Identification of Fluorescent Compounds with Non-Specific Binding Property via High Throughput Live Cell Microscopy

10.1371/journal.pone.0028802PLoS ONE71

Colocalization of coregulated genes: a steered molecular dynamics study of human chromosome 19

The connection between chromatin nuclear organization and gene activity is vividly illustrated by the observation that transcriptional coregulation of certain genes appears to be directly influenced by their spatial proximity. This fact poses the more general question of whether it is at all feasible that the numerous genes that are coregulated on a given chromosome, especially those at large genomic distances, might become proximate inside the nucleus. This problem is studied here using steered molecular dynamics simulations in order to enforce the colocalization of thousands of knowledge-based gene sequences on a model for the gene-rich human chromosome 19. Remarkably, it is found that most, ~80% gene pairs can be brought simultaneously into contact. This is made possible by the low degree of intra-chromosome entanglement and the large number of cliques in the gene coregulatory network. A clique is a set of genes coregulated all together as a group. The constrained conformations for the model chromosome 19 are further shown to be organised in spatial macrodomains that are similar to those inferred from recent HiC measurements. The findings indicate that gene coregulation and colocalization are largely compatible and that this relationship can be exploited to draft the overall spatial organization of the chromosome in vivo. The more general validity and implications of these findings could be investigated by applying to other eukaryotic chromosomes the general and transferable computational strategy introduced here

Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms

Transcription factories in the context of the nuclear and genome organization

In the eukaryotic nucleus, genes are transcribed in transcription factories. In the present review, we re-evaluate the models of transcription factories in the light of recent and older data. Based on this analysis, we propose that transcription factories result from the aggregation of RNA polymerase II-containing pre-initiation complexes assembled next to each other in the nuclear space. Such an aggregation can be triggered by the phosphorylation of the C-terminal domain of RNA polymerase II molecules and their interaction with various transcription factors. Individual transcription factories would thus incorporate tissue-specific, co-regulated as well as housekeeping genes based only on their initial proximity to each other in the nuclear space. Targeting genes to be transcribed to protein-dense factories that contain all factors necessary for transcription initiation and elongation through chromatin templates clearly favors a more economical utilization and better recycling of the transcription machinery

Activation of Estrogen-Responsive Genes Does Not Require Their Nuclear Co-Localization

The spatial organization of the genome in the nucleus plays a role in the regulation of gene expression. Whether co-regulated genes are subject to coordinated repositioning to a shared nuclear space is a matter of considerable interest and debate. We investigated the nuclear organization of estrogen receptor alpha (ERα) target genes in human breast epithelial and cancer cell lines, before and after transcriptional activation induced with estradiol. We find that, contrary to another report, the ERα target genes TFF1 and GREB1 are distributed in the nucleoplasm with no particular relationship to each other. The nuclear separation between these genes, as well as between the ERα target genes PGR and CTSD, was unchanged by hormone addition and transcriptional activation with no evidence for co-localization between alleles. Similarly, while the volume occupied by the chromosomes increased, the relative nuclear position of the respective chromosome territories was unaffected by hormone addition. Our results demonstrate that estradiol-induced ERα target genes are not required to co-localize in the nucleus

Cellular processes of v-Src transformation revealed by gene profiling of primary cells - Implications for human cancer

<p>Abstract</p> <p>Background</p> <p>Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets.</p> <p>Methods</p> <p>CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets.</p> <p>Results</p> <p>The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis.</p> <p>Conclusion</p> <p>By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.</p

Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery

Recognition of prior experiential learning in higher education : national and local variants of a theme promoted by European cooperation (England, French-speaking Belgium and France)

