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1.
Nat Cell Biol ; 21(9): 1164-1172, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481796

RESUMO

Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. Here, we present a low-cost, efficient, simultaneous indexing and tagmentation-based ChIP-seq (itChIP-seq) method, compatible with both low cellular input and single cells for profiling chromatin states. itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation within a single tube, enabling the processing of samples from tens of single cells to, more commonly, thousands of single cells per assay. We demonstrate that single-cell itChIP-seq (sc-itChIP-seq) yields ~9,000 unique reads per cell. Using sc-itChIP-seq to profile H3K27ac, we sufficiently capture the earliest epigenetic priming event during the cell fate transition from naive to primed pluripotency, and reveal the basis for cell-type specific enhancer usage during the differentiation of bipotent cardiac progenitor cells into endothelial cells and cardiomyocytes. Our results demonstrate that itChIP is a widely applicable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes.


Assuntos
Cromatina/metabolismo , Células Endoteliais/metabolismo , Processamento de Proteína Pós-Traducional/genética , Análise de Sequência de DNA , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Histonas/metabolismo , Camundongos Transgênicos , Análise de Sequência de DNA/métodos
2.
Nat Methods ; 16(9): 858-861, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406384

RESUMO

The decoding of transcription factor (TF) binding signals in genomic DNA is a fundamental problem. Here we present a prediction model called BindSpace that learns to embed DNA sequences and TF labels into the same space. By training on binding data from hundreds of TFs and embedding over 1 M DNA sequences, BindSpace achieves state-of-the-art multiclass binding prediction performance, in vitro and in vivo, and can distinguish between signals of closely related TFs.


Assuntos
Algoritmos , Biologia Computacional/métodos , DNA/metabolismo , Aprendizado de Máquina , Fatores de Transcrição/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/química , Humanos , Ligação Proteica
3.
Genes Dev ; 33(17-18): 1159-1174, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371436

RESUMO

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.


Assuntos
Nucleossomos/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo/metabolismo
4.
Nucleic Acids Res ; 47(15): 7825-7841, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31299083

RESUMO

The understanding of the multi-scale nature of molecular networks represents a major challenge. For example, regulation of a timely cell cycle must be coordinated with growth, during which changes in metabolism occur, and integrate information from the extracellular environment, e.g. signal transduction. Forkhead transcription factors are evolutionarily conserved among eukaryotes, and coordinate a timely cell cycle progression in budding yeast. Specifically, Fkh1 and Fkh2 are expressed during a lengthy window of the cell cycle, thus are potentially able to function as hubs in the multi-scale cellular environment that interlocks various biochemical networks. Here we report on a novel ChIP-exo dataset for Fkh1 and Fkh2 in both logarithmic and stationary phases, which is analyzed by novel and existing software tools. Our analysis confirms known Forkhead targets from available ChIP-chip studies and highlights novel ones involved in the cell cycle, metabolism and signal transduction. Target genes are analyzed with respect to their function, temporal expression during the cell cycle, correlation with Fkh1 and Fkh2 as well as signaling and metabolic pathways they occur in. Furthermore, differences in targets between Fkh1 and Fkh2 are presented. Our work highlights Forkhead transcription factors as hubs that integrate multi-scale networks to achieve proper timing of cell division in budding yeast.


Assuntos
Proteínas de Ciclo Celular/genética , DNA Fúngico/química , Fatores de Transcrição Forkhead/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Ontologia Genética , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
5.
Immunity ; 51(1): 10-12, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315029

RESUMO

In this issue of Immunity, Chisolm et al. (2019) issue a "three-alarm fire" warning to the immunology research community of unexpectedly widespread genetic variation in widely used congenic mouse strains and provide a simple method to identify such a variation through a re-analysis of existing RNA-seq and ChIP-seq datasets.


Assuntos
Variação Genética , Animais , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , Camundongos
6.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(6): 692-698, 2019 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-31270048

RESUMO

OBJECTIVE: To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. METHODS: Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. RESULTS: Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. CONCLUSIONS: Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação da Cromatina , DNA , Biblioteca Gênica , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNA
7.
Nat Commun ; 10(1): 2931, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270321

RESUMO

The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call 'Pseudomonas aeruginosa genomic regulatory network' (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.


Assuntos
Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Imunoprecipitação da Cromatina , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fatores de Transcrição/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Nucleic Acids Res ; 47(15): 7870-7885, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31226208

RESUMO

Long interspersed elements-1 (LINE-1, L1) are retrotransposons that hold the capacity of self-propagation in the genome with potential mutagenic outcomes. How somatic cells restrict L1 activity and how this process becomes dysfunctional during aging and in cancer cells is poorly understood. L1s are enriched at lamin-associated domains, heterochromatic regions of the nuclear periphery. Whether this association is necessary for their repression has been elusive. Here we show that the sirtuin family member SIRT7 participates in the epigenetic transcriptional repression of L1 genome-wide in both mouse and human cells. SIRT7 depletion leads to increased L1 expression and retrotransposition. Mechanistically, we identify a novel interplay between SIRT7 and Lamin A/C in L1 repression. Our results demonstrate that SIRT7-mediated H3K18 deacetylation regulates L1 expression and promotes L1 association with elements of the nuclear lamina. The failure of such activity might contribute to the observed genome instability and compromised viability in SIRT7 knockout mice. Overall, our results reveal a novel function of SIRT7 on chromatin organization by mediating the anchoring of L1 to the nuclear envelope, and a new functional link of the nuclear lamina with transcriptional repression.


