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1.
Cell Stem Cell ; 25(1): 120-136.e10, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31155483

RESUMO

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-ß gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

2.
Nature ; 571(7764): 270-274, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31207604

RESUMO

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/imunologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Humanos , Memória Imunológica , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Neoplasias/patologia , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Transcrição Genética
3.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
4.
Blood ; 132(7): e13-e23, 2018 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-29967128

RESUMO

The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring.

5.
Epigenetics Chromatin ; 11(1): 21, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29801521

RESUMO

BACKGROUND: DNA methylation in CpG context is fundamental to the epigenetic regulation of gene expression in higher eukaryotes. Changes in methylation patterns are implicated in many diseases, cellular differentiation, imprinting, and other biological processes. Techniques that enrich for biologically relevant genomic regions with high CpG content are desired, since, depending on the size of an organism's methylome, the depth of sequencing required to cover all CpGs can be prohibitively expensive. Currently, restriction enzyme-based reduced representation bisulfite sequencing and its modified protocols are widely used to study methylation differences. Recently, Agilent Technologies, Roche NimbleGen, and Illumina have ventured to both reduce sequencing costs and capture CpGs of known biological relevance by marketing in-solution custom-capture hybridization platforms. We aimed to evaluate the similarities and differences of these four methods considering each platform targets approximately 10-13% of the human methylome. RESULTS: Overall, the regions covered per platform were as expected: targeted capture-based methods covered > 95% of their designed regions, whereas the restriction enzyme-based method covered > 70% of the expected fragments. While the total number of CpG loci shared by all methods was low, ~ 24% of any platform, the methylation levels of CpGs covered by all platforms were concordant. Annotation of CpG loci with genomic features revealed roughly the same proportions of feature annotations across the four platforms. Targeted capture methods comprise similar types and coverage of annotations and, relative to the targeted methods, the restriction enzyme method covers fewer promoters (~ 9%), CpG shores (~ 8%) and unannotated loci (~ 11%). CONCLUSIONS: Although all methods are largely consistent in terms of covered CpG loci, the commercially available capture methods result in covering nearly all CpG sites in their target regions with few off-target loci and covering similar proportions of annotated CpG loci, the restriction-based enrichment results in more off-target and unannotated CpG loci. Quality of DNA is very important for restriction-based enrichment and starting material can be low. Conversely, quality of the starting material is less important for capture methods, and at least twice the amount of starting material is required. Pricing is marginally less for restriction-based enrichment, and the number of samples that can be prepared is not restricted to the number of capture reactions a kit supports. However, the advantage of capture libraries is the ability to custom design areas of interest. The choice of the technique would be decided by the number of samples, the quality and quantity of DNA available and the biological areas of interest since comparable data are obtained from all platforms.


Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Análise de Sequência de DNA/instrumentação , Linhagem Celular , Ilhas de CpG , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Análise de Sequência de DNA/economia
6.
Sci Data ; 5: 180061, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29664468

RESUMO

Driven by the recent advances of next generation sequencing (NGS) technologies and an urgent need to decode complex human diseases, a multitude of large-scale studies were conducted recently that have resulted in an unprecedented volume of whole transcriptome sequencing (RNA-seq) data, such as the Genotype Tissue Expression project (GTEx) and The Cancer Genome Atlas (TCGA). While these data offer new opportunities to identify the mechanisms underlying disease, the comparison of data from different sources remains challenging, due to differences in sample and data processing. Here, we developed a pipeline that processes and unifies RNA-seq data from different studies, which includes uniform realignment, gene expression quantification, and batch effect removal. We find that uniform alignment and quantification is not sufficient when combining RNA-seq data from different sources and that the removal of other batch effects is essential to facilitate data comparison. We have processed data from GTEx and TCGA and successfully corrected for study-specific biases, enabling comparative analysis between TCGA and GTEx. The normalized datasets are available for download on figshare.

