Polycomb Group Response Elements Research Articles

Polycomb repressive complex 1 initiates and maintains tailless repression in Drosophila embryo

Maternally-deposited morphogens specify the fates of embryonic cells via hierarchically regulating the expression of zygotic genes that encode various classes of developmental regulators. Once the cell fates are determined, Polycomb-group proteins frequently maintain the repressed state of the genes. This study investigates how Polycomb-group proteins repress the expression of tailless, which encodes a developmental regulator in Drosophila embryo. Previous studies have shown that maternal Tramtrack69 facilitates maternal GAGA-binding factor and Heat shock factor binding to the torso response element (tor-RE) to initiate tailless repression in the stage-4 embryo. Chromatin-immunoprecipitation and genetic-interaction studies exhibit that maternally-deposited Polycomb repressive complex 1 (PRC1) recruited by the tor-RE-associated Tramtrack69 represses tailless expression in the stage-4 embryo. A noncanonical Polycomb-group response element (PRE) is mapped to the tailless proximal region. High levels of Bric-a-brac, Tramtrack, and Broad (BTB)-domain proteins are fundamental for maintaining tailless repression in the stage-8 to -10 embryos. Trmtrack69 sporadically distributes in the linear BTB-domain oligomer, which recruits and retains a high level of PRC1 near the GCCAT cluster for repressing tll expression in the stage-14 embryos. Disrupting the retention of PRC1 decreases the levels of PRC1 and Pleiohomeotic protein substantially on the PRE and causes tailless derepression in the stage-14 embryo. Furthermore, the retained PRC1 potentially serves as a second foundation for assembling the well-characterized polymer of the Sterile alpha motif domain in Polyhomeotic protein, which compacts chromatin to maintain the repressed state of tailless in the embryos after stage 14.

Familial analysis reveals rare risk variants for migraine in regulatory regions

The most recent genome-wide association study of migraine increased the total number of known migraine risk loci to 38. Still, most of the heritability of migraine remains unexplained, and it has been suggested that rare gene dysregulatory variants play an important role in migraine etiology. Addressing the missing heritability of migraine, we aim to fine-map signals from the known migraine risk loci to regulatory mechanisms and associate these to downstream genic targets. We analyzed a large cohort of whole-genome sequenced patients from extended migraine pedigrees (1040 individuals from 155 families). We test for association between rare variants segregating in regulatory regions with migraine. The findings were replicated in an independent case-control cohort (2027 migraineurs, 1650 controls). We report an increased burden of rare variants in one CpG island and three polycomb group response elements near four migraine risk loci. We found that the association is independent of the common risk variants in the loci. The regulatory regions are suggested to affect different genes than those originally tagged by the index SNPs of the migraine loci. Families with familial clustering of migraine have an increased burden of rare variants in regulatory regions near known migraine risk loci, with effects that are independent of the variants in the loci. The possible regulatory targets suggest different genes than those originally tagged by the index SNPs of the migraine loci.

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

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.

Cooperativity, Specificity, and Evolutionary Stability of Polycomb Targeting in Drosophila

Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit PHO and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. Here, we use comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor PHO stabilize PHO recruitment to low-specificity sequences. Consistently, PHO recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila.

Polycomb Domain Formation Depends on Short and Long Distance Regulatory Cues

BackgroundPolycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila, cis-regulatory regions termed PcG response elements (PREs) act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). In addition to an action in cis, PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation in cis, and how long range interactions of PREs in trans affect the chromatin structure is largely unknown.Principal FindingsWe demonstrate that the insertion of a PRE-containing transgene in the Drosophila genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, trans-interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders.ConclusionsOur results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop spreading of H3K27me3.

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 (waplAG) in a screen for mutations that counteract silencing mediated by an engrailed Polycomb-group response element. waplAG 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. waplAG 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 waplAG mutant. Mutations in the cohesin-associated genes Nipped-B and pds5 suppress and enhance waplAG 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 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.

Wapl antagonizes cohesin binding and promotes Polycomb-group silencing inDrosophila

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.

