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1.
Anticancer Res ; 39(10): 5449-5459, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570439

RESUMO

BACKGROUND/AIM: Epigenetic abnormalities in microRNAs (miRNAs) have not been analyzed in samples other than pancreaticobiliary tissues in patients with pancreaticobiliary cancer (PBC). To identify miRNAs specific for PBC, the present study analyzed the methylation of tumor-suppressive miRNAs in bile from patients with pancreaticobiliary diseases. MATERIALS AND METHODS: Bile was collected endoscopically or percutaneously from 52 patients with pancreatic cancer, 26 with biliary tract cancer, and 20 with benign pancreaticobiliary diseases. Sequences encoding 16 tumor-suppressive miRNAs were amplified by polymerase chain reaction and sequenced, and their methylation rates were determined. RESULTS: The methylation rates of miR-1247 and miR-200a were significantly higher in patients with pancreatic cancer, and biliary tract cancer than in those with benign diseases, and the methylation rate of miR-200b was significantly higher in patients with pancreatic cancer than in those with benign diseases. CONCLUSION: Methylation of miR-1247, miR-200a, and miR-200b in bile may be useful for distinguishing PBC from benign diseases.


Assuntos
Neoplasias do Sistema Biliar/genética , Metilação de DNA/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Bile/metabolismo , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade
2.
Medicine (Baltimore) ; 98(32): e16782, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31393404

RESUMO

INTRODUCTION: Over the past 10 years, epilepsy genetics has made dramatic progress. This study aimed to analyze the knowledge structure and the advancement of epilepsy genetics over the past decade based on co-word analysis of medical subject headings (MeSH) terms. METHODS: Scientific publications focusing on epilepsy genetics from the PubMed database (January 2009-December 2018) were retrieved. Bibliometric information was analyzed quantitatively using Bibliographic Item Co-Occurrence Matrix Builder (BICOMB) software. A knowledge social network analysis and publication trend based on the high-frequency MeSH terms was built using VOSviewer. RESULTS: According to the search strategy, a total of 5185 papers were included. Among all the extracted MeSH terms, 86 high-frequency MeSH terms were identified. Hot spots were clustered into 5 categories including: "ion channel diseases," "beyond ion channel diseases," "experimental research & epigenetics," "single nucleotide polymorphism & pharmacogenetics," and "genetic techniques". "Epilepsy," "mutation," and "seizures," were located at the center of the knowledge network. "Ion channel diseases" are typically in the most prominent position of epilepsy genetics research. "Beyond ion channel diseases" and "genetic techniques," however, have gradually grown into research cores and trends, such as "intellectual disability," "infantile spasms," "phenotype," "exome," " deoxyribonucleic acid (DNA) copy number variations," and "application of next-generation sequencing." While ion channel genes such as "SCN1A," "KCNQ2," "SCN2A," "SCN8A" accounted for nearly half of epilepsy genes in MeSH terms, a number of additional beyond ion channel genes like "CDKL5," "STXBP1," "PCDH19," "PRRT2," "LGI1," "ALDH7A1," "MECP2," "EPM2A," "ARX," "SLC2A1," and more were becoming increasingly popular. In contrast, gene therapies, treatment outcome, and genotype-phenotype correlations were still in their early stages of research. CONCLUSION: This co-word analysis provides an overview of epilepsy genetics research over the past decade. The 5 research categories display publication hot spots and trends in epilepsy genetics research which could consequently supply some direction for geneticists and epileptologists when launching new projects.


Assuntos
Bibliometria , Epilepsia/genética , Medical Subject Headings/estatística & dados numéricos , Epigenômica/métodos , Humanos , Canais Iônicos/genética , Mutação , Testes Farmacogenômicos/métodos , Fenótipo , Convulsões/genética
3.
Food Chem Toxicol ; 131: 110529, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150784

RESUMO

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 µmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.


