Méthode d'analyse et de gestion du risque pour les ressources d'eau potable

Programmes, politiques et procédures -- Analyse du risque -- Index de vulnérabilité -- Délimitation de la zone d'intervention : rivière des prairies -- Caractéristiques des barrières -- Analyse de la vulnérabilité des prises d'eau face aux points de déficiences de la barrière réseau d'égouts -- Gestion de la performance des barrières

In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP

Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A). We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities

MRi of the knee compared to specialized radiography for measurements of articular cartilage height in knees with osteoarthritis

This study aims to evaluate and compare extremity-MRi with specialized radiography by measuring articular cartilage height in patients with knee osteoarthritis. A prospective study, including sixty patients. Measurements on MRi images, Rosenberg view, and coronal stress radiographs were performed. MRI was compared to specialized radiography. Measurements in the medial compartment showed negligible/weak correlation between MRi and Rosenber/varus stress. In the lateral compartment, MRi and the Rosenberg/valgus stress view were strongly correlated. We conclude that MRi cannot replace radiographs for the measurement of articular cartilage thickness. MRi should, however, be reserved for more unusual cases of atypical clinical findings

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates

Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3′-untranslated region (UTR), but also in the coding region (CR) and 5′-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation

Evidence for RNA synthesis in the intergenic region between enhancer and promoter and its inhibition by insulators in Drosophila melanogaster

Uncovering the nature of communication between enhancers, promoters and insulators is important for understanding the fundamental mechanisms that ensure appropriate gene expression levels. Here we describe an approach employing transient expression of genetic luciferase reporter gene constructs with quantitative RT–PCR analysis of transcription between an enhancer and Hsp70 promoter. We tested genetic constructs containing gypsy and/or Fab7 insulators in different orientations, and an enhancer from copia LTR-retroelement [(enh)copia]. A single gypsy or Fab7 insulator inserted between the promoter and enhancer in any polarity reduced enhancer action. A pair of insulators flanking the gene in any orientation exhibited increased insulation activity. We detected promoter-independent synthesis of non-coding RNA in the intergenic region of the constructs, which was induced by the enhancer in both directions and repressed by a single insulator or a pair of insulators. These results highlight the involvement of RNA-tracking mechanisms in the communications between enhancers and promoters, which are inhibited by insulators

Protein network study of human AF4 reveals its central role in RNA Pol II-mediated transcription and in phosphorylation-dependent regulatory mechanisms

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE (Fragile X E site) mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL (mixed-lineage leukaemia)-related leukaemogenesis, AF4-dependent physiological mechanisms have not been clearly defined. Proteins that interact with AF4 may also play important roles in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem MS and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology categories. Approximately 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. In the present paper we report one of the first extensive proteomic studies aimed at elucidating AF4 protein cross-talk. Moreover, we found that the AF4 residues Thr220 and Ser212 are phosphorylated, which suggests that AF4 function depends on phosphorylation mechanisms. We also mapped the AF4-interaction site with CDK9 (cyclin-dependent kinase 9), which is a direct interactor crucial for the function and regulation of the protein. The findings of the present study significantly expand the number of putative members of the multiprotein complex formed by AF4, which is instrumental in promoting the transcription/elongation of specific genes in human cells

Widespread Expression of BORIS/CTCFL in Normal and Cancer Cells

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a “cancer-testis” antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function