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1.
Mol Cell ; 81(13): 2793-2807.e8, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33979575

RESUMEN

DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.


Asunto(s)
Replicación del ADN , ADN de Hongos/biosíntesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Metilación , Metiltransferasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34168039

RESUMEN

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Asunto(s)
Coronavirus Humano OC43/fisiología , Procesamiento Postranscripcional del ARN/genética , SARS-CoV-2/fisiología , Replicación Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Línea Celular , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas de la Nucleocápside , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral/efectos de los fármacos
3.
Cell ; 150(1): 12-27, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22770212

RESUMEN

The epigenetic regulation of DNA-templated processes has been intensely studied over the last 15 years. DNA methylation, histone modification, nucleosome remodeling, and RNA-mediated targeting regulate many biological processes that are fundamental to the genesis of cancer. Here, we present the basic principles behind these epigenetic pathways and highlight the evidence suggesting that their misregulation can culminate in cancer. This information, along with the promising clinical and preclinical results seen with epigenetic drugs against chromatin regulators, signifies that it is time to embrace the central role of epigenetics in cancer.


Asunto(s)
Epigénesis Genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Ensamble y Desensamble de Cromatina , Metilación de ADN , Epigenómica , Regulación Neoplásica de la Expresión Génica , Código de Histonas , Humanos , Neoplasias/metabolismo
4.
Cell ; 151(2): 278-88, 2012 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-23063121

RESUMEN

MicroRNAs (miRNAs) regulate key biological processes and their aberrant expression may lead to cancer. The primary transcript of canonical miRNAs is sequentially cleaved by the RNase III enzymes, Drosha and Dicer, which generate 5' monophosphate ends that are important for subsequent miRNA functions. In particular, the recognition of the 5' monophosphate of pre-miRNAs by Dicer is important for precise and effective biogenesis of miRNAs. Here, we identify a RNA-methyltransferase, BCDIN3D, that O-methylates this 5' monophosphate and negatively regulates miRNA maturation. Specifically, we show that BCDIN3D phospho-dimethylates pre-miR-145 both in vitro and in vivo and that phospho-dimethylated pre-miR-145 displays reduced processing by Dicer in vitro. Consistently, BCDIN3D depletion leads to lower pre-miR-145 and concomitantly increased mature miR-145 levels in breast cancer cells, which suppresses their tumorigenic phenotypes. Together, our results uncover a miRNA methylation pathway potentially involved in cancer that antagonizes the Dicer-dependent processing of miR-145 as well as other miRNAs.


Asunto(s)
Metiltransferasas/metabolismo , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Línea Celular Tumoral , ARN Helicasas DEAD-box/metabolismo , Humanos , Células MCF-7 , Metilación , Neoplasias/metabolismo , Ribonucleasa III/metabolismo
5.
Nature ; 593(7860): 597-601, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33902106

RESUMEN

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.


Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Adenosina/análogos & derivados , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Mol Cell ; 74(6): 1278-1290.e9, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31031083

RESUMEN

7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.


Asunto(s)
Guanosina/análogos & derivados , Metiltransferasas/genética , MicroARNs/genética , Procesamiento Postranscripcional del ARN , Células A549 , Secuencia de Bases , Bioensayo , Células CACO-2 , Movimiento Celular , Proliferación Celular , Guanosina/metabolismo , Células HEK293 , Humanos , Metilación , Metiltransferasas/metabolismo , MicroARNs/metabolismo , Conformación de Ácido Nucleico
7.
Nat Rev Mol Cell Biol ; 15(11): 703-8, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25315270

RESUMEN

Post-translational modifications of histones regulate all DNA-templated processes, including replication, transcription and repair. These modifications function as platforms for the recruitment of specific effector proteins, such as transcriptional regulators or chromatin remodellers. Recent data suggest that histone modifications also have a direct effect on nucleosomal architecture. Acetylation, methylation, phosphorylation and citrullination of the histone core may influence chromatin structure by affecting histone-histone and histone-DNA interactions, as well as the binding of histones to chaperones.


Asunto(s)
Reparación del ADN , ADN/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Citrulina/metabolismo , Replicación del ADN , Histonas/genética , Humanos , Metilación , Chaperonas Moleculares/genética , Nucleosomas/química , Nucleosomas/genética , Fosforilación , Transcripción Genética
8.
Mol Cell ; 72(4): 625-635.e4, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30454561

RESUMEN

In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.


Asunto(s)
Daño del ADN , Reparación del ADN , Histonas/genética , Histonas/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal
9.
Cell ; 143(3): 470-84, 2010 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-21029866

RESUMEN

Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.


Asunto(s)
Ensamble y Desensamble de Cromatina , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Línea Celular Tumoral , Islas de CpG , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Nucleosomas/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteómica/métodos , Coloración y Etiquetado/métodos
10.
Nature ; 552(7683): 126-131, 2017 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-29186125

RESUMEN

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.


