Activation of automethylated PRC2 by dimerization on chromatin
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA983242 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24906027.fastq.gz | Fastqsanger.gz | |||
| SRR24906028.fastq.gz | Fastqsanger.gz | |||
| SRR24906029.fastq.gz | Fastqsanger.gz | |||
| SRR24906030.fastq.gz | Fastqsanger.gz | |||
| SRR24906031.fastq.gz | Fastqsanger.gz |
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Project description:Polycomb Repressive Complex 2 (PRC2) trimethylates lysine 27 of histone 3 (H3K27me3). This modification transcriptionally represses chromatin and its dynamics are essential for embryonic development. PRC2 also methylates its cofactor JARID2 and both methylated H3 tail and JARID2 allosterically activate the complex. PRC2 undergoes auto-methylation of its catalytic subunit, which stimulates its activity by an unknown mechanism. We show that automethylated PRC2 forms a dimer on substrate chromatin that results in autoactivation in trans. An inactive PRC2 protomer distal to the substrate nucleosome serves as an allosteric activator of the second PRC2 protomer, which is poised to methylate H3. Functional assays support our model of allosteric activation via dimerization, suggesting a novel mechanism of PRC2 trans-autoactivation that may be important for installation of H3K27me3 into naïve chromatin.
2023-11-13 | GSE234793 | GEO
Project description:Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit EZH2 stimulates its activity by an unknown mechanism. Here, we show that PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a novel mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification coupled dimerization in the regulation of chromatin modifying complexes.
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Project description:We report the high-throughput profiling of PRC2 core subunits chromatin states and histone modifications in two prostate cancer cell lines, androgen-dependent cells LNCaP and androgen-independent cells LNCaP-abl (abl). By obtaining over 1 billion bases of sequence from chromatin immunoprecipitated DNA, we generated the genome-wide localizations of EZH2, SUZ12 and H3K27 trimethylation in both cell lines, and found that EZH2 can be localized to a subsets of chromating sites that don't have H3K27me3 or SUZ12 signals nearby. Interestingly, these sites are enriched for the active histone marks H3K4 di/trimethylation as well as the RNA polymerase II, which suggest the potential function of these peaks in gene activation. Indeed, these sites are enriched near the transcription start sites of EZH2-activated genes. Further analysis of the transcription factor motifs enriched at these peaks revealed the enrichement of androgen receptor motif, suggesting a co-activator role for EZH2 in concert with AR. Our work demonstrated a novel funtion of EZH2 in transcriptional activation by directly binding to the chromatin sites that cooperate with AR. Study of the chromatin localizations of PRC2 complex core subunits and different histone marks in 2 cell types
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Project description:Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, LDB1 is recruited to the β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2 is sufficient, to completely restore LCR-promoter looping and transcription in LDB1 depleted cells. The looping function of the DD is unique and irreplaceable by heterologous dimerization domains. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the co-regulators FOG1 and NuRD complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1in these processes. RNA-seq in LDB1 knockdown, LDB1 delta4/5 construct, LDB1 full-length construct, and control in induced MEL cells; three replicates each.
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Project description:Preventing inappropriate gene expression in time and space is as fundamental as triggering the activation of tissue/cell-type specific factors at the correct developmental stage and in the correct cells. Here, we study the impact of Polycomb Repressive Complex 2 (PRC2) function on HoxA gene regulation. We analyze chromatin conformation of the HoxA cluster and its regulatory regions and show that in addition to the well-known role of PRC2 in silencing Hox genes via direct binding, its function is required for the changes in HoxA long-range interactions distinguishing proximal limb from distal limb. This effect stems from the differential PRC2 occupancy over the HoxA cluster and, at least in part, from the ability of PRC2-bound loci to engage in long-range contacts. Unexpectedly, PRC2 also impacts chromatin conformation in a way that promotes enhancer-promoter contacts required for proper HoxA expression, pointing to a dual role of PRC2 in gene regulation.
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Project description:In this study, we demonstrate that endosome-localized self RNA Rmrp directly binds to TLR3 and induces TLR3 dimerization in the early endosome but does not interact with endosome-localized TLR7, TLR8, TLR9 or cytoplasmic RNA sensor RIG-I under homeostatic conditions. Cryo-EM structure of Rmrp-TLR3 complex reveals a novel lapped conformation of TLR3 dimer engaged by Rmrp, which is distinct from the activation mechanism by dsRNA and the specific structural feature at the 3'-end of Rmrp is critical for its functional interaction with TLR3. Furthermore, K42 residue of TLR3 is essential for binding to Rmrp and subsequent dimerization. Rmrp dissociates from TLR3 following endosomal acidification, generating a matured TLR3 dimer which is primed for innate recognition and activation. Myeloid-cell deficiency of Rmrp reduces TLR3 dimerization and attenuates TLR3-mediated antiviral responses against influenza A both in vitro and in vivo.
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Project description: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.
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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.
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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.
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Project description:Polycomb Repressive Complex 2 (PRC2) plays crucial roles in transcriptional regulation and stem cell development. However, the context-specific functions associated with alternative subunits remain largely unexplored. Here we show that the related enzymatic subunits EZH1 and EZH2 undergo an expression switch during hematopoiesis. We examine the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and reveal the existence of an EZH1-SUZ12 sub-complex lacking EED. We provide evidence that EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin domains in the absence of H3K27me3, and positively regulate gene expression. Loss of EZH2 expression leads to global repositioning of EZH1 chromatin occupancy to EZH2 targets. Moreover, we demonstrate that an erythroid-specific enhancer mediates transcriptional activation of EZH1, and a switch from GATA2 to GATA1 controls the developmental EZH1/2 switch by differential association with EZH1 enhancers during erythropoiesis. Thus, the lineage- and developmental stage-specific regulation of PRC2 expression and subunit composition leads to a switch from canonical silencing to non-canonical PRC2 functions during blood stem cell specification. Analysis of genomic occupancy of EZH1, EZH2, EED, SUZ12, various histone marks and transcription factors in primary human fetal liver proerythroblasts by ChIP-seq. Sample GSM970262 was used as the input DNA sample.
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