RESUMEN
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
RESUMEN
Polycomb repressive complex 2 (PRC2) interacts with RNA in cells, but there is no consensus on how RNA regulates PRC2 canonical functions, including chromatin modification and the maintenance of transcription programs in lineage-committed cells. We assayed two separation-of-function mutants of the PRC2 catalytic subunit EZH2, defective in RNA binding but functional in methyltransferase activity. We find that part of the RNA-binding surface of EZH2 is required for chromatin modification, yet this activity is independent of RNA. Mechanistically, the RNA-binding surface within EZH2 is required for chromatin modification in vitro and in cells, through interactions with nucleosomal DNA. Contrarily, an RNA-binding-defective mutant exhibited normal chromatin modification activity in vitro and in lineage-committed cells, accompanied by normal gene repression activity. Collectively, we show that part of the RNA-binding surface of EZH2, rather than the RNA-binding activity per se, is required for the histone methylation in vitro and in cells, through interactions with the substrate nucleosome.
Asunto(s)
Cromatina , Proteína Potenciadora del Homólogo Zeste 2 , Histonas , Nucleosomas , ARN , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Nucleosomas/metabolismo , ARN/metabolismo , ARN/genética , Humanos , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Unión Proteica , Metilación , Animales , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/genética , Ratones , MutaciónRESUMEN
The chromatin compaction activity of Polycomb group proteins has traditionally been considered essential for transcriptional repression. However, there is very little information on how Polycomb group proteins compact chromatin at the molecular level and no causal link between the compactness of chromatin and transcriptional repression. Recently, a more complete picture of Polycomb-dependent chromatin architecture has started to emerge, owing to advanced methods for imaging and chromosome conformation capture. Discoveries into Polycomb-driven phase separation add another layer of complexity. Recent observations generally imply that Polycomb group proteins modulate chromatin structure at multiple scales to reduce its dynamics and segregate it from active domains. Hence, it is reasonable to hypothesise that Polycomb group proteins maintain the energetically favourable state of compacted chromatin, rather than actively compact it.
Asunto(s)
Cromatina , Proteínas del Grupo Polycomb , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/genética , Cromatina/metabolismo , Cromatina/química , Humanos , AnimalesRESUMEN
The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the three-dimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.
RESUMEN
RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.
Asunto(s)
Cromatina , Complejo Represivo Polycomb 2 , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Histonas/genética , ARN/genética , Heterocromatina , Ribonucleasa Pancreática , Artefactos , ADNRESUMEN
Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR-ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR-ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Leucemia , Animales , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Factores de Transcripción/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Cromatina , Leucemia/genética , Inmunoprecipitación de Cromatina , Mamíferos/genéticaRESUMEN
Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2-AEBP2-JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2-AEBP2-JARID2.
Asunto(s)
Histonas , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Ubiquitinación , ADN/química , Histonas/química , Histonas/genética , Metilación , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/genéticaRESUMEN
Polycomb group proteins predominantly exist in polycomb repressive complexes (PRCs) that cooperate to maintain the repressed state of thousands of cell-type-specific genes. Targeting PRCs to the correct sites in chromatin is essential for their function. However, the mechanisms by which PRCs are recruited to their target genes in mammals are multifactorial and complex. Here we review DNA binding by polycomb group proteins. There is strong evidence that the DNA-binding subunits of PRCs and their DNA-binding activities are required for chromatin binding and CpG targeting in cells. In vitro, CpG-specific binding was observed for truncated proteins externally to the context of their PRCs. Yet, the mere DNA sequence cannot fully explain the subset of CpG islands that are targeted by PRCs in any given cell type. At this time we find very little structural and biophysical evidence to support a model where sequence-specific DNA-binding activity is required or sufficient for the targeting of CpG-dinucleotide sequences by polycomb group proteins while they are within the context of their respective PRCs, either PRC1 or PRC2. We discuss the current knowledge and open questions on how the DNA-binding activities of polycomb group proteins facilitate the targeting of PRCs to chromatin.
Asunto(s)
Islas de CpG , ADN , Proteínas del Grupo Polycomb , Animales , Cromatina/genética , ADN/química , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.
Asunto(s)
Origen de la Vida , Proteínas/metabolismo , ARN , Ribosomas , Péptidos/metabolismo , Biosíntesis de Proteínas , ARN/metabolismo , Ribosomas/metabolismoRESUMEN
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
Asunto(s)
Genoma Viral/genética , Pliegue del ARN/genética , ARN Viral/genética , Rotavirus/crecimiento & desarrollo , Empaquetamiento del Genoma Viral/genética , Proteínas no Estructurales Virales/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Rotavirus/genética , Rotavirus/metabolismoRESUMEN
The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype altered by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.