Assuntos
Genoma , Lamina Tipo A/genética , Elementos Nucleotídeos Longos e Dispersos , Sirtuínas/genética , Transcrição Genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Lamina Tipo A/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Miocárdio/citologia , Miocárdio/metabolismo , Lâmina Nuclear/metabolismo , Lâmina Nuclear/ultraestrutura , Sirtuínas/deficiência , Sirtuínas/metabolismo , Testículo/citologia , Testículo/metabolismo
9.
Nat Immunol ; 20(7): 890-901, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209400

RESUMO

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Infecção/etiologia , Análise de Célula Única , Animais , Biomarcadores , Imunoprecipitação da Cromatina , Epigênese Genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , Infecção/metabolismo , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Fatores de Tempo , Transcriptoma
10.
Nat Genet ; 51(6): 1060-1066, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31152164

RESUMO

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.


Assuntos
Neoplasias da Mama/genética , Imunoprecipitação da Cromatina , Cromatina/genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Cromatina/metabolismo , Biologia Computacional/métodos , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Análise de Célula Única/métodos , Células Estromais , Fluxo de Trabalho
11.
Nat Commun ; 10(1): 2633, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201330

RESUMO

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal-distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética/genética , Zea mays/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Produção Agrícola , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Histonas/genética , Histonas/imunologia , Regiões Promotoras Genéticas , Locos de Características Quantitativas/genética
12.
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
13.
Nucleic Acids Res ; 47(11): 5735-5745, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31114922

RESUMO

High-occupancy target (HOT) regions are segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and consequently associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solely defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed 'hyper-ChIPable' regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpG-rich k-mers, enrichment of RNA-DNA hybrids (R-loops) and DNA tertiary structures (G-quadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies.


Assuntos
Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Motivos de Aminoácidos , Animais , Artefatos , Caenorhabditis elegans , DNA/química , Drosophila melanogaster , Reações Falso-Positivas , Quadruplex G , Genoma , Genoma Humano , Genômica , Humanos , Camundongos , Ligação Proteica , Domínios Proteicos , RNA/química , Análise de Sequência de DNA
14.
Stud Health Technol Inform ; 260: 105-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118325

RESUMO

BACKGROUND: ChIP-seq is a method to identify genome-wide transcription factor (TF) binding sites. The TF FXR is a nuclear receptor that controls gene regulation of different metabolic pathways in the liver. OBJECTIVES: To re-analyze, standardize and combine all publicly available FXR ChIP-seq data sets to create a global FXR signaling atlas. METHODS: All data sets were (re-)analyzed in a standardized manner and compared on every relevant level from raw reads to affected functional pathways. RESULTS: Public FXR data sets were available for mouse, rat and primary human hepatocytes in different treatment conditions. Standardized re-analysis shows that the data sets are surprisingly heterogeneous concerning baseline quality criteria. Combining different data sets increased the depth of analysis and allowed to recover more peaks and functional pathways. CONCLUSION: Published single FXR ChIP-seq data sets do not cover the full spectrum of FXR signaling. Combining different data sets and creating a "FXR super-signaling atlas" enhances understanding of FXR signaling capacities.


Assuntos
Análise de Dados , Regulação da Expressão Gênica , Genoma , Receptores Citoplasmáticos e Nucleares , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Bases de Dados Factuais , Humanos , Camundongos , Ratos
15.
Plant Sci ; 284: 192-202, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084872

RESUMO

In rice, OsBBX14, a B-box (BBX) transcription factor, reportedly delays heading. Here, we revealed that OsBBX14 positively regulates rice photomorphogenesis. The OsBBX14-overexpressing (OsBBX14-OX) seedlings were hypersensitive to light, especially blue light, and exhibited dwarfism, while the OsBBX14 knock-out plants (osbbx14) were taller than wild-type plants under blue light. Histological analyses indicated that the observed dwarfism was mainly due to decreased cell length. Additionally, OsBBX14 abundance (mRNA and protein levels) was influenced by different light wavelengths in a time-dependent manner. The expression levels of HY5Ls (LONG HYPOCOTYL 5 LIKE) and ELIPs (EARLY LIGHT-INDUCIBLE PROTEIN) genes, whose Arabidopsis thaliana homologs function as positive regulators in the light signaling pathway, were significantly upregulated in OsBBX14-OX lines. In contrast, the expression of genes related to cell wall organization and dwarfism was downregulated in OsBBX14-OX lines. Chromatin immunoprecipitation (ChIP) assays confirmed that OsBBX14 binds to the T/G-box of HY5L1 (LONG HYPOCOTYL 5 LIKE 1) promoter. LUC complementation imaging (LCI) results suggested that OsBBX14 had physical interaction with OsCRY2 protein. Collectively, in response to blue light, OsBBX14 promotes photomorphogenesis, probably by directly or indirectly regulating the expression of HY5L1 or other genes related to cell wall organization and dwarfism.