La reconnaissance des acquis de l'expérience fait référence à des mesures permettant d'identifier et de valoriser les acquis des apprentissages réalisés en dehors des établissements d'enseignement et de formation. À présent, les dispositions de ce type existent ou se mettent en place dans de nombreux pays, et elles sont également promues dans le cadre de la coopération européenne en éducation et formation. Toutefois, la reconnaissance des acquis de l'expérience ne se réalise pas nécessairement dans tous les secteurs de l'éducation et de la formation, sa mise en place étant particulièrement délicate dans le secteur de l'enseignement supérieur. Partant de ces éléments, cette recherche se penche sur trois systèmes d'enseignement supérieur relativement avancés en la matière : l'Angleterre, la Belgique francophone et la France. En interrogeant l'institutionnalisation et la mise en oeuvre de la reconnaissance des acquis de l'expérience dans ces trois contextes, la recherche pose la question de savoir si, sous l'influence des processus supranationaux, les dispositifs de reconnaissance des acquis de l'expérience convergent vers les mêmes modalités de réalisation. Une triple perspective est adoptée pour explorer cette question. Premièrement, il s'agit d'examiner le rôle de la coopération européenne dans la mise en place des dispositifs nationaux de reconnaissance des acquis de l'expérience. Deuxièmement, il s'agit d'étudier le cheminement de la reconnaissance des acquis de l'expérience dans les trois systèmes investigués. Troisièmement, il s'agit de mettre en lumière la façon dont se réalise la reconnaissance des acquis de l'expérience dans les établissements d'enseignement supérieur, en l'occurrence les universités. Les deux premiers volets de la recherche s'appuient principalement sur le recueil et l'analyse des sources écrites, y compris la réglementation, les lignes directrices officielles, les rapports d'analyse et les travaux de recherche. L'investigation au niveau institutionnel est centrée sur un échantillon de trois à quatre universités dans chaque système étudié, et elle s'appuie sur des visites de sites, des entretiens avec des acteurs impliqués dans la reconnaissance des acquis de l'expérience et l'information diffusée par les universités. La recherche met en évidence le fait que les dispositifs de reconnaissance des acquis de l'expérience s'élaborent en fonction de nombreux paramètres tels que l'organisation et la structuration de l'enseignement supérieur, la perception du rôle de l'enseignement supérieur ou encore la présence des acteurs individuels et collectifs aptes à promouvoir ces dispositifs. La mouvance européenne autour de la reconnaissance des acquis de l'expérience apparaît ainsi comme un cadre de référence général, prêt à se décliner différemment dans chaque système d'enseignement supérieur.The recognition of prior experiential learning refers to arrangements that aim to identify and valorise learning outcomes achieved outside education and training institutions. At present, such arrangements exist or are being introduced in a number of countries, and are also promoted in the framework of European cooperation in education and training. However, the recognition of prior experiential learning does not always apply to all sectors of education and training, its implementation being particularly challenging in the higher education sector. Starting from these elements, this research looks at three higher education systems considered relatively advanced in this field: England, French-speaking Belgium and France. By researching the institutionalisation and implementation of the recognition of prior experiential learning in the aforementioned systems, the study raises the question of whether, under the influence of supranational processes, approaches to the recognition of prior experiential learning converge towards the same model. A three-point perspective is adopted to explore this issue. First, the research examines the role of European cooperation in the development of national arrangements for the recognition of prior experiential learning. Second, the study looks at the evolution of the recognition of prior experiential learning in the investigated systems. Finally, the analysis considers how the recognition of prior experiential learning is being implemented in higher education institutions, in this case, universities. The investigation related to the first two areas mainly relies on the collection and analysis of written sources, including regulations, official guidelines, analysis reports and research. The institutional analysis focuses on a sample of three to four universities in each studied system, and is based on site visits, interviews with actors involved in the recognition of prior experiential learning and the information disseminated by universities. Research shows that the development of arrangements for the recognition of prior experiential learning is influenced by a number of factors, including the organisation and structure of higher education systems, the perception of the role of higher education, as well as the presence of individual and collective actors able to promote these arrangements. Thus, European activities in the area of the recognition of prior experiential learning appear as a general frame of reference that translates into different arrangements in each higher education system