Assuntos
Aldeídos/metabolismo , Fenóis/metabolismo , Proteômica/métodos , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Aldeídos/química , Domínio Catalítico , Ensaios Enzimáticos , Epigenômica/métodos , Ontologia Genética/estatística & dados numéricos , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Informática/métodos , Metiltransferases/química , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/metabolismo , Olea/química , Azeite de Oliva/química , Fenóis/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Software
4.
Nat Commun ; 10(1): 2581, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197173

RESUMO

Despite existing reports on differential DNA methylation in type 2 diabetes (T2D) and obesity, our understanding of its functional relevance remains limited. Here we show the effect of differential methylation in the early phases of T2D pathology by a blood-based epigenome-wide association study of 4808 non-diabetic Europeans in the discovery phase and 11,750 individuals in the replication. We identify CpGs in LETM1, RBM20, IRS2, MAN2A2 and the 1q25.3 region associated with fasting insulin, and in FCRL6, SLAMF1, APOBEC3H and the 15q26.1 region with fasting glucose. In silico cross-omics analyses highlight the role of differential methylation in the crosstalk between the adaptive immune system and glucose homeostasis. The differential methylation explains at least 16.9% of the association between obesity and insulin. Our study sheds light on the biological interactions between genetic variants driving differential methylation and gene expression in the early pathogenesis of T2D.


Assuntos
Metilação de DNA/fisiologia , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Insulina/metabolismo , Obesidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Simulação por Computador , Ilhas de CpG/genética , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética/fisiologia , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla/métodos , Homeostase/genética , Humanos , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Adulto Jovem
5.
Nat Commun ; 10(1): 2632, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201335

RESUMO

Chromatin loops connect regulatory elements to their target genes. They serve as bridges between transcriptional regulation and phenotypic variation in mammals. However, spatial organization of regulatory elements and its impact on gene expression in plants remain unclear. Here, we characterize epigenetic features of active promoter proximal regions and candidate distal regulatory elements to construct high-resolution chromatin interaction maps for maize via long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The maps indicate that chromatin loops are formed between regulatory elements, and that gene pairs between promoter proximal regions tend to be co-expressed. The maps also demonstrated the topological basis of quantitative trait loci which influence gene expression and phenotype. Many promoter proximal regions are involved in chromatin loops with distal regulatory elements, which regulate important agronomic traits. Collectively, these maps provide a high-resolution view of 3D maize genome architecture, and its role in gene expression and phenotypic variation.


Assuntos
Cromatina/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes/genética , Locos de Características Quantitativas/genética , Zea mays/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Produção Agrícola , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Epigenômica/métodos , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Mutação , Fenótipo , Regiões Promotoras Genéticas/genética
6.
Nat Commun ; 10(1): 1930, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036827

RESUMO

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.


Assuntos
Cromatina/química , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Cromatina/metabolismo , Regulação da Expressão Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/genética , Transposases/metabolismo
7.
Nat Commun ; 10(1): 2094, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064978

RESUMO

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with a modest concordance rate in monozygotic twins, which strongly argues for involvement of epigenetic factors. We observe highly similar peripheral blood mononuclear cell-based methylomes in 45 MS-discordant monozygotic twins. Nevertheless, we identify seven MS-associated differentially methylated positions (DMPs) of which we validate two, including a region in the TMEM232 promoter and ZBTB16 enhancer. In CD4 + T cells we find an MS-associated differentially methylated region in FIRRE. Additionally, 45 regions show large methylation differences in individual pairs, but they do not clearly associate with MS. Furthermore, we present epigenetic biomarkers for current interferon-beta treatment, and extensive validation shows that the ZBTB16 DMP is a signature for prior glucocorticoid treatment. Taken together, this study represents an important reference for epigenomic MS studies, identifies new candidate epigenetic markers, and highlights treatment effects and genetic background as major confounders.


Assuntos
Metilação de DNA/genética , Doenças em Gêmeos/genética , Epigênese Genética , Predisposição Genética para Doença , Esclerose Múltipla/genética , Adulto , Idoso , Biomarcadores , Doenças em Gêmeos/sangue , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Feminino , Humanos , Leucócitos Mononucleares , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , RNA Longo não Codificante/genética , Gêmeos Monozigóticos , Adulto Jovem
8.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035542

RESUMO

The etiology of cerebral palsy (CP) is complex and remains inadequately understood. Early detection of CP is an important clinical objective as this improves long term outcomes. We performed genome-wide DNA methylation analysis to identify epigenomic predictors of CP in newborns and to investigate disease pathogenesis. Methylation analysis of newborn blood DNA using an Illumina HumanMethylation450K array was performed in 23 CP cases and 21 unaffected controls. There were 230 significantly differentially-methylated CpG loci in 258 genes. Each locus had at least 2.0-fold change in methylation in CP versus controls with a FDR p-value ≤ 0.05. Methylation level for each CpG locus had an area under the receiver operating curve (AUC) ≥ 0.75 for CP detection. Using Artificial Intelligence (AI) platforms/Machine Learning (ML) analysis, CpG methylation levels in a combination of 230 significantly differentially-methylated CpG loci in 258 genes had a 95% sensitivity and 94.4% specificity for newborn prediction of CP. Using pathway analysis, multiple canonical pathways plausibly linked to neuronal function were over-represented. Altered biological processes and functions included: neuromotor damage, malformation of major brain structures, brain growth, neuroprotection, neuronal development and de-differentiation, and cranial sensory neuron development. In conclusion, blood leucocyte epigenetic changes analyzed using AI/ML techniques appeared to accurately predict CP and provided plausible mechanistic information on CP pathogenesis.


