RESUMEN
Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Elongación de la Transcripción Genética , Animales , Cromatina/química , Proteínas Cromosómicas no Histona/fisiología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/fisiología , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Ratones , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN no Traducido/biosíntesis , Sitio de Iniciación de la Transcripción , Transcripción Genética , Factores de Elongación Transcripcional/fisiologíaRESUMEN
Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter or regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a striking difference between genes that use these distinct regulatory strategies lies in the "default" chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly regulated genes whose sequences inherently disfavor nucleosome formation within the gene but favor occlusion of the promoter by nucleosomes. In contrast, housekeeping genes that lack pronounced Pol II pausing show higher nucleosome occupancy downstream, but their promoters are deprived of nucleosomes regardless of polymerase binding. Our results indicate that a key role of paused Pol II is to compete with nucleosomes for occupancy of highly regulated promoters, thereby preventing the formation of repressive chromatin architecture to facilitate further or future gene activation.
Asunto(s)
Regulación de la Expresión Génica , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Animales , Línea Celular , Ensamble y Desensamble de Cromatina , Drosophila , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Cardiomyocyte elongation and alignment, a critical step in cardiomyocyte maturation starting from the perinatal stage, is crucial for formation of the highly organized intra- and inter-cellular structures for spatially and temporally ordered contraction in adult cardiomyocytes. However, the mechanism(s) underlying the control of cardiomyocyte alignment remains elusive. Here, we report that SIRT1, the most conserved NAD+-dependent protein deacetylase highly expressed in perinatal heart, plays an important role in regulating cardiomyocyte remodeling during development. We observed that SIRT1 deficiency impairs the alignment of cardiomyocytes/myofibrils and disrupts normal beating patterns at late developmental stages in an in vitro differentiation system from human embryonic stem cells. Consistently, deletion of SIRT1 at a late developmental stage in mouse embryos induced the irregular distribution of cardiomyocytes and misalignment of myofibrils, and reduced the heart size. Mechanistically, the expression of several genes involved in chemotaxis, including those in the CXCL12/CXCR4 and CCL2/CCR2/CCR4 pathways, was dramatically blunted during maturation of SIRT1-deficient cardiomyocytes. Pharmacological inhibition of CCL2 signaling suppressed cardiomyocyte alignment. Our study identifies a regulatory factor that modulates cardiomyocyte alignment at the inter-cellular level during maturation.
Asunto(s)
Células Madre Embrionarias Humanas , Miocitos Cardíacos , Sirtuina 1 , Animales , Diferenciación Celular , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Transducción de Señal , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.
Asunto(s)
Daño del ADN/efectos de la radiación , Metilación de ADN/genética , Replicación del ADN/genética , Piel/efectos de la radiación , Metilación de ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Genoma Humano/genética , Genoma Humano/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Genómica/métodos , Humanos , Mutación INDEL/efectos de la radiación , Melanocitos/efectos de la radiación , Mutagénesis/genética , Mutagénesis/efectos de la radiación , Piel/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment.
Asunto(s)
Carcinogénesis/genética , Proteínas de Ciclo Celular/metabolismo , Elementos de Facilitación Genéticos/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Melanoma/genética , Melanoma/fisiopatología , Proteínas Nucleares/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proliferación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Silenciador del Gen , Xenoinjertos , Humanos , Subunidad 1 del Complejo Mediador/genética , Melanocitos/metabolismo , Melanoma/enzimología , Ratones , Proteínas Nucleares/genética , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.
Asunto(s)
Cromatina/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Cromatina/metabolismo , ADN sin Sentido/genética , Regulación de la Expresión Génica , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Modelos Genéticos , Nucleosomas/genética , Nucleosomas/metabolismo , Motivos de Nucleótidos/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genéticaRESUMEN
The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.
