In wild life, yeast cells are able to survive in severe conditions, without nutriment for a long period of time because the cell is able to enter in stationary phase. During this phase, the cell can transform varied sources of energy and pause its growth to preserve the cells from death. It is known that most of genes are downregulated in stationary phase and the cell activity is globally reduced to its strict minimum, while a subset of “specialized” genes is induced to promote survival in extreme conditions. We are analysing the transcriptome of exponentially grown or stationary phase yeasts, investigating different level of regulation.

The increased incidences of invasive fungal infections coupled with the paucity of available antifungal drugs for treatment have driven the search for novel agents with unique fungal targets. Studies in the pathogenic yeast Candida albicans have attempted to identify novel transcription factors that may be associated with regulating antifungal drug tolerance. In order to investigate novel regulators of cell wall permeability and antifungal responses in the pathogenic mold Aspergillus fumigatus, we employed a library composed of deletions in nearly 400 transcription factors. A screen for mutants that demonstrated altered susceptibility to congo red and calcofluor white, two cell wall binding agents, identified three hypersensitive and three resistant mutants. RNA-seq analysis is ongoing in order to identify and analyze the genes/pathways that play a role in the modification of cell wall permeability.

The recent analysis of the Cryptococcus neoformans transcriptomes revealed the presence of thousands of lncRNAs. In these yeasts, different types of lncRNAs seem to exist. The ones that are antisense of coding genes (the NATs), the ones that are located between coding genes (the lincRNAs) and some others that seem to result from alternative transcription start site selection. We  identified growth conditions under which the expression of some of them is regulated. We have also identified some genes implicated in the regulation of some of these lncRNAs. This project deals with the characterisation of these lncRNAs, the analysis of their regulation and the study of their function in the biology and virulence of this pathogenic yeast.

The aim of the project is to create a viewer that will help visualisation and correlation between genomic, transcriptomic, proteomic and metabolomic data generated by the comparison of amastigote and promastigote stages of the Leishmania donovani parasite.

Skeletal muscle stem cells constitute a population of cells with heterogeneous properties. Interestingly, muscle stem cells have a remarkable capacity to regenerate muscle fibres after regeneration. We are performing a molecular analysis of these stem cells.

The post-translational modification by SUMO is an essential regulatory mechanism of protein function that is involved in most challenges faced by eukaryotic cells. Gene expression is particularly regulated by sumoylation as many SUMO substrates are transcription factors and chromatin-associated proteins, including histones. The emerging paradigm for the proposed work is that sumoylation controls multiple aspects of chromatin structure and function in response to external cues. According to this view, sumoylation is expected to impact both global and specific transcriptional programs thereby affecting constitutive and inducible expression of both coding and non coding genes. Recently, we found SUMO as an integral and instructive component of chromatin in cell growth and senescence, thus establishing sumoylation as a new and paradigmatic chromatin modification. This work now paves the way for detailed understanding of the contribution of SUMO as a multifaceted modifier of chromatin.

Leptospiral promoter regions are poorly characterized: experimentally proven transcription factor binding sites have not been described in the literature and promoter prediction algorithms and E. coli consensus sequences of DNA motifs are not appropriate for Leptospira. Identification of Transcription Start Sites (TSS) and promoters on a global scale will provide essential information on DNA motifs that are targets of RNA polymerases, sigma factors and transcription factors. RNA sequencing will also provide information on small regulatory RNAs. These small (~30-500 nt) non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators which play a variety of important roles in many biological processes. Studies on sRNA regulation of gene expression in Leptospira are currently in their infancy. Our results will provide new insights into the transcriptional landscape of L. interrogans, including the repertoire of sRNA, and it will establish the foundation for future experimental work on gene regulation.    

Les cyanobactéries sont des microorganismes qui prolifèrent dans de nombreux plans d’eau et perturbent leurs fonctionnements et leurs usages car elles sont capables de produire des toxines dangereuses pour la santé humaine et animale. Si la réglementation sanitaire est basée, pour l’instant, sur la surveillance d’une seule toxine, il est désormais connu que ces microorganismes sont capables d’en synthétiser un grand nombre qu’il conviendrait de mieux prendre en compte dans le futur. C’est pourquoi, dans le but de mieux connaître le potentiel toxique des cyanobactéries, ma thèse s'applique, par des études sur leur génome et par une approche de chimie, à caractériser les gènes impliqués dans la synthèse de ces métabolites ainsi que les métabolites produits par ces gènes, à déterminer sur des souches de culture et dans des échantillons naturels provenant de plans d’eau d’Ile de France quel est le potentiel de production de ces métabolites et à mieux comprendre les facteurs environnementaux qui favorisent cette production. Deux équipes de Paris (Pasteur et iEES) sont associées sur ce travail qui implique également des collaborations étrangères. S'il est désormais bien connu qu'une part importante du métabolisme des cyanobactéries qui sont des microorganismes photosynthétiques, est régulée en fonction des phases de lumière et d'obscurité, les connaissances disponibles sur la synthèse des métabolites secondaires sont en revanche beaucoup plus limitées. Ces métabolites ont pourtant un double intérêt puisque certains sont toxiques pour l'Homme alors que d'autres ont un intérêt pharmaceutique potentiel. Leur synthèse repose sur l'expression de clusters de gènes pouvant être de très grande taille (jusqu’à 100 kb par région).

