprecipitation studies have shown that, in vivo, most PcG proteins are specifically bound at the PREs of target genes. However, the mechanisms by which these PcG protein complexes are recruited to PREs and how they repress transcription are still poorly understood. Recent studies challenge earlier models that invoke covalent histone modifications and chromatin binding as the key steps in the recruitment of PcG proteins to PREs. The available evidence suggests that PREs are largely devoid of nucleosomes and that PRE DNA serves as an assembly platform for many different PcG protein complexes through DNA-protein and protein-protein interactions. The emerging picture suggests that the binding and modification of chromatin by PcG proteins is needed for interaction of PRE-tethered PcG protein complexes with nucleosomes in the flanking chromatin in order to maintain a Polycomb-repressed chromatin state at promoters and coding regions of target genes.

Defining the Boundaries of Polycomb Domains in Drosophila.

Polycomb group (PcG) proteins are an important group of transcriptional repressors that act by modifying chromatin. PcG target genes are covered by the repressive chromatin mark H3K27me3. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is responsible for generating H3K27me3. In Drosophila, PRC2 is recruited by Polycomb Response Elements (PREs) and then trimethylates flanking nucleosomes, spreading the H3K27me3 mark over large regions of the genome, the "Polycomb domains." What defines the boundary of a Polycomb domain? There is experimental evidence that insulators, PolII, and active transcription can all form the boundaries of Polycomb domains. Here we divide the boundaries of larval Polycomb domains into six different categories. In one category, genes are transcribed toward the Polycomb domain, where active transcription is thought to stop the spreading of H3K27me3. In agreement with this, we show that introducing a transcriptional terminator into such a transcription unit causes an extension of the Polycomb domain. Additional data suggest that active transcription of a boundary gene may restrict the range of enhancer activity of a Polycomb-regulated gene.

Homolog Pairing at the Push of a Button.

Homologous chromosomes pair in somatic cells in Drosophila, but how this occurs is poorly understood. In this issue of Developmental Cell, Viets et al. (2019) show that proteins and chromatin structure mediate pairing and argue against a DNA sequence-based mechanism.