Deciphering “Immaturity-Stemness” in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status

Genome-wide location of yeast RNA polymerase III transcription machinery

RNA polymerase III (Pol III) transcribes a large set of genes encoding small untranslated RNAs like tRNAs, 5S rRNA, U6 snRNA or RPR1 RNA. To get a global view of class III (Pol III-transcribed) genes, the distribution of essential components of Pol III, TFIIIC and TFIIIB was mapped across the yeast genome. During active growth, most class III genes and few additional loci were targeted by TFIIIC, TFIIIB and Pol III, indicating that they were transcriptionally active. SNR52, which encodes a snoRNA, was identified as a new class III gene. During the late growth phase, TFIIIC remained bound to most class III genes while the recruitment of Pol III and, to a lesser extent, of TFIIIB was down regulated. This study fixes a reasonable upper bound to the number of class III genes in yeast and points to a global regulation at the level of Pol III and TFIIIB recruitment

Modulation of Yeast Genome Expression in Response to Defective RNA Polymerase III-Dependent Transcription

We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNA(Met). Initiator tRNA(Met) depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes

Mapping to 1q23 of the human gene (NDUFS2) encoding the 49-kDa subunit of the mitochondrial respiratory Complex I and immunodetection of the mature protein in mitochondria

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Novel pathway for megakaryocyte production after in vivo conditional eradication of integrin αIIb-expressing cells

International audienceOur knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet alphaIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains. In this context, we identified and characterized a novel population of Lin-Sca-1hi c-Kit- cells, with a lymphoid-like expression pattern, potentially involved in the reconstitution process. Our study revealed novel transcriptional cross talk between myeloid and lymphoid lineages and identified gene expression modifications that occur in vivo under these particular stress conditions, opening important prospects for therapeutic applications

Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases

RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 μg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes

Impairment of Base Excision Repair in Dermal Fibroblasts Isolated From Nevoid Basal Cell Carcinoma Patients

International audienceThe nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600–256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome

SUMATECS : Gestion durable des sols contaminés par les éléments traces : état de l'art et besoins de recherche

Le développement des technologies de remédiation in situ dites "douces" comme la phytoremédiation ou la stabilisation in situ, fait l'objet de recherches intenses depuis plusieurs dizaines d'années (Fig 1). D'importants progrès ont été réalisés au niveau expérimental mais l'application de ces technologies en pratique n'en est qu'à ses débuts. Le projet européen SUMATECS a pour objectif de faire un état de l'art à partir de la littérature et des projets disponibles, y compris les projets nationaux et les procédures locales, pour identifier le statut actuel de la recherche et des applications en Europe. Ce projet a également pour objectif de proposer des scénarios de remédiation incluant les impacts potentiels sur l'environnement local et de définir les futurs besoins de recherche. Plus particulièrement, le projet s'est attaché à (a) réaliser un état de l'art de la recherche et de la mise en pratique des technologies douces de remédiation, (b) réaliser un état de l'art des aspects environnementaux et socio-économiques associées à ces technologies, (c) de rassembler et d'évaluer les méthodes existantes pour déterminer les fractions d'éléments traces (métaux et non-métaux) pertinentes pour leur écotoxicologie (c'est à dire la fraction biodisponible), et (d) de réaliser un état de l'art des outils d'aide à la décision existants. En plus de l'approche bibliographique, des essais en laboratoire ayant pour objectif la valorisation de la biomasse contaminée ont été conduits. Une synthèse des résultats obtenus ainsi que les besoins de recherche identifiés seront présentés lors du colloque