RESUMEN
Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.
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Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.
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Proteínas de Drosophila , Complejo Represivo Polycomb 1 , Animales , Sistema Nervioso Central/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Larva/metabolismo , Larva/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT.
Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , Animales , Sistemas CRISPR-Cas , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas del Grupo Polycomb/genéticaRESUMEN
Polycomb (PcG) and Trithorax (TrxG) group proteins are evolutionarily conserved chromatin-modifying factors originally identified as part of an epigenetic cellular memory system that maintains repressed or active gene expression states. Recently, they have been shown to globally control a plethora of cellular processes. This functional diversity is achieved by their ability to regulate chromatin at multiple levels, ranging from modifying local chromatin structure to orchestrating the three-dimensional organization of the genome. Understanding this system is a fascinating challenge of critical relevance for biology and medicine, since misexpression or mutation of multiple PcG components, as well as of TrxG members of the COMPASS family and of the SWI/SNF complex, is implicated in cancer and other diseases.
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Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo Polycomb/metabolismo , Animales , Proteínas Cromosómicas no Histona/historia , Células Madre Embrionarias/metabolismo , Genoma , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Neoplasias/metabolismo , Proteínas del Grupo Polycomb/historiaRESUMEN
Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.
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Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
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Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Genes de Insecto , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas del Grupo Polycomb/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental regulatory genes. PcG proteins are recruited to specific regulatory elements to modify the chromatin surrounding them. In addition, they regulate the organization of their target genes in the 3D space of the nucleus, and this regulatory function of the 3D genome architecture is involved in cell differentiation and the maintenance of cellular memory. In this review we discuss recent advances in our understanding of how PcG proteins are recruited to chromatin to induce local and global changes in chromosome conformation and regulate their target genes.
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Genoma , Proteínas del Grupo Polycomb/metabolismo , Animales , Cromatina/metabolismo , Epigénesis Genética , Humanos , Modelos Biológicos , Proteínas del Grupo Polycomb/químicaRESUMEN
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.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Polycomb 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 FINDINGS: We 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. CONCLUSIONS: Our 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.
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Cromatina/genética , Histona Demetilasas/genética , Proteínas del Grupo Polycomb/genética , Animales , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína/genética , Elementos de Respuesta/genéticaRESUMEN
The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.
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Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Proteínas de Drosophila , Neurogénesis/genética , Complejo Represivo Polycomb 1 , Factores de Transcripción , Animales , Diferenciación Celular , Linaje de la Célula/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.
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Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética , Animales , ADN/metabolismo , Daño del ADN , Humanos , Unión Proteica , Transducción de SeñalRESUMEN
In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.
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Drosophila/genética , Drosophila/metabolismo , Genes Homeobox , Proteínas Represoras/metabolismo , Animales , Proteína con Homeodominio Antennapedia/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Silenciador del Gen , Proteínas del Grupo Polycomb , Elementos Reguladores de la TranscripciónRESUMEN
Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology.
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Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Elementos Aisladores/fisiología , Elementos de Respuesta/fisiología , Animales , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , Complejo Represivo Polycomb 1RESUMEN
Many Polycomb group (PcG) proteins assemble into complexes containing histone-modifying enzymes that act in concert to control developmental regulators. In a recent study in Nature, Scheuermann et al. report the identification of a PcG complex with histone H2A-specific deubiquitinase activity that may be a key player in PcG-target gene regulation.
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Polycomb group (PcG) protein complexes dynamically define cellular identity through the regulation of key developmental genes. Important advances in the PcG field have come from genome-wide mapping studies in a variety of tissues and cell types that have analyzed PcG protein complexes, their associated histone marks and putative mechanisms of PcG protein recruitment. We review how these analyses have contributed to our understanding of PcG protein complex targeting to chromatin and consider the importance of diverse PcG protein complex composition for gene regulation. Finally, we focus on the dynamics of PcG protein complex action during cell fate transitions and on the implications of histone modifications for cell lineage commitment.
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Diferenciación Celular , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Humanos , Proteínas del Grupo PolycombRESUMEN
Polycomb Group (PcG) proteins silence critical developmental genes and modulate cell proliferation. Using the Drosophila melanogaster eye as a model system, we show that cells with mutations in the gene locus (ph) that encodes the PcG protein Polyhomeotic (PH) overproliferate and lose both the ability to differentiate and their normal polarity. They invade the neighboring tissues and, when combined with an activated form of the Ras proto-oncogene, they trigger the formation of metastases. PcG proteins bind to multiple genes in the Notch pathway and control their transcription as well as Notch signaling. The massive cell-autonomous overproliferation of ph mutant cell clones can be rescued by ectopic expression of a dominant negative form of Notch or by RNA interference (RNAi)-mediated repression of Notch. Conversely, overexpression of ph induces a small-eye phenotype that is rescued by activation of Notch signaling. These data show that ph is a tumor suppressor locus that controls cellular proliferation by silencing multiple Notch signaling components.
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Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nucleoproteínas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Polaridad Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ojo/citología , Ojo/metabolismo , Genes Supresores de Tumor , Microscopía Electrónica de Rastreo , Mutación , Nucleoproteínas/genética , Complejo Represivo Polycomb 1 , Interferencia de ARN , Receptores Notch/genéticaRESUMEN
Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.
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Cromatina/fisiología , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Animales , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica/fisiología , Complejo Represivo Polycomb 1 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Metilación de ADN , Drosophila/genética , Drosophila/metabolismo , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Polycomb group (PcG) and trithorax group (trxG) proteins are critical regulators of numerous developmental genes. To silence or activate gene expression, respectively, PcG and trxG proteins bind to specific regions of DNA and direct the posttranslational modification of histones. Recent work suggests that PcG proteins regulate the nuclear organization of their target genes and that PcG-mediated gene silencing involves noncoding RNAs and the RNAi machinery.
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Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica/genética , Interferencia de ARN/fisiología , Proteínas Represoras/genética , Animales , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Evolución Molecular , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas Represoras/metabolismoRESUMEN
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.