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Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila (ChIP-seq)

ABSTRACT: 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 H2K27me3 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE202870 | GEO | 2023/04/19

REPOSITORIES: GEO

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Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila (RNA-seq)

Project description: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 H2K27me3 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.

2023-04-19 | GSE202832 | GEO

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

Project description: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, GAF and many others. 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 two 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, two 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.

2023-05-08 | GSE227553 | GEO

In embryo analysis of H3K27 trimethylation suggests a two-step process involving PRC2 interacting sequences and high GC content

Project description:ABSTRACT Polycomb group (PcG) proteins are essential for the repression of key developmental regulators during development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined DNA sequences termed polycomb response elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to try and map DNA sequences of importance for the proper recruitment of polycomb proteins within the genetically well-characterized HoxD genomic locus. Here, we report that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. These elements synergize, when clustered together, to form a fully functional repressive domain. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters, in embryonic stem cells, for their subsequent coordinated transcriptional activation during development. ChIP-chip using K27 antibodies

2015-01-27 | E-GEOD-43915 | biostudies-arrayexpress

High-resolution mapping defines the cooperative architecture of Polycomb Response Elements

Project description:Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb Response Elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of non-histone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal cooperative interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor Adf1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished at multiple steps in transcription, including inactivation of distant enhancers, enhanced Polycomb recruitment to PREs and target promoters, and elevated stalling of RNAPII in repressed genes. These results suggest that the stability of complexes bridging promoters, enhancers, and PREs is a crucial aspect of developmentally regulated gene expression. Native chromatin immunoprecipitation of histones, transcription factors and Polycomb protein in Drosophila cell lines.

2014-02-08 | E-GEOD-47829 | biostudies-arrayexpress

Global changes of H3K27me3 domains and PcG protein distribution in the absence of the PcG recruiters Spps or Pho

Project description: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.

2018-03-07 | GSE102339 | GEO

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)

2011-08-15 | E-GEOD-22623 | biostudies-arrayexpress

High-resolution mapping defines the cooperative architecture of Polycomb Response Elements

2014-02-08 | GSE47829 | GEO

In embryo analysis of H3K27 trimethylation suggests a two-step process involving PRC2 interacting sequences and high GC content

2015-01-27 | GSE43915 | GEO

ChIP-Seq with wild type mESC and NPC

Project description:Master regulatory genes require stable silencing by the Polycomb-Group (PcG) to prevent improper expression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in mouse ES cells and neural progenitors using high-resolution 5C technology and super-resolution microscopy. The genomic loci of repressed PcG target genes formed discrete, small domains of tight interaction that corresponded to locations bound by canonical Polycomb Repressive Complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1, and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in numerous aspectsá. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher order structure.

2017-02-03 | GSE89929 | GEO

RNA-Seq with wild type mESC, wild type NPC and Phc1 knock-out ESC

Project description:Master regulatory genes require stable silencing by the Polycomb-Group (PcG) to prevent improper expression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in mouse ES cells and neural progenitors using high-resolution 5C technology and super-resolution microscopy. The genomic loci of repressed PcG target genes formed discrete, small domains of tight interaction that corresponded to locations bound by canonical Polycomb Repressive Complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1, and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in numerous aspects . These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher order structure.

2017-02-03 | GSE89945 | GEO

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