RESUMEN
A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Asunto(s)
Cromatina/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Nucleosomas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica/fisiología , Proteómica/métodos , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/metabolismoRESUMEN
The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Ratones , Células Madre Embrionarias de Ratones , Proteínas Nucleares/metabolismo , Unión Proteica , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.
Asunto(s)
Péptido Hidrolasas , Ubiquitina , Humanos , Ubiquitina/genética , Ubiquitina/metabolismo , Péptido Hidrolasas/metabolismo , Ubiquitinación , Procesamiento Proteico-Postraduccional , Ubiquitinas/genética , Ubiquitinas/metabolismo , Daño del ADN , Endopeptidasas/metabolismo , Inestabilidad GenómicaRESUMEN
After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSLMMS22L homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSLMMS22L binds new histones H3H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSLMMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Cromatina/genética , Inestabilidad Genómica , Histonas/química , Recombinación Homóloga , Humanos , Lisina/metabolismo , Metilación , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
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, the 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.
Asunto(s)
Cromatina/metabolismo , Complejo Represivo Polycomb 2/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Animales , Células Cultivadas , Exones , Regulación de la Expresión Génica , Intrones , Ratones , Células Madre Embrionarias de Ratones/fisiología , Nucleosomas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Estabilidad del ARNRESUMEN
The regulation of chromatin by epigenetic mechanisms plays a central role in gene expression and is essential for development and maintenance of cell identity and function. Aberrant chromatin regulation is observed in many diseases where it leads to defects in epigenetic gene regulation resulting in pathological gene expression programmes. These defects are caused by inherited or acquired mutations in genes encoding enzymes that deposit or remove DNA and histone modifications and that shape chromatin architecture. Chromatin deregulation often results in neurodevelopmental disorders and intellectual disabilities, frequently linked to physical and developmental abnormalities, but can also cause neurodegenerative diseases, immunodeficiency, or muscle wasting syndromes. Epigenetic diseases can either be of monogenic origin or manifest themselves as complex multifactorial diseases such as in congenital heart disease, autism spectrum disorders, or cancer in which mutations in chromatin regulators are contributing factors. The environment directly influences the epigenome and can induce changes that cause or predispose to diseases through risk factors such as stress, malnutrition or exposure to harmful chemicals. The plasticity of chromatin regulation makes targeting the enzymatic machinery an attractive strategy for therapeutic intervention and an increasing number of small molecule inhibitors against a variety of epigenetic regulators are in clinical use or under development. In this review, we will give an overview of the molecular lesions that underlie epigenetic diseases, and we will discuss the impact of the environment and prospects for epigenetic therapies.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/enzimología , Epigénesis Genética , Animales , Trastorno del Espectro Autista/genética , Cromatina/efectos de los fármacos , Cromatina/genética , ADN/metabolismo , Contaminantes Ambientales/farmacología , Femenino , Predisposición Genética a la Enfermedad , Cardiopatías/genética , Histonas/metabolismo , Humanos , Masculino , Mutación , Neoplasias/genética , Enfermedades Neurodegenerativas/genéticaRESUMEN
The therapeutic targeting of DNA repair pathways is an emerging concept in cancer treatment. Compounds that target specific DNA repair processes, such as those mending DNA double-strand breaks (DSBs), are therefore of therapeutic interest. UNC3866 is a small molecule that targets CBX4, a chromobox protein, and a SUMO E3 ligase. As a key modulator of DNA end resection-a prerequisite for DSB repair by homologous recombination (HR)-CBX4 promotes the functions of the DNA resection factor CtIP. Here, we show that treatment with UNC3866 markedly sensitises HR-deficient, NHEJ-hyperactive cancer cells to ionising radiation (IR), while it is non-toxic in selected HR-proficient cells. Consistent with UNC3866 targeting CtIP functions, it inhibits end-resection-dependent DNA repair including HR, alternative end joining (alt-EJ), and single-strand annealing (SSA). These findings raise the possibility that the UNC3866-mediated inhibition of end resection processes we define highlights a distinct vulnerability for the selective killing of HR-ineffective cancers.
RESUMEN
Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , Precursores del ARN/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , G-Cuádruplex , Histonas/metabolismo , Humanos , Ratones , Nucleosomas/metabolismo , Unión Proteica , Precursores del ARN/química , Activación TranscripcionalRESUMEN
Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.
Asunto(s)
Proteína BRCA1/metabolismo , Cromátides/metabolismo , Histonas/metabolismo , Recombinación Homóloga , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Línea Celular Tumoral , Cromátides/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Fase G2/genética , Células HCT116 , Células HeLa , Humanos , Lisina/metabolismo , Metilación , Fase S/genética , Homología de Secuencia de Aminoácido , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein-DNA and protein-nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Marcadores de Afinidad/química , Cromatografía de Afinidad , Proteínas de Unión al ADN/química , Ligandos , Nucleosomas/metabolismoRESUMEN
In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.