RESUMEN
GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.
RESUMEN
Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.
Asunto(s)
ADN Metiltransferasa 3A , Histonas , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , Metilasas de Modificación del ADN/genética , Histonas/metabolismo , Enfermedades NeuroinflamatoriasRESUMEN
Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.
Asunto(s)
Neoplasias Encefálicas/genética , Carcinogénesis/genética , Glioma/genética , Histonas/genética , Interneuronas/metabolismo , Mutación/genética , Células-Madre Neurales/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Neoplasias Encefálicas/patología , Carcinogénesis/patología , Linaje de la Célula , Reprogramación Celular/genética , Cromatina/metabolismo , Embrión de Mamíferos/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Glioma/patología , Histonas/metabolismo , Lisina/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Clasificación del Tumor , Oligodendroglía/metabolismo , Regiones Promotoras Genéticas/genética , Prosencéfalo/embriología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transcripción Genética , Transcriptoma/genéticaRESUMEN
Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.
Asunto(s)
Acetatos/metabolismo , Glucosa/metabolismo , Ácido Pirúvico/metabolismo , ATP Citrato (pro-S)-Liasa/fisiología , Acetilcoenzima A/biosíntesis , Acetilcoenzima A/metabolismo , Acetilación , Animales , Femenino , Glucólisis/fisiología , Lipogénesis/fisiología , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Oxidorreductasas , Piruvato Descarboxilasa/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Social insects are emerging models to study how gene regulation affects behavior because their colonies comprise individuals with the same genomes but greatly different behavioral repertoires. To investigate the molecular mechanisms that activate distinct behaviors in different castes, we exploit a natural behavioral plasticity in Harpegnathos saltator, where adult workers can transition to a reproductive, queen-like state called gamergate. Analysis of brain transcriptomes during the transition reveals that corazonin, a neuropeptide homologous to the vertebrate gonadotropin-releasing hormone, is downregulated as workers become gamergates. Corazonin is also preferentially expressed in workers and/or foragers from other social insect species. Injection of corazonin in transitioning Harpegnathos individuals suppresses expression of vitellogenin in the brain and stimulates worker-like hunting behaviors, while inhibiting gamergate behaviors, such as dueling and egg deposition. We propose that corazonin is a central regulator of caste identity and behavior in social insects.
Asunto(s)
Hormigas/metabolismo , Proteínas de Insectos/metabolismo , Neuropéptidos/metabolismo , Animales , Hormigas/genética , Hormigas/crecimiento & desarrollo , Conducta Animal , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Conducta SocialRESUMEN
The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4)2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4)2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.
Asunto(s)
Proteínas de Ciclo Celular , Microscopía por Crioelectrón , Chaperonas de Histonas , Histonas , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/química , Histonas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Multimerización de Proteína , Sitios de Unión , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.
Asunto(s)
Metiltransferasas , Proteínas Ribosómicas , Ribosomas , Neoplasias Gástricas , Animales , Femenino , Humanos , Ratones , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Lisina/metabolismo , Metilación/efectos de los fármacos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/deficiencia , Metiltransferasas/metabolismo , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Biosíntesis de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of â¼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.
Asunto(s)
ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Iniciación de la Transcripción Genética , Microscopía por Crioelectrón , Regulación Fúngica de la Expresión Génica , Modelos Moleculares , Regiones Promotoras Genéticas , Conformación Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Relación Estructura-Actividad , Factores de Tiempo , Elongación de la Transcripción Genética , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Proteínas F-Box , N-Metiltransferasa de Histona-Lisina , Histona Demetilasas con Dominio de Jumonji , Proteínas del Grupo Polycomb , Proteína SMARCB1 , Cromatina/genética , Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patología , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Células Tumorales Cultivadas/metabolismoRESUMEN
Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.
Asunto(s)
Acetilación , Cromatina , Lisina , Metilación , Procesamiento Proteico-Postraduccional , Sitio de Iniciación de la Transcripción , Animales , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Péptidos/química , Péptidos/metabolismo , Histona Desacetilasas/metabolismoRESUMEN
Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.
Asunto(s)
Carcinogénesis/genética , ADN Helicasas/genética , Proteínas Intrínsecamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Animales , Secuencia de Bases , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patología , ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Péptidos/genética , Péptidos/metabolismo , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Transporte de Proteínas , Transducción de Señal , Análisis de Supervivencia , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.
Asunto(s)
Condensados Biomoleculares , Virus , Fosforilación , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.
Asunto(s)
COVID-19 , Epigénesis Genética , Histonas , Interacciones Microbiota-Huesped , Imitación Molecular , SARS-CoV-2 , Proteínas Virales , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Epigenoma/genética , Histonas/química , Histonas/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.
Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Heterocromatina/metabolismo , ADN Ribosómico/metabolismo , Epigénesis Genética/fisiología , Eucromatina/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteómica/métodos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.
Asunto(s)
Células Madre Embrionarias/metabolismo , Código de Histonas , Histonas/metabolismo , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Fibroblastos/metabolismo , Células HeLa , Histonas/química , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Grupo Polycomb/metabolismo , Regiones Promotoras Genéticas , Procesamiento Proteico-PostraduccionalRESUMEN
H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Epigénesis Genética , Histonas/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Silenciador del Gen , Histonas/química , Activación de Linfocitos/genética , Masculino , Metilación , Ratones , Ratones NoqueadosRESUMEN
Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.
Asunto(s)
Proliferación Celular , Senescencia Celular , Ensamble y Desensamble de Cromatina , Cromatina/enzimología , Fibroblastos/enzimología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Proliferación Celular/genética , Senescencia Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Represión Epigenética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/genética , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.
Asunto(s)
Acilcoenzima A/metabolismo , Metabolismo Energético , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIID/metabolismo , Metabolismo Energético/genética , Ácidos Grasos/metabolismo , Histonas/genética , Homeostasis , Lisina , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factor de Transcripción TFIID/genética , Transcripción GenéticaRESUMEN
Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.