Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Apparent RNA bridging between PRC2 and chromatin is an artifact of non-specific chromatin precipitation upon RNA degradation

ABSTRACT: Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain repression of genes specific for other cell lineages and is essential for development. In vitro, RNA inhibits PRC2 catalytic activity and interaction with nucleosomes but the effect of RNA on PRC2 in cells has been unclear, with studies concluding that RNA either antagonises or promotes the association of PRC2 with chromatin. Suggesting the existence of an RNA bridge that connects PRC2 to chromatin, treatment of formaldehyde-crosslinked, sonicated nuclear extracts with RNase A reduces co-precipitation of polycomb target sites with PRC2. Here, we show that this apparent loss of PRC2 chromatin association after RNase A treatment is due to non-specific chromatin precipitation. RNA degradation causes chromatin to precipitate out of solution, thereby reducing the enrichment of specific DNA sequences in chromatin immunoprecipitation reactions. Maintaining chromatin solubility by the addition of poly-L-glutamic acid (PGA) rescues detection of PRC2 chromatin association upon RNA degradation. These findings undermine support for the model that RNA bridges PRC2 and chromatin in cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE240380 | GEO | 2024/02/22

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion

Project description:RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favour and against this idea. RNase treatment of chromatin is a prevalent tool for the study of RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. One of the most studied chromatin modifiers in the context of RNA-mediated regulation is the H3K27me3 methyltransferase Polycomb Repressive Complex 2 (PRC2). RNase A treatment during chromatin immunoprecipitation (rChIP) reduces the occupancy of PRC2 on chromatin. This led to suggestions of an “RNA bridge" between PRC2 and chromatin. Here we show that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including both PRC2 and its H3K27me3 mark genome-wide. This phenomenon persists in mouse embryonic stem cells, human cancer cells and human-induced pluripotent stem cells. We track this observation to a global gain of chromatin that artificially reduces ChIP signals from facultative heterochromatin. Our results point to substantial limitations in RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

2024-02-21 | GSE240079 | GEO

Mapping RNA interactions of human ATRX with RepA fragment of Xist

Project description:Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.

2020-03-09 | PXD017806 | Pride

Chip-chip from WT and Polycomb Component Kock Out Mouse ES cells for H2AZ, H3K27me3, EZH2 and Ring1B.

Project description:The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner. Array design includes 2 biological replicates for all samples and technical replication (dye swaps for H3K27me3_WT_ES, EZH2_WT_ES, EZH2_RING1b_KO_ES, H2AZ_WT_ES(2), H2AZ_Eed_KO_ES and H2AZ_Suz12_KO_ES). Ring1B_WT_ES is represented by a single replicate.

2012-04-15 | E-GEOD-36999 | biostudies-arrayexpress

The interaction of PRC2 with RNA or chromatin is mutually antagonistic

Project description:Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3’UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

2016-05-06 | GSE66831 | GEO

Identification of JARID2/PRC2 interactome in human embryonic stem cells

Project description:JARID2 is an integral subunit of the chromatin modifier PRC2 (Polycomb Repressive Complex 2). PRC2 methylates lysine 27 in histone H3 and is required for human development. Mutaitons of PRC2 subunits have been frequently found in a variety of adult and pediatric cancer, suggesting that PRC2 critically regulates normal and cancer development. The goal of this project is to identify new regulator or co-factors of PRC2 and study their molecular interactions and activities in order to better understand function and regulation of PRC2 in normal and cancer development.

2020-08-04 | PXD015670 | Pride

VAL1 acts as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC

Project description:Polycomb (PcG) silencing is crucial for development, but how targets are specified remains incompletely understood. The cold-induced Polycomb Repressive Complex 2 (PRC2) silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate PcG regulation. Association of the DNA binding protein VAL1 to the PcG nucleationregion at FLC is an important step. VAL1 interacts with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and PRC1. Here, we show that ASAP and PRC1 are necessary for co-transcriptional repression and chromatin regulation during FLC silencing. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulates at FLC nucleation region during cold, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2Aub thus marks the transition to epigenetic silencing, while H3K27me3 is necessary for long-term epigenetic memory. Overall, our work highlights the importance of VAL1 as an assembly platform co-ordinating the co-transcriptional repression and chromatin regulation necessary for the epigenetic silencing of FLC.

2022-08-15 | PXD036057 |

Chip-chip from WT and Polycomb Component Knock Out Mouse ES cells for H2AZ, H3K27me3, EZH2 and Ring1B.

2012-04-16 | GSE36999 | GEO

Dynamic protein interactions of the Polycomb Repressive Complex 2

Project description:Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12 and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency dataset reflecting the endogenous state of PRC2 was produced that included all previously reported ‘core’ and ‘associated’ PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that favoured either one or other state. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, while PCL1 and SMAD3 more associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors found that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.

2016-09-27 | PXD004462 | Pride

Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation

Project description:The Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin in order to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb Repressive Complex 2 (PRC2) component PHF19 binds the active chromatin mark H3K36me3 via its tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and is required to recruit the PRC2 complex and NO66 to stem cells genes during differentiation, leading to PRC2 mediated H3K27 tri-methylation, loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during ES cell differentiation to transiently bind the H3K36me3 mark via its tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state. Examination of PHF19 genome-wide binding in mouse embryonic stem cells

2012-11-17 | E-GEOD-41609 | biostudies-arrayexpress

The role of early chromatin changes in X chromosome inactivation

Project description:During development, transcriptional and chromatin modification changes co-occur but the order and causality of events often remain unclear. We explore the interrelationship of these processes using the paradigm of X-chromosome inactivation (XCI). We initiate XCI in female, mouse embryonic stem cells by inducing Xist expression and monitor changes in transcription and chromatin by allele-specific TT-seq and ChIP-seq respectively. An unprecedented temporal resolution enabled identification of the earliest chromatin alterations during XCI. We demonstrate that HDAC3 interacts with both NCOR1 and NCOR2 and is pre-bound on the X chromosome where it deacetylates histones to promote efficient gene silencing. We also reveal the choreography of polycomb accumulation following Xist RNA coating, with PRC1-associated H2AK119Ub preceding PRC2-associated H3K27me3. Furthermore, polycomb-associated marks accumulate initially at large, intergenic domains and then spreads into genes but only in the context of gene silencing. Our results provide the hierarchy of chromatin events during XCI and demonstrate that some chromatin changes play key roles in mediating transcriptional silencing.

2019-01-17 | PXD011344 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data