Asunto(s)
Adenosina/análogos & derivados , Regulación Neoplásica de la Expresión Génica/genética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Metiltransferasas/metabolismo , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Adenosina/genética , Adenosina/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Femenino , Genes Relacionados con las Neoplasias/genética , Humanos , Leucemia Mieloide Aguda/patología , Metiltransferasas/química , Metiltransferasas/deficiencia , Metiltransferasas/genética , Ratones , Biosíntesis de Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Sitio de Iniciación de la Transcripción
12.
Gut ; 2022 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-35012996

RESUMEN

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

13.
Mol Cell ; 53(5): 806-18, 2014 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-24582497

RESUMEN

Amplification of the EMSY gene in sporadic breast and ovarian cancers is a poor prognostic indicator. Although EMSY has been linked to transcriptional silencing, its mechanism of action is unknown. Here, we report that EMSY acts as an oncogene, causing the transformation of cells in vitro and potentiating tumor formation and metastatic features in vivo. We identify an inverse correlation between EMSY amplification and miR-31 expression, an antimetastatic microRNA, in the METABRIC cohort of human breast samples. Re-expression of miR-31 profoundly reduced cell migration, invasion, and colony-formation abilities of cells overexpressing EMSY or haboring EMSY amplification. We show that EMSY is recruited to the miR-31 promoter by the DNA binding factor ETS-1, and it represses miR-31 transcription by delivering the H3K4me3 demethylase JARID1b/PLU-1/KDM5B. Altogether, these results suggest a pathway underlying the role of EMSY in breast cancer and uncover potential diagnostic and therapeutic targets in sporadic breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Animales , Secuencia de Bases , Movimiento Celular , Estudios de Cohortes , Femenino , Silenciador del Gen , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , MicroARNs/genética , Datos de Secuencia Molecular , Células 3T3 NIH , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Oncogenes/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/metabolismo
14.
Nature ; 525(7570): 538-42, 2015 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-26367796

RESUMEN

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/ß-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.


Asunto(s)
Benzodiazepinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Células Clonales/patología , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Triazoles/farmacología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo
16.
Exp Cell Res ; 381(1): 129-138, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31077711

RESUMEN

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.


Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/genética
17.
Nature ; 505(7484): 564-8, 2014 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-24352239

RESUMEN

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.


Asunto(s)
Glutamina/metabolismo , Histonas/química , Histonas/metabolismo , ARN Polimerasa I/metabolismo , Alanina/genética , Alanina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Nucléolo Celular/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Ribosómico/genética , Epistasis Genética , Humanos , Metilación , Metiltransferasas/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Transcripción Genética
18.
Nature ; 507(7490): 104-8, 2014 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-24463520

RESUMEN

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Citrulina/metabolismo , Histonas/química , Histonas/metabolismo , Células Madre Pluripotentes/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Arginina/química , Arginina/metabolismo , Sitios de Unión , Reprogramación Celular/genética , Cromatina/química , ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Hidrolasas/metabolismo , Ratones , Células Madre Pluripotentes/citología , Unión Proteica , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Proteómica , Especificidad por Sustrato , Transcripción Genética
19.
Genes Dev ; 24(7): 647-52, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20299449

RESUMEN

Chromodomain proteins (Chp1/Chp2/Swi6/Clr4) bind to methylated H3K9 (H3K9me) and regulate pericentric heterochromatin in fission yeast. Chp1 and Clr4 (H3K9-HMT), bind transcriptionally active heterochromatin, whereas Chp2/Swi6 (HP1 homologs) are recruited during the inactive state. We show that H3K4 acetylation (H3K4ac) plays a role in the transition of dimethylated H3K9 (H3K9me2) occupancy from Chp1/Clr4 to Chp2/Swi6. H3K4ac, mediated by Mst1, is enriched at pericentromeres concomitantly with heterochromatin reassembly. H3K4R (Lys --> Arg) mutation increases Chp1 and decreases Chp2/Swi6 pericentric occupancy and exhibits centromeric desilencing. Consistent with structural data, H3K4ac specifically reduces Chp1/Clr4 affinity to H3K9me. We propose that H3K4ac mediates a chromodomain switch from Chp1/Clr4 to Swi6/Chp2 to allow heterochromatin reassembly.


Asunto(s)
Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Acetilación , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Centrosoma , Histonas/genética , Lisina/metabolismo , Mutación/genética , Secuencias Repetitivas de Ácidos Nucleicos
20.
Nature ; 478(7370): 529-33, 2011 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-21964340

RESUMEN

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-associated factor complex (PAFc), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.


Asunto(s)
Cromatina/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica/efectos de los fármacos , Proteómica , Transcripción Genética/efectos de los fármacos
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