RESUMO
Two studies in this issue, Kristariyanto et al. (2015) and Michel et al. (2015), describe innovative ways to produce large quantities of atypical K29 and K33 ubiquitin chains and report structures and mechanisms of chain-specific recognition.
Assuntos
Endopeptidases/química , Lisina/química , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , HumanosRESUMO
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.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Cromatina/genética , Epigênese Genética , Genoma/genética , Humanos , Metilação , Complexo Repressor Polycomb 2/genética , Ligação ProteicaRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
Assuntos
Dano ao DNA , Histonas/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Humanos , Lisina/metabolismo , Estrutura Terciária de Proteína , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.
Assuntos
Adenosina Trifosfatases/química , Bacillus subtilis/enzimologia , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Reparo de Erro de Pareamento de DNA , Endonucleases/química , Ativação Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Zinco/metabolismoRESUMO
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.
Assuntos
Cromatina , Proteínas do Grupo Polycomb , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Cromatina/metabolismo , Cromatina/química , Humanos , AnimaisRESUMO
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.
Assuntos
Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Histonas , Nucleossomos , RNA , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Nucleossomos/metabolismo , RNA/metabolismo , RNA/genética , Humanos , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Ligação Proteica , Metilação , Animais , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Camundongos , MutaçãoRESUMO
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.
RESUMO
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.
Assuntos
Histonas , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Ubiquitinação , DNA/química , Histonas/química , Histonas/genética , Metilação , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genéticaRESUMO
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.
Assuntos
Regulação Alostérica/fisiologia , DNA/metabolismo , Nucleossomos/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Animais , Catálise , Diferenciação Celular , Linhagem Celular , Histonas/metabolismo , Humanos , Camundongos , Complexo Repressor Polycomb 2/genética , Ligação ProteicaRESUMO
Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.
RESUMO
Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.
Assuntos
Proteases Específicas de Ubiquitina/metabolismo , Animais , Ensaios Enzimáticos/métodos , Humanos , Cinética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Especificidade por Substrato , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/isolamento & purificaçãoRESUMO
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.
Assuntos
Histonas/química , Ubiquitina-Proteína Ligases/química , Diferenciação Celular , Reparo do DNA , Histonas/metabolismo , Humanos , Modelos Moleculares , Domínios Proteicos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , UbiquitinaçãoRESUMO
BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.
Assuntos
Proteína BRCA1/metabolismo , Histonas/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Proteína BRCA1/genética , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Células HeLa , Humanos , Cinética , Camundongos Knockout , Interferência de RNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
DNA double strand breaks need to be repaired in an organized fashion to preserve genomic integrity. In the organization of faithful repair, histone ubiquitination plays a crucial role. Recent findings suggest an integrated model for DNA repair regulation through site-specific histone ubiquitination and crosstalk to other posttranslational modifications. Here we discuss how site-specific histone ubiquitination is achieved on a molecular level and how different multi-protein complexes work together to integrate different histone ubiquitination states. We propose a model where site-specific H2A ubiquitination organizes the spatio-temporal recruitment of DNA repair factors which will ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Histonas/metabolismo , Reparo de DNA por Recombinação , Animais , DNA/metabolismo , Histonas/química , Humanos , Transdução de Sinais , UbiquitinaçãoRESUMO
During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13-15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13-15 ubiquitination reaction during the complex signalling process at DNA damage sites.