Assuntos
Inteligência Artificial , Ácidos Nucleicos Livres , Paralisia Cerebral/genética , Aprendizado Profundo , Epigênese Genética , Estudos de Casos e Controles , Paralisia Cerebral/sangue , Paralisia Cerebral/metabolismo , Ilhas de CpG , Metilação de DNA , Epigenômica/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Recém-Nascido , Curva ROC
9.
Nord J Psychiatry ; 73(4-5): 257-263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31070508

RESUMO

Background: Prenatal maternal stress increases the risk of offspring developmental and psychological difficulties. The biological mechanisms behind these associations are mostly unknown. One explanation suggests that exposure of the fetus to maternal stress may influence DNA methylation. However, this hypothesis is largely based on animal studies, and human studies of candidate genes from single timepoints. Aim: The aim of this study was to investigate if prenatal maternal stress, in the form of maternal depressive symptoms, was associated with variation in genome-wide DNA methylation at two timepoints. Methods: One-hundred and eighty-four mother-child dyads were selected from a population of pregnant women in the Little-in-Norway study. The Edinburgh Postnatal Depression Scale (EPDS) measured maternal depressive symptoms. It was completed by the pregnant mothers between weeks 17 and 32 of gestation. DNA was obtained from infant saliva cells at two timepoints (age 6 weeks and 12 months). DNA methylation was measured in 274 samples from 6 weeks (n = 146) and 12 months (n = 128) using the Illumina Infinium HumanMethylation 450 BeadChip. Linear regression analyses of prenatal maternal depressive symptoms and infant methylation were performed at 6 weeks and 12 months separately, and for both timepoints together using a mixed model. Results: The analyses revealed no significant genome-wide association between maternal depressive symptoms and infant DNA methylation in the separate analyses and for both timepoints together. Conclusions: This sample of pregnant women and their infants living in Norway did not reveal associations between maternal depressive symptoms and infant DNA methylation.


Assuntos
Metilação de DNA/fisiologia , Depressão/psicologia , Epigenômica/métodos , Complicações na Gravidez/psicologia , Efeitos Tardios da Exposição Pré-Natal/psicologia , Adulto , Animais , Depressão/epidemiologia , Depressão/genética , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Recém-Nascido , Estudos Longitudinais , Mães/psicologia , Noruega/epidemiologia , Gravidez , Complicações na Gravidez/epidemiologia , Complicações na Gravidez/genética , Efeitos Tardios da Exposição Pré-Natal/epidemiologia , Efeitos Tardios da Exposição Pré-Natal/genética , Adulto Jovem
10.
BMC Genomics ; 20(1): 366, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088362

RESUMO

BACKGROUND: There has been a steady increase in the number of studies aiming to identify DNA methylation differences associated with complex phenotypes. Many of the challenges of epigenetic epidemiology regarding study design and interpretation have been discussed in detail, however there are analytical concerns that are outstanding and require further exploration. In this study we seek to address three analytical issues. First, we quantify the multiple testing burden and propose a standard statistical significance threshold for identifying DNA methylation sites that are associated with an outcome. Second, we establish whether linear regression, the chosen statistical tool for the majority of studies, is appropriate and whether it is biased by the underlying distribution of DNA methylation data. Finally, we assess the sample size required for adequately powered DNA methylation association studies. RESULTS: We quantified DNA methylation in the Understanding Society cohort (n = 1175), a large population based study, using the Illumina EPIC array to assess the statistical properties of DNA methylation association analyses. By simulating null DNA methylation studies, we generated the distribution of p-values expected by chance and calculated the 5% family-wise error for EPIC array studies to be 9 × 10- 8. Next, we tested whether the assumptions of linear regression are violated by DNA methylation data and found that the majority of sites do not satisfy the assumption of normal residuals. Nevertheless, we found no evidence that this bias influences analyses by increasing the likelihood of affected sites to be false positives. Finally, we performed power calculations for EPIC based DNA methylation studies, demonstrating that existing studies with data on ~ 1000 samples are adequately powered to detect small differences at the majority of sites. CONCLUSION: We propose that a significance threshold of P < 9 × 10- 8 adequately controls the false positive rate for EPIC array DNA methylation studies. Moreover, our results indicate that linear regression is a valid statistical methodology for DNA methylation studies, despite the fact that the data do not always satisfy the assumptions of this test. These findings have implications for epidemiological-based studies of DNA methylation and provide a framework for the interpretation of findings from current and future studies.