Asunto(s)
Células Madre Embrionarias/enzimología , Mamíferos/crecimiento & desarrollo , ARN Polimerasa III/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
While multiple factors are associated with cardiovascular disease (CVD), many environmental exposures that may contribute to CVD have not been examined. To understand environmental effects on cardiovascular health, we performed an exposome-wide association study (ExWAS), a hypothesis-free approach, using survey data on endogenous and exogenous exposures at home and work and data from health and medical histories from the North Carolina-based Personalized Environment and Genes Study (PEGS) (n = 5015). We performed ExWAS analyses separately on six cardiovascular outcomes (cardiac arrhythmia, congestive heart failure, coronary artery disease, heart attack, stroke, and a combined atherogenic-related outcome comprising angina, angioplasty, atherosclerosis, coronary artery disease, heart attack, and stroke) using logistic regression and a false discovery rate of 5%. For each CVD outcome, we tested 502 single exposures and built multi-exposure models using the deletion-substitution-addition (DSA) algorithm. To evaluate complex nonlinear relationships, we employed the knockoff boosted tree (KOBT) algorithm. We adjusted all analyses for age, sex, race, BMI, and annual household income. ExWAS analyses revealed novel associations that include blood type A (Rh-) with heart attack (OR[95%CI] = 8.2[2.2:29.7]); paint exposures with stroke (paint related chemicals: 6.1[2.2:16.0], acrylic paint: 8.1[2.6:22.9], primer: 6.7[2.2:18.6]); biohazardous materials exposure with arrhythmia (1.8[1.5:2.3]); and higher paternal education level with reduced risk of multiple CVD outcomes (stroke, heart attack, coronary artery disease, and combined atherogenic outcome). In multi-exposure models, trouble sleeping and smoking remained important risk factors. KOBT identified significant nonlinear effects of sleep disorder, regular intake of grapefruit, and a family history of blood clotting problems for multiple CVD outcomes (combined atherogenic outcome, congestive heart failure, and coronary artery disease). In conclusion, using statistics and machine learning, these findings identify novel potential risk factors for CVD, enable hypothesis generation, provide insights into the complex relationships between risk factors and CVD, and highlight the importance of considering multiple exposures when examining CVD outcomes.
Asunto(s)
Enfermedades Cardiovasculares , Enfermedad de la Arteria Coronaria , Exposoma , Insuficiencia Cardíaca , Infarto del Miocardio , Accidente Cerebrovascular , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Humanos , Factores de Riesgo , Accidente Cerebrovascular/epidemiología , Encuestas y CuestionariosRESUMEN
Retinoid homeostasis is critical for normal embryonic development. Both the deficiency and excess of these compounds are associated with congenital malformations. Here we demonstrate that SIRT1, the most conserved mammalian NADâº-dependent protein deacetylase, contributes to homeostatic retinoic acid (RA) signaling and modulates mouse embryonic stem cell (mESC) differentiation in part through deacetylation of cellular retinoic acid binding protein II (CRABPII). We show that RA-mediated acetylation of CRABPII at K102 is essential for its nuclear accumulation and subsequent activation of RA signaling. SIRT1 interacts with and deacetylates CRABPII, regulating its subcellular localization. Consequently, SIRT1 deficiency induces hyperacetylation and nuclear accumulation of CRABPII, enhancing RA signaling and accelerating mESC differentiation in response to RA. Consistently, SIRT1 deficiency is associated with elevated RA signaling and development defects in mice. Our findings reveal a molecular mechanism that regulates RA signaling and highlight the importance of SIRT1 in regulation of ESC pluripotency and embryogenesis.
Asunto(s)
Células Madre Embrionarias/metabolismo , Receptores de Ácido Retinoico/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Tretinoina/farmacología , Acetilación/efectos de los fármacos , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Interacción Gen-Ambiente , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Transducción de Señal/efectos de los fármacosRESUMEN
Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.
Asunto(s)
Desarrollo Embrionario/genética , Epigénesis Genética , Metionina Adenosiltransferasa/genética , Metionina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Sirtuina 1/genética , Acetilación , Animales , Apoptosis , Diferenciación Celular , Embrión de Mamíferos , Histonas/genética , Histonas/metabolismo , Metabolómica , Metionina/administración & dosificación , Metionina Adenosiltransferasa/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Células Madre Embrionarias de Ratones/citología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , S-Adenosilmetionina/metabolismo , Sirtuina 1/deficienciaRESUMEN
High-throughput sequencing has become ubiquitous in biomedical sciences. As new technologies emerge and sequencing costs decline, the diversity and volume of available data increases exponentially, and successfully navigating the data becomes more challenging. Though datasets are often hosted by public repositories, scientists must rely on inconsistent annotation to identify and interpret meaningful data. Moreover, the experimental heterogeneity and wide-ranging quality of high-throughput biological data means that even data with desired cell lines, tissue types, or molecular targets may not be readily interpretable or integrated. We have developed ORSO (Online Resource for Social Omics) as an easy-to-use web application to connect life scientists with genomics data. In ORSO, users interact within a data-driven social network, where they can favorite datasets and follow other users. In addition to more than 30,000 datasets hosted from major biomedical consortia, users may contribute their own data to ORSO, facilitating its discovery by other users. Leveraging user interactions, ORSO provides a novel recommendation system to automatically connect users with hosted data. In addition to social interactions, the recommendation system considers primary read coverage information and annotated metadata. Similarities used by the recommendation system are presented by ORSO in a graph display, allowing exploration of dataset associations. The topology of the network graph reflects established biology, with samples from related systems grouped together. We tested the recommendation system using an RNA-seq time course dataset from differentiation of embryonic stem cells to cardiomyocytes. The ORSO recommendation system correctly predicted early data point sources as embryonic stem cells and late data point sources as heart and muscle samples, resulting in recommendation of related datasets. By connecting scientists with relevant data, ORSO provides a critical new service that facilitates wide-ranging research interests.
