RESUMEN
Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.
Asunto(s)
ARN Largo no Codificante/fisiología , Procesos de Crecimiento Celular/genética , Movimiento Celular/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Canales de Potasio KCNQ/metabolismo , Anotación de Secuencia Molecular , Oligonucleótidos Antisentido , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , ARN Interferente PequeñoRESUMEN
CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+), Chd7(+/-), and Chd7(-/-) ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Animales , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Ratones , Ratones Noqueados , Unión ProteicaRESUMEN
De novo mutation of the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7) is the primary cause of CHARGE syndrome, a complex developmental disorder characterized by the co-occurrence of a specific set of birth defects. Recent studies indicate that CHD7 functions as a transcriptional regulator in the nucleoplasm. Here, we report based on immunofluorescence and western blotting of subcellular fractions that CHD7 is also constitutively localized to the nucleolus, the site of rRNA transcription. Standard chromatin immunoprecipitation (ChIP) assays indicate that CHD7 physically associates with rDNA, a result that is also observable upon alignment of whole-genome CHD7 ChIP coupled with massively parallel DNA sequencing data to the rDNA reference sequence. ChIP-chop analyses demonstrate that CHD7 specifically associates with hypomethylated, active rDNA, suggesting a role as a positive regulator of rRNA synthesis. Consistent with this hypothesis, siRNA-mediated depletion of CHD7 results in hypermethylation of the rDNA promoter and a concomitant reduction of 45S pre-rRNA levels. Accordingly, cells overexpressing CHD7 show increased levels of 45S pre-rRNA compared with control cells. Depletion of CHD7 also reduced cell proliferation and protein synthesis. Lastly, compared with wild-type ES cells, the levels of 45S pre-rRNA are reduced in both Chd7(+/-) and Chd7(-/-) mouse ES cells, as well as in Chd7(-/-) whole mouse embryos and multiple tissues dissected from Chd7(+/-) embryos. Together with previously published studies, these results indicate that CHD7 dually functions as a regulator of both nucleoplasmic and nucleolar genes and provide a novel avenue for investigation into the pathogenesis of CHARGE syndrome.
Asunto(s)
Síndrome CHARGE/metabolismo , Nucléolo Celular/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , ARN Ribosómico/genética , Animales , Síndrome CHARGE/genética , Línea Celular , Nucléolo Celular/genética , ADN Helicasas/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Humanos , Ratones , Ratones Noqueados , Unión Proteica , Transporte de Proteínas , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismoRESUMEN
Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Atrofia Muscular Espinal/enzimología , Ribosomas/enzimología , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Extractos Celulares , Línea Celular Tumoral , ADN Helicasas/química , Proteínas de Unión al ADN/química , Activación Enzimática , Humanos , Ratones , Proteínas Mutantes/metabolismo , Unión Proteica , Ribonucleoproteínas/metabolismo , Factores de Transcripción/químicaRESUMEN
MOTIVATION: ChIP-seq is becoming the main approach to the genome-wide study of protein-DNA interactions and histone modifications. Existing informatics tools perform well to extract strong ChIP-enriched sites. However, two questions remain to be answered: (i) to which extent is a ChIP-seq experiment able to reveal the weak ChIP-enriched sites? (ii) are the weak sites biologically meaningful? To answer these questions, it is necessary to identify the weak ChIP signals from background noise. RESULTS: We propose a linear signal-noise model, in which a noise rate was introduced to represent the fraction of noise in a ChIP library. We developed an iterative algorithm to estimate the noise rate using a control library, and derived a library-swapping strategy for the false discovery rate estimation. These approaches were integrated in a general-purpose framework, named CCAT (Control-based ChIP-seq Analysis Tool), for the significance analysis of ChIP-seq. Applications to H3K4me3 and H3K36me3 datasets showed that CCAT predicted significantly more ChIP-enriched sites that the previous methods did. With the high sensitivity of CCAT prediction, we revealed distinct chromatin features associated to the strong and weak H3K4me3 sites. AVAILABILITY: http://cmb.gis.a-star.edu.sg/ChIPSeq/tools.htm.