Currently there is increased focus for developing novel antibacterial strategies. The demand is driven by the rise in antibiotic resistance bacteria and many Gram-negative bacteria are on the list of increasingly drug resistant agents. A major target of successful antibiotics is the bacterial cell wall. The target of these drugs is often defined but what is much less understood is the off-target impact of these very important antibiotics. We designed and executed a mutli-omics strategy centered on the Gram-negative pathogen H. pylori. Our goal is to identify potential 'therapeutically susceptible' pathways associated with the physiological response to cell wall stress.

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, T-cell response and type I interferon (IFN-I) are important for an effective response to LASV infection. Among dendritic cells (DC), myeloid DC can induce T-cell activation and plasmacytoid DC are specialized in IFN-I response. DC are also the first target of LASV during the infection of a new host, and studies on in vitro differentiated DC suggest a role of DC in T-cell response to LASV infection. Therefore, plasmacytoid and myeloid DC could be important for an efficient response to LASV infection.

Over the past three decades Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields including virulence-factor regulation, cell biology, and bacterial pathophysiology. More recently, the number of Listeria “omics” data produced has increased exponentially, not only in term of number, but also in term of heterogeneity of data. There are now more than 40 published Listeria genomes, around 400 different transcriptomics data and 10 proteomics studies available. The capacity to analyze these data through a systems biology approach and generate tools for biologists to analyze these data themselves is a challenge for bioinformaticians. To tackle these challenges we are developing a web-based platform named Listeriomics which integrates different type of tools for “omics” data manipulation, the two most important being: 1) a genome viewer for displaying gene expression array, tiling array, and RNASeq data along with proteomics and genomics data. 2) An expression atlas, which is a query based tool which connects every genomics elements (genes, smallRNAs, antisenseRNAs) to the most relevant “omics” data. Our platform integrates already all genomics, and transcriptomics data ever published on Listeria and will thus allow biologists to analyze dynamically all these data, and bioinformaticians to have a central database for network analysis. Finally, it has been used already several times in our laboratory for different types of studies, including transcriptomics analysis in different biological conditions, and whole genome analysis of Listeria proteins N-termini. This project is funded by an ANR Investissement d'avenir: BACNET  10-BINF-02-01

Measles virus protein C interplay with cellular apoptotic pathways; applications for cancer treatment.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation Associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, recent data showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected. The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) determine whether Tac4 could also interact with other mRNA region, such as the 5'-UTR region, 4) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in tra

Analysing the transcriptome of exponentially grown or stationary phase yeasts in a genetic background that stabilises pervasive transcipts, we identified a first subset of ≈ 140 antisense transcripts anti-correlated with gene transcripts that are specifically expressed in quiescence. We are further investigating whether these genes are subject to a transcriptional interference and what are the mechanisms underlying this regulation. More in detail, we would like to analyse the loci where an antisense ncRNA are detected.

Insect vectors durably transmit many important human and animal diseases. Insects are mobile, adaptable and difficult to control, which makes them efficient vehicles for disease emergence, spread and maintenance. Genomic tools have been applied to the study of vectors and vector control, and some insects such as the African malaria vector Anopheles gambiae have now become new model organisms for natural host-pathogen interactions. We study mosquito vectors of malaria and arboviruses, which generates four main kinds of large-scale data that requires dedicated bioinformatics expertise: i) functional dissection of mosquito immune signaling pathways by RNAseq transcriptome profiling to detect responses to pathogen infection and/or silencing of target genes, including mRNA as well as small and other non-coding RNAs, ii) next-gen genetic linkage mapping by deep sequencing of index-tagged phenotyped individual mosquitoes, iii) population genomic analysis by whole-genome sequencing of hundreds of individual 250Mb mosquito genomes, iv) metagenomic analysis of the mosquito microbiome and pathogen susceptibility, and of ecological metagenomic communities in field samples of mosquito vectors.