Assuntos
Metilação de DNA , Epigenômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ilhas de CpG , Epigênese Genética , Estudo de Associação Genômica Ampla , Humanos , Modelos Lineares
11.
Hum Genet ; 138(6): 563-590, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31115652

RESUMO

Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeat (the CRISPR) system has been increasingly applied to therapeutic genome editing. Employment of several viral and non-viral vectors has enabled efficient delivery of the CRISPR system to target cells or tissues. In addition, the CRISPR system is able to modulate the target gene's expression in various ways, such as mutagenesis, gene integration, epigenome regulation, chromosomal rearrangement, base editing and mRNA editing. However, there are still limitations hindering an ideal application of the system: inefficient delivery, dysregulation of the delivered gene, the immune response against the CRISPR system, the off-target effects or the unintended on-target mutations. In addition, there are recent discoveries that have not been yet applied to CRISPR-mediated therapeutic genome editing. Here, we review the overall principles related to the therapeutic application of the CRISPR system, along with new strategies for the further application and prospects to overcome the limitations.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Transferência de Genes , Mutagênese , Epigenômica/métodos , Rearranjo Gênico , Terapia Genética/métodos , Humanos , Reprodutibilidade dos Testes
12.
Genes Cells ; 24(6): 449-457, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974043

RESUMO

To study the epigenetic gene silencing, yeast is an excellent model organism. Sir proteins are required for the formation of silent heterochromatin. Sir2 couples histone deacetylation and NAD hydrolysis to generate an endogenous epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). AAR is involved in the conformational change of SIR complexes, modulates the formation of SIR-nucleosome preheterochromatin and contributes to the spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin regions. Here, we show that AAR is capable of enhancing the chromatin silencing effect under either an extra exogenous AAR or a defect AAR metabolic enzyme situation, but decreasing the chromatin silencing effect under a defect AAR synthetic enzyme state. Our results provide an evidence of biological function importance of AAR. It is indicated that AAR does not only function in vitro but also play a role in vivo to increase the effect of heterochromatin epigenetic gene silencing. However, further investigations of AAR are warranted to expand our knowledge of epigenetics and associated small molecules.


Assuntos
Cromatina/genética , O-Acetil-ADP-Ribose/genética , O-Acetil-ADP-Ribose/metabolismo , Cromatina/fisiologia , Epigênese Genética/genética , Epigenômica/métodos , Inativação Gênica/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , O-Acetil-ADP-Ribose/fisiologia , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo
13.
BMC Genomics ; 20(Suppl 2): 193, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30967126

RESUMO

MOTIVATION: Quantitative detection of histone modifications has emerged in the recent years as a major means for understanding such biological processes as chromosome packaging, transcriptional activation, and DNA damage. However, high-throughput experimental techniques such as ChIP-seq are usually expensive and time-consuming, prohibiting the establishment of a histone modification landscape for hundreds of cell types across dozens of histone markers. These disadvantages have been appealing for computational methods to complement experimental approaches towards large-scale analysis of histone modifications. RESULTS: We proposed a deep learning framework to integrate sequence information and chromatin accessibility data for the accurate prediction of modification sites specific to different histone markers. Our method, named DeepHistone, outperformed several baseline methods in a series of comprehensive validation experiments, not only within an epigenome but also across epigenomes. Besides, sequence signatures automatically extracted by our method was consistent with known transcription factor binding sites, thereby giving insights into regulatory signatures of histone modifications. As an application, our method was shown to be able to distinguish functional single nucleotide polymorphisms from their nearby genetic variants, thereby having the potential to be used for exploring functional implications of putative disease-associated genetic variants. CONCLUSIONS: DeepHistone demonstrated the possibility of using a deep learning framework to integrate DNA sequence and experimental data for predicting epigenomic signals. With the state-of-the-art performance, DeepHistone was expected to shed light on a variety of epigenomic studies. DeepHistone is freely available in https://github.com/QijinYin/DeepHistone .


