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1.
Methods ; 170: 33-37, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283985

RESUMO

Genome organization is now understood to be tightly linked to all genomic functions. Thus, the high-resolution mapping of higher-order chromosomal structures via 3C-based approaches has become an integral tool for studying transcriptional and cell cycle regulation, signaling effects or disease onset. Nonetheless, 3C-based protocols are not without caveats, like dependencies on fixation conditions, restriction enzyme pervasiveness in crosslinked chromatin and ligation efficiency. To address some of these caveats, we describe here the streamlined iHi-C 2.0 protocol that allows for the genome-wide interrogation of native spatial chromatin contacts without a need for chemical fixation. This approach improves ligation efficiency and presents minimal material losses, and is thus suitable for analysing samples with limiting cell numbers. Following high throughput sequencing, iHi-C 2.0 generates high signal-to-noise and focal maps of the interactions within and between mammalian chromosomes under native conditions.

2.
Mol Cell ; 75(2): 267-283.e12, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31202576

RESUMO

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.


Assuntos
DNA Topoisomerases Tipo II/genética , Instabilidade Genômica/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Translocação Genética/genética , Fator de Ligação a CCCTC/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Humanos , Leucemia/genética , Leucemia/patologia
3.
Mol Cell ; 70(4): 730-744.e6, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29706538

RESUMO

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Genoma Humano , Proteína HMGB2/metabolismo , Fator de Ligação a CCCTC/genética , Proliferação de Células , Senescência Celular , Cromatina/genética , Proteína HMGB2/genética , Células Endoteliais da Veia Umbilical Humana , Humanos
4.
Genome Res ; 26(11): 1478-1489, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27633323

RESUMO

Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κΒ and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κΒ that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.


Assuntos
Sequência Consenso , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Células Cultivadas , Cromatina/metabolismo , Edição de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , NF-kappa B/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
5.
Nat Methods ; 13(4): 303-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26901649

RESUMO

DNase-seq allows nucleotide-level identification of transcription factor binding sites on the basis of a computational search of footprint-like DNase I cleavage patterns on the DNA. Frequently in high-throughput methods, experimental artifacts such as DNase I cleavage bias affect the computational analysis of DNase-seq experiments. Here we performed a comprehensive and systematic study on the performance of computational footprinting methods. We evaluated ten footprinting methods in a panel of DNase-seq experiments for their ability to recover cell-specific transcription factor binding sites. We show that three methods--HINT, DNase2TF and PIQ--consistently outperformed the other evaluated methods and that correcting the DNase-seq signal for experimental artifacts significantly improved the accuracy of computational footprints. We also propose a score that can be used to detect footprints arising from transcription factors with potentially short residence times.


Assuntos
Biologia Computacional/métodos , Pegada de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Software , Fatores de Transcrição/metabolismo , Algoritmos , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Desoxirribonuclease I/metabolismo , Humanos , Células K562 , Ligação Proteica
6.
Nucleic Acids Res ; 43(20): 9680-93, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26476451

RESUMO

Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Multipotent progenitors (MPP) are committed to DC restricted common DC progenitors (CDP), which differentiate into specific DC subsets, classical DC (cDC) and plasmacytoid DC (pDC). To determine epigenetic states and regulatory circuitries during DC differentiation, we measured consecutive changes of genome-wide gene expression, histone modification and transcription factor occupancy during the sequel MPP-CDP-cDC/pDC. Specific histone marks in CDP reveal a DC-primed epigenetic signature, which is maintained and reinforced during DC differentiation. Epigenetic marks and transcription factor PU.1 occupancy increasingly coincide upon DC differentiation. By integrating PU.1 occupancy and gene expression we devised a transcription factor regulatory circuitry for DC commitment and subset specification. The circuitry provides the transcription factor hierarchy that drives the sequel MPP-CDP-cDC/pDC, including Irf4, Irf8, Tcf4, Spib and Stat factors. The circuitry also includes feedback loops inferred for individual or multiple factors, which stabilize distinct stages of DC development and DC subsets. In summary, here we describe the basic regulatory circuitry of transcription factors that drives DC development.


Assuntos
Células Dendríticas/metabolismo , Epigênese Genética , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo
7.
Clin Epigenetics ; 7: 19, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25763115

RESUMO

BACKGROUND: Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome. RESULTS: In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes. CONCLUSIONS: Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