7.
Clin Colorectal Cancer ; 16(4): 293-299.e6, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29033218

RESUMO

BACKGROUND: Early-onset colorectal cancer (E-CRC) is increasing in incidence, unlike traditional CRC (T-CRC). We sought to characterize differences between E-CRC and T-CRC. MATERIALS AND METHODS: Data sources included the Surveillance, Epidemiology, and End Results database, the Behavioral Risk Factor Surveillance Survey, and The Cancer Genome Atlas (TCGA). We compared demographics, tumor characteristics, and incidence of CRC in subjects aged 20 to 49 years (E-CRC) with those aged ≥ 50 years (T-CRC). We correlated the incidence of E-CRC and T-CRC to CRC risk factors and age-dependent genomic characteristics of CRC using TCGA. RESULTS: A total of 369,796 CRCs were identified (2000-2011). E-CRC incidence has risen 1.4% per year, whereas T-CRC has declined 3.1% per year (P < .05). The incidence of E-CRC increases in a step-wise fashion from the ascending colon to rectum (P < 2.2e-16). E-CRC is more prevalent in male (53.7% vs. 46.4%; P < .001), Black (14.6% vs. 11.0%; P < .001), and Hispanic (14.7% vs. 8.3%; P < .001) patients. E-CRC presents with aggressive histology, including high-grade (1.5% vs. 1.3%; P < .001), signet ring cell (1.9% vs. 0.9%; P < .001), and mucinous carcinomas (8.9% vs. 8.1%; P < .001), and more often with distant disease (24.4% vs. 18.8%; P < .001). The geographic distribution of E-CRC mirrors United States counties with higher Black population densities. Unlike T-CRC, E-CRC prevalence is not correlated with known CRC risk factors. E-CRC is associated with a lower rate of mutations than traditional CRC. Limitations of this study include E-CRC sample size for the TCGA analysis, as well as lack of comorbidity information and family history. CONCLUSION: E-CRC tumors are clinically, pathologically, and molecularly distinct from T-CRC. Further evaluation of genetic and molecular differences is necessary to understand the pathophysiology of E-CRC and to help target treatment/surveillance strategies.


Assuntos
Adenocarcinoma Mucinoso/patologia , Carcinoma de Células em Anel de Sinete/patologia , Neoplasias Colorretais/patologia , Adenocarcinoma Mucinoso/epidemiologia , Adulto , Fatores Etários , Idade de Início , Carcinoma de Células em Anel de Sinete/epidemiologia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Fatores de Risco , Programa de SEER , Adulto Jovem
8.
Stem Cell Reports ; 9(1): 355-365, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28602613

RESUMO

Human pluripotent stem cells (PSCs) provide an unlimited cell source for cell therapies and disease modeling. Despite their enormous power, technical aspects have hampered reproducibility. Here, we describe a modification of PSC workflows that eliminates a major variable for nearly all PSC experiments: the quality and quantity of the PSC starting material. Most labs continually passage PSCs and use small quantities after expansion, but the "just-in-time" nature of these experiments means that quality control rarely happens before use. Lack of quality control could compromise PSC quality, sterility, and genetic integrity, which creates a variable that might affect results. This method, called CryoPause, banks PSCs as single-use, cryopreserved vials that can be thawed and immediately used in experiments. Each CryoPause bank provides a consistent source of PSCs that can be pre-validated before use to reduce the possibility that high levels of spontaneous differentiation, contamination, or genetic integrity will compromise an experiment.


Assuntos
Criopreservação/métodos , Células-Tronco Pluripotentes/citologia , Animais , Bancos de Espécimes Biológicos , Diferenciação Celular , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Edição de Genes , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante
9.
Hematol Oncol Clin North Am ; 31(3): 389-408, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28501083

RESUMO

Gastric malignancies are a leading cause of cancer-related death worldwide. At least 2 microbial species are currently linked to carcinogenesis and the development of cancer within the human stomach. These include the bacterium Helicobacter pylori and the Epstein-Barr virus. In recent years, there has been increasing evidence that within the human gastrointestinal tract it is not only pathogenic microbes that impact human health but also the corresponding autochthonous microbial communities. This article reviews the gastrointestinal microbiome as it relates primarily to mechanisms of disease and carcinogenesis within the upper gastrointestinal tract.


Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Microbioma Gastrointestinal , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Herpesvirus Humano 4/metabolismo , Neoplasias Gástricas , Animais , Infecções por Vírus Epstein-Barr/patologia , Infecções por Helicobacter/patologia , Humanos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-28503670

RESUMO

BACKGROUND: Despite our understanding of the significance of the prefrontal cortex in the consolidation of long-term memories (LTM), its role in the encoding of LTM remains elusive. Here we investigated the role of new protein synthesis in the mouse medial prefrontal cortex (mPFC) in encoding contextual fear memory. METHODS: Because a change in the association of mRNAs to polyribosomes is an indicator of new protein synthesis, we assessed the changes in polyribosome-associated mRNAs in the mPFC following contextual fear conditioning (CFC) in the mouse. Differential gene expression in mPFC was identified by polyribosome profiling (n = 18). The role of new protein synthesis in mPFC was determined by focal inhibition of protein synthesis (n = 131) and by intra-prelimbic cortex manipulation (n = 56) of Homer 3, a candidate identified from polyribosome profiling. RESULTS: We identified several mRNAs that are differentially and temporally recruited to polyribosomes in the mPFC following CFC. Inhibition of protein synthesis in the prelimbic (PL), but not in the anterior cingulate cortex (ACC) region of the mPFC immediately after CFC disrupted encoding of contextual fear memory. Intriguingly, inhibition of new protein synthesis in the PL 6 hours after CFC did not impair encoding. Furthermore, expression of Homer 3, an mRNA enriched in polyribosomes following CFC, in the PL constrained encoding of contextual fear memory. CONCLUSIONS: Our studies identify several molecular substrates of new protein synthesis in the mPFC and establish that encoding of contextual fear memories require new protein synthesis in PL subregion of mPFC.

11.
PLoS One ; 11(9): e0163699, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27685844

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are potential biomarkers in various malignancies. We aim to characterize miRNA expression in intrahepatic cholangiocarcinoma (ICC) and identify circulating plasma miRNAs with potential diagnostic and prognostic utility. METHODS: Using deep-sequencing techniques, miRNA expression between tumor samples and non-neoplastic liver parenchyma were compared. Overexpressed miRNAs were measured in plasma from an independent cohort of patients with cholangiocarcinoma using RT-qPCR and compared with that healthy volunteers. The discriminatory ability of the evaluated plasma miRNAs between patients and controls was evaluated with receiving operating characteristic (ROC) curves. RESULTS: Small RNAs from 12 ICC and 11 tumor-free liver samples were evaluated. Unsupervised hierarchical clustering using the miRNA expression data showed clear grouping of ICC vs. non-neoplastic liver parenchyma. We identified 134 down-regulated and 128 upregulated miRNAs. Based on overexpression and high fold-change, miR21, miR200b, miR221, and miR34c were measured in plasma from an independent cohort of patients with ICC (n = 25) and healthy controls (n = 7). Significant overexpression of miR-21 and miR-221 was found in plasma from ICC patients. Furthermore, circulating miR-21 demonstrated a high discriminatory ability between patients with ICC and healthy controls (AUC: 0.94). CONCLUSION: Among the differentially expressed miRNAs in ICC, miR-21 and miR-221 are overexpressed and detectable in the circulation. Plasma expression levels of these miRNAs, particularly miR-21, accurately differentiates patients with ICC from healthy controls and could potentially serve as adjuncts in diagnosis. Prospective validation and comparison with other hepatobiliary malignancies is required to establish their potential role as diagnostic and prognostic biomarkers.

13.
Genome Biol ; 16: 265, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26614063

RESUMO

Identifying the microbiome composition from primary tissues directly affords an opportunity to study the causative relationships between the host microbiome and disease. However, this is challenging due the low abundance of microbial DNA relative to the host. We present a systematic evaluation of microbiome profiling directly from endoscopic biopsies by whole genome sequencing. We compared our methods with other approaches on datasets with previously identified microbial composition. We applied this approach to identify the microbiome from 27 stomach biopsies, and validated the presence of Helicobacter pylori by quantitative PCR. Finally, we profiled the microbial composition in The Cancer Genome Atlas gastric adenocarcinoma cohort.