Assuntos
Cromatina/química , Aprendizado Profundo , Epigenômica/métodos , Regulação da Expressão Gênica , Histonas/química , Polimorfismo de Nucleotídeo Único , Cromatina/genética , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Histonas/genética , Humanos , Processamento de Proteína Pós-Traducional
14.
Methods Mol Biol ; 1979: 235-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028642

RESUMO

DNA methylation at cytosine is a major epigenetic mark, heavily implicated in controlling key cellular processes such as development and differentiation, cellular memory, or carcinogenesis. Bisulfite treatment in conjunction with next generation sequencing has been a powerful tool for studying this modification in a quantitative manner in the context of the whole genome and with a single nucleotide resolution. This chapter describes a protocol for bisulfite sequencing adapted to a single-cell format that allows for capturing the methylation signal from up to 50% CpG nucleotides in each cell.


Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Animais , Ilhas de CpG , Epigênese Genética , Epigenômica/métodos , Humanos , Reação em Cadeia da Polimerase/métodos , Sulfitos/química
15.
Methods Mol Biol ; 1979: 363-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028648

RESUMO

Single-cell transcriptome and single-cell methylome analysis have successfully revealed the heterogeneity in transcriptome and DNA methylome between single cells, and have become powerful tools to understand the dynamics of transcriptome and DNA methylome during the complicated biological processes, such as differentiation and carcinogenesis.Inspired by the success of using these single-cell -omics methods to understand the regulation of a particular "-ome," more interests have been put on elucidating the regulatory relationship among multiple-omics at single-cell resolution. The simultaneous profiling of multiple-omics from the same single cell would provide us the ultimate power to understand the relationship among different "-omes," but this idea is not materialized for decades due to difficulties to assay extremely tiny amount of DNA or RNA in a single cell.To address this technical challenge, we have recently developed a novel method named scMT-seq that can simultaneously profile both DNA methylome and RNA transcriptome from the same cell. This method enabled us to measure, from a single cell, the DNA methylation status of the most informative 0.5-1 million CpG sites and mRNA level of 10,000 genes, of which 3200 genes can be further analyzed with both promoter DNA methylation and RNA transcription. Using the scMT-seq data, we have successfully shown the regulatory relationship between DNA methylation and transcriptional level in a single dorsal root ganglion neuron (Hu et al., Genome Biol 17:88, 2016). We believe the scMT-seq would be a powerful technique to uncover the regulatory mechanism between transcription and DNA methylation, and would be of wide interest beyond the epigenetics community.


Assuntos
Metilação de DNA , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Análise de Célula Única/métodos , Animais , Ilhas de CpG , DNA/genética , Epigênese Genética , Humanos , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de RNA/métodos , Software , Transcriptoma
16.
SAR QSAR Environ Res ; 30(4): 265-277, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31012353

RESUMO

The growing interest in epigenetic probes and drug discovery, as revealed by several epigenetic drugs in clinical use or in the lineup of the drug development pipeline, is boosting the generation of screening data. In order to maximize the use of structure-activity relationships there is a clear need to develop robust and accurate models to understand the underlying structure-activity relationship. Similarly, accurate models should be able to guide the rational screening of compound libraries. Herein we introduce a novel approach for epigenetic quantitative structure-activity relationship (QSAR) modelling using conformal prediction. As a case study, we discuss the development of models for 11 sets of inhibitors of histone deacetylases (HDACs), which are one of the major epigenetic target families that have been screened. It was found that all derived models, for every HDAC endpoint and all three significance levels, are valid with respect to predictions for the external test sets as well as the internal validation of the corresponding training sets. Furthermore, the efficiencies for the predictions are above 80% for most data sets and above 90% for four data sets at different significant levels. The findings of this work encourage prospective applications of conformal prediction for other epigenetic target data sets.


Assuntos
Descoberta de Drogas , Epigenômica/métodos , Inibidores de Histona Desacetilases/química , Conformação Molecular , Relação Quantitativa Estrutura-Atividade
17.
Nat Methods ; 16(5): 397-400, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962623

RESUMO

We present cisTopic, a probabilistic framework used to simultaneously discover coaccessible enhancers and stable cell states from sparse single-cell epigenomics data ( http://github.com/aertslab/cistopic ). Using a compendium of single-cell ATAC-seq datasets from differentiating hematopoietic cells, brain and transcription factor perturbations, we demonstrate that topic modeling can be exploited for robust identification of cell types, enhancers and relevant transcription factors. cisTopic provides insight into the mechanisms underlying regulatory heterogeneity in cell populations.