Cryptococcus neoformans is a ubiquitous yeast present in the environment that is able to interact closely with numerous organisms including amoeba, paramecium or nematodes. The interaction with these organisms shaped its virulence with acquisition of infectious properties as a consequence especially in mammals . The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and during infection, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation upon immunosuppression, leading to a life threatening  disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease's pathogenesis. It is well known in bacteria (tuberculosis), parasites (Plasmodium, Toxoplasma). In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Indeed, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions, altered growth capacity, and the ability to resuscitate upon adequate stimulus. Dormant cells are known to have increased mitochondrial masse and activity justifying a screening strategy of a collection of KO mutants for mitochondrial proteins. In parallel the whole proteome, transcriptome and secretome will be obtain with the ambition to correlate these parameters. Our current project aims at exploring the metabolism of the dormant yeast to have a comprehensive picture of the pathways that are required for the maintenance of dormancy and fo exit from dormancy.  

We study the regulation of type I interferon (IFN) response in humans and in particular the functioning of a key negative feedback regulator, USP18. A recent article reported on a predicted 897nt-long LincRNA (long intergenic non coding RNA) that may target USP18. We have collected information for this LincRNA (genomic locus, putative transcript variants, sequence similarities with USP18 RNA, predicted ORFs, homologies with other genes, etc). To draw a guideline for wet lab experiments, we need to mine databases on this LincRNA. We expect to find it up-regulated in conditions of inflammation or in specific immune cell subsets, like macrophages. We wish to interrogate existing RNA-seq and Chip-seq human databases to obtain information on expression, transcriptional regulation, function, tissue-specificity or relation with pathological conditions of this LincRNA.

The genome of the yellow fever mosquito (Aedes Aegypti) is not fully annoyed, and this project aims at discovering novel transcripts using RNAseq data.

The three HP1 proteins (Heterochromatin Protein 1 alpha, -beta, -gamma) are epigenetic markers of heterochromatin, the condensed, repressed form of chromatin. They are typically known to associate to the di-/tri-methylated lysine 9 of histone H3 (H3-K9me2/3), a repressive histone mark, HP1s are therefore linked to chromatin silencing. But on the other hand, HP1s are also linked to activated transcription, for example HP1g has been shown to localize within the body of coding genes in correlation with their transcriptional activity. We focus on the mechanisms linking HP1gamma to regulation of chromatin and transcription in response to cellular stimuli. We are dissecting the functional links between HP1 proteins and chromatin-associated RNA. For this purpose, have identified all RNA populations associated with HP1gamma factor in immortalized cell lines derived from MEF (mouse embryonic fibroblast) with or without stimuli. We are now analysing the functional impact on gene expression levels. This should allow us to understand to what extend HP1gamma is associated to transcriptional processes on the chromatin.

Chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, spondyloarthritis (SpA) and psoriasis cause significant morbidity and are a substantial burden for the affected individuals and the society. An important obstacle to early diagnosis and the development of more specific and effective therapies is the very limited understanding of the pathogenesis of these diseases. In the past years genome-wide association studies have identified many genes that were not known to be involved in pathogenesis, and have linked several genes in immune pathways to inflammatory diseases, indicating that the immune system plays an essential role in the pathogenesis of these diseases. The current challenge is to correlate these genetic variants with the effector mechanisms implicated in pathogenesis, to allow translation of the genetic data into relevant diagnostics and innovative treatment strategies. To meet this challenge, we have designed a clinical study that examines the immune signaling pathways, the transcriptional networks and the genotype in the same SpA patient, in order to establish a link between genetic variation, cellular phenotype and function, and pathology. This approach will advance our understanding of the pathogenic mechanisms, and identify novel and relevant diagnostic tools, therapeutic targets and biomarkers. The long-term outcome of this strategy will be the rational design of specific therapies tailored to the genotype of the patient.

Entamoeba histolytica is a protozoan parasite and an amitochondriate pathogenic amoeba, which causes amoebiasis (dysentery and liver abscess) in humans. In addition to E. histolytica several species infect the human intestine although these do not cause disease and include in most of cases E. dispar and ocassionnally E. moshkovskii. A phylogenetically close Entamoeba, E. invadens infecting snails, is used as cellular model for Entamoeba cyst formation.

Supported by the National Agency for Research (ANR-10-GENM-0011) we developed a project to firstly study the transcriptional landscape of pathogenic E. histolytica. Among the results we discovered that 60% of ORFs present anti-sense RNAs (NATs) that map to the 3‘ end of genes. Their regulation is modified upon environmental changes. The regulation of NATs is basically governed by genomic sequences within the very short intragenic region of the amoeba genome. Secondly, we have started to conduct comparative genomics and transcriptomics approaches to understand phenotypic differences between Entamoeba species, in particular with respect to virulence.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

Enhancers of transcription are regulatory sequences enabling gene expression from a distance. The landscape of active enhancers is cell-type specific and provides extensive information on the transcription factors at play. Currently, enhancers are mostly mapped based on the histone modifications positioned on their DNA sequence. This type of data is abundantly available for tumor-derived tissue culture cells, but difficult to obtain when the biological material has a limited availability. As an alternative approach, it is possible to detect enhancers as sites of divergent transcription. The objective of this project will be to develop tools allowing detection of sites of divergent transcription in transcriptome and run-on data, to evaluate the quality of the prediction by comparing the outcome with existing enhancer maps, and then ultimately use this approach to identify changes in the enhancer landscape between patients with multiple sclerosis and healthy controls.