Assuntos
Genoma Humano , Helicobacter pylori/genética , Microbiota/genética , Neoplasias Gástricas/genética , Biópsia , Projeto HapMap , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
15.
Nat Genet ; 47(7): 766-75, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26029871

RESUMO

Polycistronic microRNA (miRNA) clusters are a common feature of vertebrate genomes. The coordinated expression of miRNAs belonging to different seed families from a single transcriptional unit suggests functional cooperation, but this hypothesis has not been experimentally tested. Here we report the characterization of an allelic series of genetically engineered mice harboring selective targeted deletions of individual components of the miR-17 ∼ 92 cluster. Our results demonstrate the coexistence of functional cooperation and specialization among members of this cluster, identify a previously undescribed function for the miR-17 seed family in controlling axial patterning in vertebrates and show that loss of miR-19 selectively impairs Myc-driven tumorigenesis in two models of human cancer. By integrating phenotypic analysis and gene expression profiling, we provide a genome-wide view of how the components of a polycistronic miRNA cluster affect gene expression in vivo. The reagents and data sets reported here will accelerate exploration of the complex biological functions of this important miRNA cluster.


Assuntos
MicroRNAs/genética , Animais , Apoptose , Linfócitos B/fisiologia , Carcinogênese/genética , Células Cultivadas , Pálpebras/anormalidades , Frequência do Gene , Genes Letais , Estudo de Associação Genômica Ampla , Deficiência Intelectual/genética , Deformidades Congênitas dos Membros/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/genética , Família Multigênica , Mutação , Fístula Traqueoesofágica/genética
16.
PLoS One ; 10(6): e0129350, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26066343

RESUMO

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos
17.
J Vis Exp ; (96): e52246, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25742437

RESUMO

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.


Assuntos
Metilação de DNA , Análise de Sequência de DNA/métodos , Pareamento de Bases , Sequência de Bases , Ilhas de CpG , Citosina/análise , Citosina/química , Enzimas de Restrição do DNA/metabolismo , Humanos , Dados de Sequência Molecular , Sulfitos/química
18.
Genome Biol ; 14(9): R95, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24020486

RESUMO

A large number of computational methods have been developed for analyzing differential gene expression in RNA-seq data. We describe a comprehensive evaluation of common methods using the SEQC benchmark dataset and ENCODE data. We consider a number of key features, including normalization, accuracy of differential expression detection and differential expression analysis when one condition has no detectable expression. We find significant differences among the methods, but note that array-based methods adapted to RNA-seq data perform comparably to methods designed for RNA-seq. Our results demonstrate that increasing the number of replicate samples significantly improves detection power over increased sequencing depth.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Proteínas do Tecido Nervoso/genética , RNA/genética , Análise de Sequência de RNA/estatística & dados numéricos , Software , Química Encefálica , Linhagem Celular , Conjuntos de Dados como Assunto , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA/métodos , Razão Sinal-Ruído
19.
Methods ; 58(2): 94-105, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22926237

RESUMO

miRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable tools for the characterization of posttranscriptional gene regulation. Here we present a step-by-step protocol and guidelines for the computational analysis for the large-scale identification of miRNA target sites in cultured cells by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) of AGO proteins.


Assuntos
Proteínas Argonauta , MicroRNAs , RNA Mensageiro , Ribonucleosídeos , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica , Genoma , Camundongos , MicroRNAs/química , MicroRNAs/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonucleosídeos/química , Ribonucleosídeos/isolamento & purificação
20.
Cell Cycle ; 11(8): 1517-23, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22436490

RESUMO

MicroRNA (miRNA) has been shown to be essential for regulating cell fate and pluripotency; however, our knowledge of miRNA function in stem cells is incomplete due to experimental limitations and difficulties in identifying their physiological targets. Recent studies implicated hESC-expressed miRNAs (miR­302-367 and miR­371-373 clusters) in regulating BMP signaling and promoting pluripotency, suggesting that low levels of BMP signaling may promote pluripotency by preventing neural induction. A comprehensive list of miR­302-367 targets recently identified by genome-wide approaches suggests a number of additional cellular processes and signaling pathways whose regulation by miR­302-367 may promote pluripotency and reprogramming, such as cell cycle, epigenetic changes, metabolism and vesicular transfer.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , MicroRNAs/metabolismo , Animais , Sequência de Bases , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Neurônios/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
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