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Inmunoprecipitación de Cromatina/métodos , Biología Computacional/métodos , Regulación de la Expresión Génica , Algoritmos , Sitios de Unión , Simulación por Computador , Genoma , Histonas/química , Modelos Estadísticos , Distribución de Poisson , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughput approach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recent development in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneous capture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA and that can be used to amplify these features prior to sequencing. However, because the technology is in its infancy, there is little information available on how such experimental parameters can be optimized. To overcome this, we varied the capture sequence, capture sequence position, and gRNA backbone to identify an optimal gRNA scaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with our observations and recommendations for future use.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Análisis de la Célula Individual/métodos , Células Madre Embrionarias Humanas , Humanos , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Distal spinal muscular atrophy type 1 (DSMA1) is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene. Patients with DSMA1 present between 6 weeks and 6 months of age with progressive muscle weakness and respiratory failure due to diaphragmatic palsy. Contrary to this "classic" infantile disease, we have previously described a DSMA1 patient with juvenile disease onset. In this paper, we present (1) a second juvenile case and (2) the first study of DSMA1 on protein level in patients with infantile (n = 3) as well as juvenile (n = 2) disease onset observing elevated residual steady-state IGHMBP2 protein levels in the patients with late onset DSMA1 as compared to those with classic DSMA1. Mutation screening in IGHMBP2 revealed two patients compound heterozygous for a novel missense mutation (c.1478C-->T; p.T493I) and another previously described mutation. In lymphoblastoid cells of both patients, steady-state IGHMBP2 protein levels were reduced. In comparison to wild-type IGHMBP2, the p.T493I variant protein had an increased tendency to aggregate and spontaneously degrade in vitro. We verified a change in the physicochemical properties of the p.T493I variant which may explain the pathogenicity of this mutation. Our data further suggest that the age of onset of DSMA1 is variable, and we discuss the effect of residual IGHMBP2 protein levels on the clinical course and the severity of the disease.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación Missense , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adulto , Edad de Inicio , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Embarazo , Nacimiento PrematuroRESUMEN
The need to derive and culture diverse cell or tissue types in vitro has prompted investigations on how changes in culture conditions affect cell states. However, the identification of the optimal conditions (e.g., signaling molecules and growth factors) required to maintain cell types or convert between cell types remains a time-consuming task. Here, we developed EpiMogrify, an approach that leverages data from â¼100 human cell/tissue types available from ENCODE and Roadmap Epigenomics consortia to predict signaling molecules and factors that can either maintain cell identity or enhance directed differentiation (or cell conversion). EpiMogrify integrates protein-protein interaction network information with a model of the cell's epigenetic landscape based on H3K4me3 histone modifications. Using EpiMogrify-predicted factors for maintenance conditions, we were able to better potentiate the maintenance of astrocytes and cardiomyocytes in vitro. We report a significant increase in the efficiency of astrocyte and cardiomyocyte differentiation using EpiMogrify-predicted factors for conversion conditions.
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Predicción/métodos , Histonas/genética , Transducción de Señal/inmunología , Astrocitos , Diferenciación Celular/inmunología , Diferenciación Celular/fisiología , Cromatina/metabolismo , Metilación de ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Código de Histonas/genética , Histonas/metabolismo , Humanos , Miocitos Cardíacos , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.
Asunto(s)
Azepinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Histonas/química , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Triazoles/farmacología , Acetilación/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Modelos Moleculares , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.
Asunto(s)
Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Animales , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular/genética , Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Células Madre Embrionarias/citología , Genes Homeobox , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Transgénicos , Dominios ProteicosRESUMEN
Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. However, little is known about CTCF-associated higher-order chromatin structures at a global scale. Here we applied chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, we identified 1,480 cis- and 336 trans-interacting loci with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive cross-talk between promoters and regulatory elements. This highly complex nuclear organization offers insights toward the unifying principles that govern genome plasticity and function.
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Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Genes Reguladores , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/química , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Epigenómica , Regulación de la Expresión Génica , Hibridación Fluorescente in Situ , Ratones , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.
Asunto(s)
Arginina/química , Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Proteína-Arginina N-Metiltransferasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas Represoras/química , Secuencia de Aminoácidos , Arginina/análogos & derivados , Proteína Tirosina Quinasa CSK , Metilación de ADN , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/metabolismo , Etanolaminas/química , Células HeLa , Humanos , Espectrometría de Masas , Metilación , Microscopía Fluorescente , Datos de Secuencia Molecular , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , ARN/química , Proteínas Recombinantes/química , Sefarosa/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Madre/metabolismo , Transcripción Genética , Transfección , Familia-src QuinasasRESUMEN
RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific double-stranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.