Assuntos
Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Modelos Teóricos , Análise de Célula Única/métodos , Animais , Células Sanguíneas/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Análise por Conglomerados , Redes Reguladoras de Genes/genética , Humanos , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA , Fluxo de Trabalho
18.
BMC Genomics ; 20(1): 205, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866818

RESUMO

BACKGROUND: Plants adapted to diverse environments on Earth throughout their evolutionary history, and developed mechanisms to thrive in a variety of terrestrial habitats. When plants are grown in the novel environment of spaceflight aboard the International Space Station (ISS), an environment completely outside their evolutionary history, they respond with unique alterations to their gene expression profile. Identifying the genes important for physiological adaptation to spaceflight and dissecting the biological processes and pathways engaged by plants during spaceflight has helped reveal spaceflight adaptation, and has furthered understanding of terrestrial growth processes. However, the underlying regulatory mechanisms responsible for these changes in gene expression patterns are just beginning to be explored. Epigenetic modifications, such as DNA methylation at position five in cytosine, has been shown to play a role in the physiological adaptation to adverse terrestrial environments, and may play a role in spaceflight as well. RESULTS: Whole Genome Bisulfite Sequencing of DNA of Arabidopsis grown on the ISS from seed revealed organ-specific patterns of differential methylation compared to ground controls. The overall levels of methylation in CG, CHG, and CHH contexts were similar between flight and ground DNA, however, thousands of specifically differentially methylated cytosines were discovered, and there were clear organ-specific differences in methylation patterns. Spaceflight leaves had higher methylation levels in CHG and CHH contexts within protein-coding genes in spaceflight; about a fifth of the leaf genes were also differentially regulated in spaceflight, almost half of which were associated with reactive oxygen signaling. CONCLUSIONS: The physiological adaptation of plants to spaceflight is likely nuanced by epigenomic modification. This is the first examination of differential genomic methylation from plants grown completely in the spaceflight environment of the ISS in plant growth hardware developed for informing exploration life support strategies. Yet even in this optimized plant habitat, plants respond as if stressed. These data suggest that gene expression associated with physiological adaptation to spaceflight is regulated in part by methylation strategies similar to those engaged with familiar terrestrial stress responses. The differential methylation maps generated here provide a useful reference for elucidating the layers of regulation of spaceflight responses.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Metilação de DNA , Perfilação da Expressão Gênica/métodos , Adaptação Fisiológica , Arabidopsis/genética , Epigenômica/métodos , Regulação da Expressão Gênica de Plantas , Especificidade de Órgãos , Folhas de Planta/genética , Voo Espacial , Sequenciamento Completo do Genoma
19.
Nat Immunol ; 20(5): 546-558, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30911105

RESUMO

Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.


Assuntos
Epigênese Genética/imunologia , Epigenômica/métodos , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Transativadores/imunologia , Animais , Candida albicans/imunologia , Candida albicans/fisiologia , Candidíase/genética , Candidíase/imunologia , Candidíase/microbiologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Análise de Sobrevida , Transativadores/deficiência , Transativadores/genética , Transcriptoma/genética , Transcriptoma/imunologia
20.
Analyst ; 144(10): 3172-3189, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-30849139

RESUMO

As the basic units of life, cells present dramatic heterogeneity which, although crucial to an organism's behavior, is undetected by bulk analysis. Recently, much attention has been paid to reveal cellular types and states at the single-cell level including genome, transcriptome, epigenome or proteome-based on sequencing and immunological methods, etc. Among these approaches, transcriptomic analysis provides knowledge of the molecular linkages between genetic information and the proteome, leading to a comprehensive understanding of biological processes and diseases. Compared to single-dimensional inspection, multi-dimensional analysis combines the transcriptome with other "omics" to enable a comprehensive understanding of single-cell processes and functions. Moreover, compared to separate observations or single snapshots, spatial dimension and lineage time tracing can provide a more multifaceted understanding of the micro-environment and dynamic processes. In this review, we will introduce current transcriptomic analysis methods, as well as their combination with other omics methods including genomic, proteomic, and epigenetic approaches. Multi-dimensional analysis using these approaches for spatial positioning and lineage tracing applications will be reviewed. The future perspectives of single-cell multi-omics analysis based on the transcriptome will also be discussed.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Transcriptoma , Linhagem Celular Tumoral , Epigenômica/métodos , Genoma , Humanos , Proteoma , Proteômica/métodos
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