Asunto(s)
Sistemas de Administración de Bases de Datos , Bases de Datos Genéticas , Genómica , Redes Sociales en Línea , Investigadores/organización & administración , Genómica/métodos , Genómica/organización & administración , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Medios de Comunicación SocialesRESUMEN
Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.
Asunto(s)
Proteínas de Drosophila/genética , ARN Polimerasa II/fisiología , ARN Citoplasmático Pequeño/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Estabilidad del ARN , Transducción de Señal , Elongación de la Transcripción GenéticaRESUMEN
p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.
Asunto(s)
Proteínas de Unión al ADN/genética , Genoma Humano/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , ADN Intergénico/genética , Bases de Datos Genéticas , Regulación de la Expresión Génica/genética , Genes/genética , Humanos , Linfocitos , Biosíntesis de ProteínasRESUMEN
The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , ARN Polimerasa II/fisiología , Activación Transcripcional , Animales , Drosophila melanogaster/inmunología , Células Madre Embrionarias/metabolismo , Inmunidad/genética , Quinasas Janus/metabolismo , Ratones , ARN Polimerasa II/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT1 is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T522 on SIRT1 is crucial for tissue-specific regulation of SIRT1 activity in mice. Dephosphorylation of T522 is critical for repression of its activity during adipogenesis. The phospho-T522 level is reduced during adipogenesis. Knocking-in a constitutive T522 phosphorylation mimic activates the ß-catenin/GATA3 pathway, repressing PPARγ signaling, impairing differentiation of white adipocytes, and ameliorating high-fat diet-induced dyslipidemia in mice. In contrast, phosphorylation of T522 is crucial for activation of hepatic SIRT1 in response to over-nutrition. Hepatic SIRT1 is hyperphosphorylated at T522 upon high-fat diet feeding. Knocking-in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high-fat diet feeding. In addition, the T522 dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT1 phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T522 is a critical element in tissue-specific regulation of SIRT1 activity in vivo.
Asunto(s)
Adipogénesis , Metabolismo Energético , Sirtuina 1/metabolismo , Treonina/química , Adipocitos/fisiología , Animales , Diferenciación Celular , Dieta Alta en Grasa , Dislipidemias/genética , Dislipidemias/fisiopatología , Hígado Graso/genética , Hígado Graso/fisiopatología , Femenino , Factor de Transcripción GATA3/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Fosforilación , Sirtuina 1/genéticaRESUMEN
Established and emerging next generation sequencing (NGS)-based technologies allow for genome-wide interrogation of diverse biological processes. However, accessibility of NGS data remains a problem, and few user-friendly resources exist for integrative analysis of NGS data from different sources and experimental techniques. Here, we present Online Resource for Integrative Omics (ORIO; https://orio.niehs.nih.gov/), a web-based resource with an intuitive user interface for rapid analysis and integration of NGS data. To use ORIO, the user specifies NGS data of interest along with a list of genomic coordinates. Genomic coordinates may be biologically relevant features from a variety of sources, such as ChIP-seq peaks for a given protein or transcription start sites from known gene models. ORIO first iteratively finds read coverage values at each genomic feature for each NGS dataset. Data are then integrated using clustering-based approaches, giving hierarchical relationships across NGS datasets and separating individual genomic features into groups. In focusing its analysis on read coverage, ORIO makes limited assumptions about the analyzed data; this allows the tool to be applied across data from a variety of experiments and techniques. Results from analysis are presented in dynamic displays alongside user-controlled statistical tests, supporting rapid statistical validation of observed results. We emphasize the versatility of ORIO through diverse examples, ranging from NGS data quality control to characterization of enhancer regions and integration of gene expression information. Easily accessible on a public web server, we anticipate wide use of ORIO in genome-wide investigations by life scientists.
