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

Zaragosi Laure-Emmanuelle

The study of the airway epithelium in vitro is routinely performed using air-liquid culture (ALI) models from nasal or bronchial basal cells. These 3D experimental models allow to follow the regeneration steps of fully differentiated mucociliary epithelium and to study gene function by performing gene invalidation. Recent progress made with CRISPR-based techniques has overcome the experimental difficulty of this approach, by a direct transfection of ribonucleoprotein complexes combining a mix of synthetic small guide RNAs (sgRNAs) and recombinant Cas9. The approach shows more than 95% efficiency and does not require any selection step. A limitation of this approach is that it generates cell populations that contain heterogeneous deletions, which makes the evaluation of invalidation efficiency difficult. We have successfully used Flongle sequencing (Nanopore) to quantify the number of distinct deletions. We describe the use of CRISPR-Cas9 RNP in combination with single-cell RNA sequencing to functionally characterize the impact of gene invalidation in ALI cultures. The complex ecosystem of the airway epithelium, composed of many cell types, makes single-cell approaches particularly relevant to study cell type, or cell state-specific events. This protocol describes the invalidation of FOXJ1 in ALI cultures through the following steps: (1) Establishment of basal cell cultures from nasal turbinates, (2) CRISPR-Cas9 RNP invalidation of FOXJ1, (3) Quantification of FOXJ1 invalidation efficiency by Nanopore sequencing, (4) Dissociation of ALI cultures and single-cell RNAseq, (5) Analysis of single-cell RNAseq data from FOXJ1-invalidated cells.We confirm here that FOXJ1 invalidation impairs the final differentiation step of multiciliated cells and provides a framework to explore other gene functions.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication.

Rationale: The respiratory tract constitutes an elaborated line of defense that is based on a unique cellular ecosystem. Single-cell profiling methods enable the investigation of cell population distributions and transcriptional changes along the airways. Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree. Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%), but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuro-endocrine (PNEC) and brush cells, and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells, and with squamous cells expressing SCEL, SPRR1A/B. Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

The upper airway epithelium, mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate upon aggressions. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs.Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells that we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFβ pathways can be established. Confirmations of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

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.

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5' selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3' selective approaches which just provide internal sequences close to the 3' end. The only currently existing 5' selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5' selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

Data from next generation sequencing technologies uncovered the existence of many classes of small RNAs. Recent studies reported that small RNAs are released by cells and can be detected in the blood. In this report, we aimed to discover the occurrence of novel circulating small RNAs in coronary artery disease (CAD). METHODS: We used high-throughput sequencing of small RNAs from human and mouse apoptotic primary macrophages, and analyzed the data by empirical Bayes moderated t-statistics to assess differential expression and the Benjamini and Hochberg method to control the false discovery rate. Results were then confirmed by Northern blot and RT-qPCR in foam cells and in two animal models for atherosclerosis, namely ApoE(-/-) and Ldlr(-/-) mouse lines. Quantitative RT-PCR to detect identified small RNAs, the RNY-derived small RNAs, was performed using sera of 263 patients with CAD compared to 514 matched healthy controls; the Student t-test was applied to statistically assess differences. Associations of small RNAs with clinical characteristics and biological markers were tested using Spearman's rank correlations, while multivariate logistic regressions were performed to test the statistical association of small RNA levels with CAD. RESULTS: Here, we report that, in macrophages stimulated with pro-apoptotic or pro-atherogenic stimuli, the Ro-associated non-coding RNAs, called RNYs or Y-RNAs, are processed into small RNAs (~24-34 nt) referred to as small-RNYs (s-RNYs), including s-RNY1-5p processed from RNY1. A significant upregulation of s-RNY expression was found in aortic arches and blood plasma from ApoE(-/-) and Ldlr(-/-) mice and in serum from CAD patients (P <0.001). Biostatistical analysis revealed a positive association of s-RNY1-5p with hs-CRP and ApoB levels; however, no statistical interaction was found between either of these two markers and s-RNY1-5p in relation to the CAD status. Levels of s-RNY1-5p were also independent from statin and fibrate therapies. CONCLUSION: Our results position the s-RNY1-5p as a relevant novel independent diagnostic biomarker for atherosclerosis-related diseases. Measurement of circulating s-RNY expression would be a valuable companion diagnostic to monitor foam cell apoptosis during atherosclerosis pathogenesis and to evaluate patient's responsiveness to future therapeutic strategies aiming to attenuate apoptosis in foam cells in advanced atherosclerotic lesions.

Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.

ABCC4 is a member of the ATP-binding cassette transporter family known to transport prostaglandin E2 and other molecules across cellular membranes. A mutation in ABCC4 is now shown to cause defects in ciliogenesis, revealing a role for prostaglandin signalling in regulating cilia dynamics.

Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.

BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

OBJECTIVE: Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS: Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS: INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPβ-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS: Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.