Expertise

La plateforme de génomique fonctionnelle de Nice Sophia Antipolis existe depuis 1999. Initialement orientée vers la conception, la fabrication et l'analyse de puces à ADN, elle a contribué à ouvrir cette nouvelle technologie à une large communauté, mettant à cette occasion en place un système d'information performant (Mediante), capable de gérer de grandes masses de données, et fonctionnant en production depuis plus de 10 ans.

Tout en fournissant encore aujourd'hui un service d'analyse de puces à ADN s'appuyant sur la technologie développée par Agilent, son activité s'est principalement réorientée vers des services de séquencage à haut-débit (Illumina NextSeq500), offrant dans ce contexte de nombreux types d'analyses des acides nucléiques, et une capacité pour analyser de grandes collections d'échantillons, y compris au niveau de la cellule unique. L'activité de routine concerne des applications comme le RNA-seq, le smallRNA-seq, le CHiP-seq, le CLIP-seq, le reséquencage, mais des projets spécifiques peuvent aussi etre mis en place dans des domaines moins standards, comme le séquencage de novo de génomes, ou certains protocoles particuliers : riboSeq, capSeq,... La plateforme se compose de 4 ingénieurs wet lab et de 4 bio-informaticiens.

Equipements

1. Pré-séquencage : Nanodrop, Bioanalyzer, Qubit, CovarisS2, Ion Chef, NeoPrep, Blue pippin
2. Analyse Single Cell : 10x Genomics Chromium, Fluidigm C1, Fluidigm Biomark
3. Séquencage : NextSeq500 Illumina, MinION et PromethION Oxford Nanopore Technology, Chromium 10X Genomics
4. Puces à ADN : High-Resolution Microarray Scanner Agilent, Station Affymetrix

Les résultats sont stockés automatiquement sur le portail d'informations de la plateforme Mediante. Cela concerne notamment les fichiers .BAM d'alignement, les fichiers .BW de couverture et l'ensemble des fichiers de l'analyse secondaire et des analyses statistiques conduites en partenariat avec le collaborateur. Sur demande l'ensemble des données brutes sont également mises à disposition et une aide est fournit pour la soumission des données vers la base de données publiques GEO (Gene Expression Omnibus).

Related publications

Popa Alexandra

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson-Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12 dimethylbenz[a]anthracene [DMBA] and 12-O-tetradecanoylphorbol-13-acetate [TPA], respectively) is associated with the upregulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically downregulating the expression of genes of the late-cornified-envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16. Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5'-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5'-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5'-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.

The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, we have developed 'RiboProfiling', a new Bioconductor open-source package. 'RiboProfiling' provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R. This article illustrates how to identify codon-motifs accumulating ribosome footprints, based on data from Escherichia coli.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

BACKGROUND: Open reading frames are common in long noncoding RNAs (lncRNAs) and 5'UTRs of protein coding transcripts (uORFs). The question of whether those ORFs are translated was recently addressed by several groups using ribosome profiling. Most of those studies concluded that certain lncRNAs and uORFs are translated, essentially based on computational analysis of ribosome footprints. However, major discrepancies remain on the scope of translation and the translational status of individual ORFs. In consequence, further criteria are required to reliably identify translated ORFs from ribosome profiling data. RESULTS: We examined the effect of the translation inhibitors pateamine A, harringtonine and puromycin on murine ES cell ribosome footprints. We found that pateamine A, a drug that targets eIF4A, allows a far more accurate identification of translated sequences than previously used drugs and computational scoring schemes. Our data show that at least one third but less than two thirds of ES cell lncRNAs are translated. We also identified translated uORFs in hundreds of annotated coding transcripts including key pluripotency transcripts, such as dicer, lin28, trim71, and ctcf. CONCLUSION: Pateamine A inhibition data clearly increase the precision of the detection of translated ORFs in ribosome profiling experiments. Our data show that translation of lncRNAs and uORFs in murine ES cells is rather common although less pervasive than previously suggested. The observation of translated uORFs in several key pluripotency transcripts suggests that translational regulation by uORFs might be part of the network that defines mammalian stem cell identity.

Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. RESULTS: Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 (-/-) MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 (-/-) MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 (-/-) MEF tumors grew faster than wild-type MEF tumors. CONCLUSIONS: Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.