Asunto(s)
Genómica/estadística & datos numéricos , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Histonas/genética , Sitio de Iniciación de la Transcripción , Interfaz Usuario-Computador , Animales , Inmunoprecipitación de Cromatina , Interpretación Estadística de Datos , Elementos de Facilitación Genéticos , Genómica/métodos , Histonas/metabolismo , Humanos , Internet , Ratones , Análisis de Secuencia de ADNRESUMEN
Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.
Asunto(s)
Elementos sin Sentido (Genética)/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Transcripción Genética , Elementos sin Sentido (Genética)/biosíntesis , Cromatina/genética , Islas de CpG/genética , Regulación Fúngica de la Expresión Génica , Genómica , Código de Histonas/genética , Histonas/genética , Humanos , Proteínas Nucleares/biosíntesis , Nucleosomas/genética , Unión Proteica/genética , Alineación de SecuenciaRESUMEN
Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 60013,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.
Asunto(s)
Daño del ADN/genética , Genoma Humano/genética , Mutación/efectos de la radiación , Neoplasias/genética , Piel/efectos de la radiación , Biopsia , Células Clonales/efectos de la radiación , Daño del ADN/efectos de la radiación , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Genoma Humano/efectos de la radiación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis/genética , Mutación/genética , Tasa de Mutación , Neoplasias/etiología , Neoplasias/patología , Análisis de la Célula Individual , Piel/patología , Luz Solar/efectos adversosRESUMEN
BACKGROUND: Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. RESULTS: Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. CONCLUSIONS: Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.
Asunto(s)
Genoma Fúngico , Acumulación de Mutaciones , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Biología Computacional , Padre , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Tasa de Mutación , Estándares de ReferenciaRESUMEN
BACKGROUND & AIMS: Intestinal epithelial homeostasis is maintained by complex interactions among epithelial cells, commensal gut microorganisms, and immune cells. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD), but the mechanisms of this process are not clear. We investigated how Sirtuin 1 (SIRT1), a conserved mammalian NAD+-dependent protein deacetylase, senses environmental stress to alter intestinal integrity. METHODS: We performed studies of mice with disruption of Sirt1 specifically in the intestinal epithelium (SIRT1 iKO, villin-Cre+, Sirt1flox/flox mice) and control mice (villin-Cre-, Sirt1flox/flox) on a C57BL/6 background. Acute colitis was induced in some mice by addition of 2.5% dextran sodium sulfate to drinking water for 5-9 consecutive days. Some mice were given antibiotics via their drinking water for 4 weeks to deplete their microbiota. Some mice were fed with a cholestyramine-containing diet for 7 days to sequester their bile acids. Feces were collected and proportions of microbiota were analyzed by 16S rRNA amplicon sequencing and quantitative PCR. Intestines were collected from mice and gene expression profiles were compared by microarray and quantitative PCR analyses. We compared levels of specific mRNAs between colon tissues from age-matched patients with ulcerative colitis (n=10) vs without IBD (n=8, controls). RESULTS: Mice with intestinal deletion of SIRT1 (SIRT1 iKO) had abnormal activation of Paneth cells starting at the age of 5-8 months, with increased activation of NF-κB, stress pathways, and spontaneous inflammation at 22-24 months of age, compared with control mice. SIRT1 iKO mice also had altered fecal microbiota starting at 4-6 months of age compared with control mice, in part because of altered bile acid metabolism. Moreover, SIRT1 iKO mice with defective gut microbiota developed more severe colitis than control mice. Intestinal tissues from patients with ulcerative colitis expressed significantly lower levels of SIRT1 mRNA than controls. Intestinal tissues from SIRT1 iKO mice given antibiotics, however, did not have signs of inflammation at 22-24 months of age, and did not develop more severe colitis than control mice at 4-6 months. CONCLUSIONS: In analyses of intestinal tissues, colitis induction, and gut microbiota in mice with intestinal epithelial disruption of SIRT1, we found this protein to prevent intestinal inflammation by regulating the gut microbiota. SIRT1 might therefore be an important mediator of host-microbiome interactions. Agents designed to activate SIRT1 might be developed as treatments for IBDs.