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1.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
2.
Sci Total Environ ; 763: 142983, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131849

RESUMO

In environmental health, vulnerability reflecting the cumulative harmful constraints and nuisances to which populations are subjected and resilience defined as the capacity of a territory to cope with health inequalities have been little extensively investigated together with the same importance. Besides the diversity of factors involved, there is no consensual framework to develop composite indices, one recognized methodology to deal with a multifaceted issue. Therefore, this research aims to establish a new transferable approach to assess the spatial heterogeneity of territorial inequalities. This new strategy relies on the simultaneous evaluation of resilience and vulnerability and the joint analysis based on the cross-interpretation of the spatialized composite indices of resilience and vulnerability. A case study was conducted to demonstrate the feasibility of this methodology, using the municipality as a spatial unit of analysis within a region in the north of France. To provide the most holistic description possible of the 3817 studied municipalities, 50 variables related to the economic, environment, policy, health, services and social dimensions were used to develop the composite indices. The vulnerability Index has a median value of 0.151 with an IQR of [0.126-0.180] and the Resilience Index has a median value of 0.341 with an IQR of [0.273-0.401]. The joint analysis was conducted to classify each municipality among four defined typologies: 1687 municipalities (44.2%) belong to the "To monitor" category, 1646 (43.1%) to the "Resilient" category, 329 (8.6%) to the "Have resources" category and 155 (4.1%) to the "Territorial blackspot" category. The methodology herein may be a diagnostic tool to identify and prioritize municipalities that could benefit from the implementation of specifically tailored public health policies.

3.
Mol Brain ; 13(1): 156, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203444

RESUMO

Long-term memory formation is supported by functional and structural changes of neuronal networks, which rely on de novo gene transcription and protein synthesis. The modulation of the neuronal transcriptome in response to learning depends on transcriptional and post-transcriptional mechanisms. DNA methylation writers and readers regulate the activity-dependent genomic program required for memory consolidation. The most abundant DNA methylation reader, the Methyl CpG binding domain protein 2 (MeCP2), has been shown to regulate alternative splicing, but whether it establishes splicing events important for memory consolidation has not been investigated. In this study, we identified the alternative splicing profile of the mouse hippocampus in basal conditions and after a spatial learning experience, and investigated the requirement of MeCP2 for these processes. We observed that spatial learning triggers a wide-range of alternative splicing events in transcripts associated with structural and functional remodeling and that virus-mediated knockdown of MeCP2 impairs learning-dependent post-transcriptional responses of mature hippocampal neurons. Furthermore, we found that MeCP2 preferentially affected the splicing modalities intron retention and exon skipping and guided the alternative splicing of distinct set of genes in baseline conditions and after learning. Lastly, comparative analysis of the MeCP2-regulated transcriptome with the alternatively spliced mRNA pool, revealed that MeCP2 disruption alters the relative abundance of alternatively spliced isoforms without affecting the overall mRNA levels. Taken together, our findings reveal that adult hippocampal MeCP2 is required to finetune alternative splicing events in basal conditions, as well as in response to spatial learning. This study provides new insight into how MeCP2 regulates brain function, particularly cognitive abilities, and sheds light onto the pathophysiological mechanisms of Rett syndrome, that is characterized by intellectual disability and caused by mutations in the Mecp2 gene.

4.
J Mol Biol ; 432(15): 4270-4286, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32320688

RESUMO

Repetitive RNA (repRNA) sequences emerge as important regulators of the dynamic organization of genomic loci into membrane-less subcompartments with distinct nuclear functions. These domains include sites of active transcription like the nucleolus as well as (peri)centromeric and telomeric satellite repeats. Recent studies point to an important role of repRNAs in complex with proteins to promote a phase separation-driven formation of chromatin domains. We review how key features of the phase separation process can be revealed by different experimental approaches and discuss the associated structure-function relationships for chromatin subcompartments that involve repRNA.


Assuntos
Cromatina/metabolismo , RNA/metabolismo , Animais , Nucléolo Celular/metabolismo , Humanos , Sequências Repetitivas de Ácido Nucleico , Sítio de Iniciação de Transcrição
5.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
6.
Sci Rep ; 7(1): 2265, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28536419

RESUMO

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.


Assuntos
Ciclo Celular/genética , Mitose/genética , RNA Longo não Codificante/genética , Processamento Alternativo , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/genética , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Especificidade de Órgãos/genética , Proteômica/métodos , Interferência de RNA , Transporte de RNA , Imagem com Lapso de Tempo
7.
J AOAC Int ; 97(6): 1638-50, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25334093

RESUMO

Romer Labs , Inc. developed an immunochromatographic lateral flow assay for the qualitative detection of gluten in raw ingredients, processed foods, finished food products, and environmental surfaces, using the G12 antibody developed by Belén Morón. The G12 antibody targets a 33-mer peptide which is resistant to enzymatic digestion and heat denatiuration, as well as being the fragment of the gliadin protein.to which celiac disease sufferers react, making it a reliable analytical marker. This study was performed to validate the AgraStrip® GlutenG12 assay method under the guidance of the AOAC Peiformance Tested MethodsSM (PTM) program against AOAC Official Method of AnalysisSM 2012.01 in rice flour, bread, cookie, ice cream, and chocolate matrixes spiked with either purified gliadin or wheat gluten standard at 0, 3, 8, 15, and 25 ppm concentrations and tested at the 5, 10, and 20 ppm assay thresholds, as well as on, environmental surfaces. Stability, robustness, variation, and lot consistency studies were performed by spiking wheat gluten into a rice flour matrix at 0 and 15 ppm concentrations. The AgraStrip Gluten G12 assay was rigorously evaluated during this study and demonstrates its suitability as an AOAC PTM-certified gluten detection method.


Assuntos
Cromatografia de Afinidade/métodos , Análise de Alimentos/métodos , Glutens/análise , Anticorpos Imobilizados/química , Pão/análise , Cacau/química , Farinha/análise , Gliadina/análise , Sorvetes/análise , Oryza/química
8.
Nat Commun ; 5: 3056, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24445999

RESUMO

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.


Assuntos
Autofagia/fisiologia , Modelos Animais de Doenças , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Proteína 5 Relacionada à Autofagia , Progressão da Doença , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia
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