Asunto(s)
Regulación Alostérica/fisiología , ADN/metabolismo , Nucleosomas/metabolismo , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Animales , Catálisis , Diferenciación Celular , Línea Celular , Histonas/metabolismo , Humanos , Ratones , Complejo Represivo Polycomb 2/genética , Unión ProteicaRESUMEN
PRC2 deposits the H3K27me3 repressive mark, which facilitates transcription repression of developmental genes. The decision of whether a particular gene is silenced at a given point during development is heavily dependent on the chromatin context. More than just a simple epigenetic writer, PRC2 employs several distinct chromatin reading capabilities to sense the local chromatin environment and modulate the H3K27me3 writer activity in a context-dependent manner. Here we discuss the complex interplay of PRC2 with the hallmarks of active and repressive chromatin, how it affects H3K27me3 deposition and how it guides transcriptional activity.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Cromatina/genética , Epigénesis Genética , Genoma/genética , Humanos , Metilación , Complejo Represivo Polycomb 2/genética , Unión ProteicaRESUMEN
Histone lysine methyltransferases (HKMTs) are key regulators of many cellular processes. By definition, HKMTs catalyse the methylation of lysine residues in histone proteins. The enzymatic activities of HKMTs are under precise control, with their allosteric regulation emerging as a prevalent paradigm. We review the molecular mechanisms of allosteric regulation of HKMTs using well-studied histone H3 (K4, K9, K27 and K36) methyltransferases as examples. We discuss the current advances and future potential in targeting allosteric sites of HKMTs for drug development.
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Regulación Alostérica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Desarrollo de Medicamentos/métodos , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Humanos , Lisina/química , Metilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
SUMMARY: Unbiased detection of protein-protein and protein-RNA interactions within ribonucleoprotein complexes are enabled through crosslinking followed by mass spectrometry. Yet, different methods detect different types of molecular interactions and therefore require the usage of different software packages with limited compatibility. We present crisscrosslinkeR, an R package that maps both protein-protein and protein-RNA interactions detected by different types of approaches for crosslinking with mass spectrometry. crisscrosslinkeR produces output files that are compatible with visualization using popular software packages for the generation of publication-quality figures. AVAILABILITY AND IMPLEMENTATION: crisscrosslinkeR is a free and open-source package, available through GitHub: github.com/egmg726/crisscrosslinker. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
RESUMEN
Ubiquitination of chromatin by modification of histone H2A is a critical step in both regulation of DNA repair and regulation of cell fate. These very different outcomes depend on the selective modification of distinct lysine residues in H2A, each by a specific E3 ligase. While polycomb PRC1 complexes modify K119, resulting in gene silencing, the E3 ligase RNF168 modifies K13/15, which is a key event in the response to DNA double-strand breaks. The molecular origin of ubiquitination site specificity by these related E3 enzymes is one of the open questions in the field. Using a combination of NMR spectroscopy, crosslinking mass-spectrometry, mutagenesis and data-driven modelling, here we show that RNF168 binds the acidic patch on the nucleosome surface, directing the E2 to the target lysine. The structural model highlights the role of E3 and nucleosome in promoting ubiquitination and provides a basis for understanding and engineering of chromatin ubiquitination specificity.
Asunto(s)
Histonas/química , Ubiquitina-Proteína Ligasas/química , Diferenciación Celular , Reparación del ADN , Histonas/metabolismo , Humanos , Modelos Moleculares , Dominios Proteicos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identity during development in multicellular organisms by marking repressed genes and chromatin domains. In addition to four core subunits, PRC2 comprises multiple accessory subunits that vary in their composition during cellular differentiation and define two major holo-PRC2 complexes: PRC2.1 and PRC2.2. PRC2 binds to RNA, which inhibits its enzymatic activity, but the mechanism of RNA-mediated inhibition of holo-PRC2 is poorly understood. Here we present in vivo and in vitro protein-RNA interaction maps and identify an RNA-binding patch within the allosteric regulatory site of human and mouse PRC2, adjacent to the methyltransferase center. RNA-mediated inhibition of holo-PRC2 is relieved by allosteric activation of PRC2 by H3K27me3 and JARID2-K116me3 peptides. Both holo-PRC2.1 and holo-PRC2.2 bind RNA, providing a unified model to explain how RNA and allosteric stimuli antagonistically regulate the enzymatic activity of PRC2.
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Histonas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Animales , Sitios de Unión/fisiología , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Metilación , Ratones , Mapas de Interacción de Proteínas/fisiologíaRESUMEN
Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the ß hairpin of ribosomal protein uL22, which is rather distal to the erythromycin binding site, also generate resistance to the antibiotic. We have determined the crystal structure of the large ribosomal subunit from Deinococcus radiodurans with a three amino acid insertion within the ß hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the ß hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within nascent chains trigger conformational rearrangements in the exit tunnel.
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Proteínas Bacterianas/química , Proteínas Ribosómicas/química , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Deinococcus/química , Eritromicina/química , Eritromicina/farmacología , Mutación , Unión Proteica , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that trimethylates H3K27, a mark of repressed chromatin. Mammalian PRC2 binds RNA promiscuously, with thousands of target transcripts in vivo. But what does PRC2 recognize in these RNAs? Here we show that purified human PRC2 recognizes G > C,U â« A in single-stranded RNA and has a high affinity for folded guanine quadruplex (G4) structures but little binding to duplex RNAs. Importantly, G-tract motifs are significantly enriched among PRC2-binding transcripts in vivo. DNA sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on chromatin and H3K27me3-modified nucleosomes. Collectively, the abundance of PRC2-binding RNA motifs rationalizes the promiscuous RNA binding of PRC2, and their enrichment at Polycomb target genes provides a means for RNA-mediated regulation.