Assuntos
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Tabaco , Técnicas do Sistema de Duplo-Híbrido
16.
J Biotechnol ; 300: 63-69, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31129070

RESUMO

While chromatin immunoprecipitation has become a widely-used method in the field of transcription regulation studies, serious limitations connected to the complexity and relatively little standardization of the method serve as obstacles for its use in clinical research. In this paper we introduce a method for developing bacteriophage-based controls for the better standardization of the chromatin immunoprecipitation reactions. Random phage display libraries were selected with ChIP-grade antibodies for several rounds and individual monoclonal phages were isolated. These monoclonal phages can be propagated, characterized, capillary sequenced and if needed later cloned from in-silico data. Using such control tools allows for a better characterization of the immunoprecipitation stage needed for further clinical research in the field of chromatin-immunoprecipitation-based studies.


Assuntos
Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Bacteriófagos/genética , Imunoprecipitação da Cromatina/normas , Células HEK293 , Humanos , Biblioteca de Peptídeos , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
Methods Mol Biol ; 1965: 389-403, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069688

RESUMO

Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500 bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.


Assuntos
Encéfalo/embriologia , Imunoprecipitação da Cromatina/métodos , DNA/metabolismo , Histonas/metabolismo , Acetilação , Animais , Sítios de Ligação , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA/química , Epigênese Genética , Metilação , Camundongos , Processamento de Proteína Pós-Traducional , Reação em Cadeia da Polimerase em Tempo Real
18.
Methods Mol Biol ; 1966: 39-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041738

RESUMO

Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) is a powerful tool to map context-dependent genome-wide binding of nuclear hormone receptors and their coregulators. This information can provide important mechanistic insight into where, when and how DNA-protein interactions are linked to target gene regulation. Here we describe a simple, yet reliable ChIP-seq method, including nuclear isolation from frozen tissue samples, cross-linking DNA-protein complexes, chromatin shearing, immunoprecipitation, and purification of ChIP DNA. We also include a standard ChIP-seq data analysis pipeline to elaborate and analyze raw single-end or paired-end sequencing data, including quality control steps, peak calling, annotation, and motif enrichment.


Assuntos
Imunoprecipitação da Cromatina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , DNA/metabolismo , Humanos , Análise de Sequência de DNA/métodos
19.
BMC Genomics ; 20(1): 344, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064321

RESUMO

BACKGROUND: Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. RESULTS: Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5' end extension, 3' end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3'-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in > 1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data. CONCLUSIONS: These validated results show significant improvement over current pig genome annotations.


Assuntos
Processamento Alternativo , Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Animais , Sus scrofa
20.
Genes Cells ; 24(7): 511-517, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31095817

RESUMO

Centromeres play crucial roles in faithful chromosome segregation and genome integrity. In simian primates, centromeres possess tandem array of alpha satellite DNA (also referred to as alphoid DNA). Average sizes of alpha satellite repeat units vary between species, for example, 171 bp in human and 343-344 bp in many platyrrhini species (New World monkeys). Interestingly, Azara's owl monkey (Aotus azarae), a platyrrhini species, possesses alpha satellite DNA of two distinct unit sizes, OwlAlp1 (185 bp) and OwlAlp2 (344 bp), both of which present as megasatellite DNAs in the genome. It is, however, unknown which repeat sequence is responsible for functional centromere formation. To investigate the localization of centromeres in vivo, we carried out chromatin immunoprecipitation (ChIP) assay using Azara's owl monkey cells. We found that CENP-A, a histone H3 variant essential for centromere formation, was enriched at OwlAlp1, but not at OwlAlp2. Moreover, CENP-A was detected only at constricted regions of chromosomes by immunofluorescent microscopy. In contrast, trimethylation of histone H3-K9 (H3K9me3), a marker of heterochromatin, was enriched at both OwlAlp1 and OwlAlp2. Our results show that the shorter alpha satellite repeat, OwlAlp1, is selectively used for centromere formation in this monkey.


Assuntos
Aotidae/genética , Proteína Centromérica A/metabolismo , Centrômero , DNA Satélite , Heterocromatina , Animais , Células Cultivadas , Proteína Centromérica A/genética , Proteína Centromérica A/imunologia , Imunoprecipitação da Cromatina , Histonas/genética , Humanos
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