8.
Bioinformatics ; 30(22): 3143-51, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25086003

RESUMO

MOTIVATION: The identification of active transcriptional regulatory elements is crucial to understand regulatory networks driving cellular processes such as cell development and the onset of diseases. It has recently been shown that chromatin structure information, such as DNase I hypersensitivity (DHS) or histone modifications, significantly improves cell-specific predictions of transcription factor binding sites. However, no method has so far successfully combined both DHS and histone modification data to perform active binding site prediction. RESULTS: We propose here a method based on hidden Markov models to integrate DHS and histone modifications occupancy for the detection of open chromatin regions and active binding sites. We have created a framework that includes treatment of genomic signals, model training and genome-wide application. In a comparative analysis, our method obtained a good trade-off between sensitivity versus specificity and superior area under the curve statistics than competing methods. Moreover, our technique does not require further training or sequence information to generate binding location predictions. Therefore, the method can be easily applied on new cell types and allow flexible downstream analysis such as de novo motif finding. AVAILABILITY AND IMPLEMENTATION: Our framework is available as part of the Regulatory Genomics Toolbox. The software information and all benchmarking data are available at http://costalab.org/wp/dh-hmm. CONTACT: ivan.costa@rwth-aachen.de or eduardo.gusmao@rwth-aachen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Imunoprecipitação da Cromatina , Desoxirribonuclease I , Histonas/metabolismo , Elementos Reguladores de Transcrição , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Sítios de Ligação , Genômica , Humanos , Células K562 , Cadeias de Markov , Pegadas de Proteínas
9.
Nucleic Acids Res ; 42(11): 6901-20, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24782528

RESUMO

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Metilação , Proteínas Nucleares/antagonistas & inibidores , Regiões Promotoras Genéticas , Fatores de Transcrição/antagonistas & inibidores
10.
Ciênc. agrotec., (Impr.) ; 32(4): 1073-1079, jul.-ago. 2008. graf, tab
Artigo em Português | LILACS-Express | ID: lil-489938

RESUMO

Estudos de biometria de frutos e sementes são importantes para o entendimento da variabilidade existente nas espécies nativas. Apesar da importância ecológica e econômica de Talisia esculenta, não se conhecem as características biométricas dos frutos e não há informações sobre a emergência das plântulas. Objetivou-se caracterizar fisicamente frutos e sementes e estudar os efeitos do armazenamento sobre a emergência das plântulas. Analisou-se a biometria de 260 frutos e sementes. A emergência de plântulas foi avaliada a partir de sementes recém-coletadas e armazenadas por 15, 30 e 60 dias. As médias de massa de matéria fresca, comprimento e diâmetro dos frutos foram, respectivamente, 15,02 g, 32,59 mm e 26,33 mm e para as sementes 4,49 g, 25,08 mm e 13,62 mm. A massa de matéria fresca do fruto foi proporcional à quantidade de polpa (rS = 0,826; P < 0,05), indicando potencial para a seleção e melhoramento genético da espécie. A emergência das plântulas de Talisia esculenta obtidas de sementes recém-colhidas foi de 88 por cento. As sementes possuem longevidade curta, com ausência de germinação após 30 e 60 dias de armazenamento.


Studies of biometrical characteristics of fruits and seeds are important to understand the variability of native species. In spite of the ecological and economical importance of Talisia esculenta, the biometrical characteristics of fruits and information about seedling emergence are unknown. The aim of this paper was to characterize fruits and seeds biometrically and to determine the effects of storage on seedling emergence. The biometry of 260 fruits and seeds was analyzed. The seedling emergence of newly-collected seeds and after 15, 30, and 60 days of storage was evaluated. The means for the fruits fresh mass, length, and diameter were, respectively, 15.02 g, 32.59 mm, and 26.33 mm and for the seeds 4.49 g, 25.08 mm, and 13.62 mm. The fresh mass of the fruit was proportional to the pulp amount (rS = 0.826; P < 0.05). These values indicated the potential for the selection and genetic improvement of the species. The seedling emergence of newly-collected seeds was 88 percent. The seeds of Talisia esculenta have a short viability period, with absence of germination at 30 and 60 days of storage.

11.
Esc. Anna Nery Rev. Enferm ; 11(3): 530-536, set. 2007. graf
Artigo em Português | LILACS | ID: lil-478307

RESUMO

Trata da experiência de oito anos de implantação do Programa "Fábrica de Cuidados - um espaço para criar modelos e tecnologias em saúde". Questão norteadora: quanto tempo é necessário para a criação de relações seguras/sustentáveis entre docentes e comunidade e que problemas/dificuldades são vividos no cotidiano de gerenciar um programa comunitário?...


Assuntos
Humanos , Relações Interpessoais , Medicina de Família e Comunidade/métodos , Medicina de Família e Comunidade/organização & administração , Organização e Administração
12.
Esc. Anna Nery Rev. Enferm ; 11(3): 530-536, set. 2007. graf
Artigo em Português | BDENF - Enfermagem | ID: bde-15397

RESUMO

Trata da experiência de oito anos de implantação do Programa "Fábrica de Cuidados - um espaço para criar modelos e tecnologias em saúde". Questão norteadora: quanto tempo é necessário para a criação de relações seguras/sustentáveis entre docentes e comunidade e que problemas/dificuldades são vividos no cotidiano de gerenciar um programa comunitário?...(AU)


Assuntos
Humanos , Relações Interpessoais , Organização e Administração , Medicina de Família e Comunidade/métodos , Medicina de Família e Comunidade/organização & administração
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