Regulation of the Potential Marker for Intestinal Cells, Bmi1, by β-Catenin and the Zinc Finger Protein KLF4

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

P-Element Homing Is Facilitated by engrailed Polycomb-Group Response Elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5kb deletion of engrailed DNA (enΔ1.5) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the enΔ1. 5 chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing.

Progressive Polycomb Assembly on H3K27me3 Compartments Generates Polycomb Bodies with Developmentally Regulated Motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. Here, we studied the nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH). Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, we could analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing.

The pleiohomeotic functions as a negative regulator of Drosophila even-skipped gene during embryogenesis.

Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs. In this study, we characterized the effects of a pho mutation on embryonic segmentation. pho maternal mutant embryos showed various segmental defects including pair-rule gene mutant patterns. Our results indicated that engrailed and even-skipped genes were misexpressed in pho mutant embryos, which caused embryonic segment defects.

Modulation of the Activity of a Polycomb-Group Response Element in Drosophila by a Mutation in the Transcriptional Activator Woc

Polycomb group response elements (PRE) are cis-regulatory elements that bind Polycomb group proteins. We are studying a 181-bp PRE from the Drosophila engrailed gene. This PRE causes pairing-sensitive silencing of mini-white in transgenes. Here we show that the 181-bp PRE also represses mini-white expression in flies with only one copy of the transgene. To isolate mutations that alter the activity of the 181-bp PRE, we screened for dominant suppressors of PRE-mediated mini-white repression. Dominant suppressors of mini-white repression were rare; we recovered only nine mutations out of 68,274 progeny screened. Two of the nine mutations isolated are due to the same single amino acid change in the transcriptional activator Woc (without children). Reversion experiments show that these are dominant gain-of-function mutations in woc. We suggest that Woc can interfere with the activity of the PRE. Our data have implications for how Polycomb group proteins act to either partially repress or completely silence their target genes.

Spps, aDrosophilaSp1/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.

Enhancer-promoter communication at theDrosophila engrailedlocus

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 ofengrailedtranscription inDrosophila

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.

Heritable chromatin states induced by the Polycomb and trithorax group genes.

In Drosophila the Polycomb group (PcG) and trithorax group (trxG) genes are required to maintain differential expression patterns of many important developmental regulatory genes. The PcG is responsible for heritable silencing throughout development. At target genes PcG response elements (PREs) attract PcG protein complexes and induce the formation of higher-order chromatin structures. We have mapped the distribution of Polycomb and other PcG members at various target genes by using an improved formaldehyde cross-linking and chromatin immunoprecipitation technique. We find that Polycomb spreads locally from PREs over several kilobases, thereby probably stabilizing the silencing complexes. Members of the trxG co-localize at PREs. GAGA factor was found to be constitutively bound to PREs independently of gene activity. PREs associated with active genes appear to have increased amounts of bound GAGA. We have developed a system capable of switching a PRE between the on/off modes. PREs and trxG-regulated elements are common chromosomal elements through which the proteins of the PcG/trxG exert their maintenance function on adjacent chromatin structures.

PRE-Mediated Bypass of Two Su(Hw) Insulators Targets PcG Proteins to a Downstream Promoter

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter.

Chromosomal Distribution of PcG Proteins during Drosophila Development

Polycomb group (PcG) proteins are able to maintain the memory of silent transcriptional states of homeotic genes throughout development. In Drosophila, they form multimeric complexes that bind to specific DNA regulatory elements named PcG response elements (PREs). To date, few PREs have been identified and the chromosomal distribution of PcG proteins during development is unknown. We used chromatin immunoprecipitation (ChIP) with genomic tiling path microarrays to analyze the binding profile of the PcG proteins Polycomb (PC) and Polyhomeotic (PH) across 10 Mb of euchromatin. We also analyzed the distribution of GAGA factor (GAF), a sequence-specific DNA binding protein that is found at most previously identified PREs. Our data show that PC and PH often bind to clustered regions within large loci that encode transcription factors which play multiple roles in developmental patterning and in the regulation of cell proliferation. GAF co-localizes with PC and PH to a limited extent, suggesting that GAF is not a necessary component of chromatin at PREs. Finally, the chromosome-association profile of PC and PH changes during development, suggesting that the function of these proteins in the regulation of some of their target genes might be more dynamic than previously anticipated.