This research project focuses on the characterization of the role of small RNAs and their associated Argonaute proteins in transcriptional and post-transcriptional regulation of germline gene expression. Using the nematode C. elegans, we have recently showed that one of the germline-expressed Argonaute protein, CSR-1, promotes germline transcription. However, CSR-1 also possess an endonucleolytic activity that might participate in post-transcriptional silencing. Therefore, two possible functions of the protein might regulate the germline transcriptome. 1) CSR-1 promotes specific germline transcription programs in the nucleus, and 2) negatively regulates expression of target transcripts in the cytoplasm. To gain mechanistic insights into these two functions, we aim to use RNA-seq, sRNA-seq, ChIP-seq, GRO-seq, Ribo-seq, RIP-seq, iCLIP in wild type worms, knock out and catalytic inactive mutants of CSR-1 protein at different times of germline development.

The project is to identify differentially modified genes and pathways in epithelia cells when challenged with Streptococcus pneumoniae serotypes. S. pneumoniae has >90 different serotypes designated as either “carrier” or “invasive”. Some serotypes of S. pneumoniae are naturally carried within the nasopharynx (carrier), or are opportunistic serotypes that escape the nasopharynx causing disease within the host (invasive). In the case of a carrier serotype, the host is able to clear/control the infection, and develop a memory response to infection. In contrast, an invasive strain subverts the host immune response, leading to disease progression and potentially lethality. These works will extend our knowledge of genes/pathways induced between carriage and invasive serotypes of S. pneumoniae.

Cellular senescence is a complex stress response that durable (yet not irreversibly) arrests cell proliferation and is accompanied by widespread changes in chromatin structure, metabolism and gene expression including the production and secretion of a plethora of inflammatory factors. Cellular senescence plays beneficial roles during embryonic development, tissue regeneration, and tumor suppression. Paradoxically, it is also considered a major contributor to aging and age-related diseases, the latter mostly through its inflammatory phenotype, the so-called SASP (senescence-associated secretory phenotype). The proposed work aims at integrating time-resolved transcriptome, ChIP-seq, and ATAC-seq datasets into a comprehensive understanding of senescence-associated gene regulation.

Identification of alternative 5'UTR in an eukaryotic transcripts

DNA Topoisomerase 1 (Top1) is a major regulator of gene expression with great impact on genome stability. Similarly, Guanine quadruplexes (G4) have recently emerged as critical elements for transcription regulation and potential threats to the genome integrity. Studies of Top1 and G4 have many medical implications since their mutations or deregulations are associated with several pathologies, such as cancers, neurodegenerative diseases or viral infections. A few studies, including ours, have revealed physical and functional interactions between these two elements and their involvement in transcriptional regulation of viral and cellular genes. Our research hypothesis is that this Top1-G4 interaction is involved in a new mechanism of transcriptional regulation. This project aims to test this hypothesis and to determine, genome-wide, the contribution of this interaction on human gene transcriptional regulation. This will be tested using genomic and transcriptomic data obtained on the Top1-dependent transcriptional regulation of cellular genes (RNA-seq data), on the localization of Top1 along the cellular genome (ChIP data) and on the presence of G4 structures at specific foci of this genome (G4Hunter prediction and ChIP data). Results obtained by this bio-informatics analyses, should help us to design new experiments to test our hypothesis, on both retroviral and cellular promoters. In long-term perspectives, this project should reveal new mechanisms of gene transcriptional regulation that could serve for the development of new therapeutic strategies.

Despite the success of anti-retroviral therapy (ART) to treat HIV-1-infected individuals, the persistence of a viral reservoir remains an obstacle to a cure. Characterization of the antibody repertoire in patients led to the identification of broadly neutralizing antibodies (bNAbs) targeting the viral envelope glycoproteins and suppressing HIV-1 infectivity with unprecedented potency. Passive administration of bNAbs in animal models or in infected humans revealed their capacity to decrease viral loads and to delay viral rebound after ART cessation. bNAbs also activate the immune system and mediate functions that go well beyond neutralization, such as killing of infected cells or enhancement of T and B cell responses. Immune cells with cytotoxic, phagocytic or immunomodulatory activities express Fc Receptors (FcR) that recognize the constant Fc region of antibodies. The complement cascade is also initiated through binding to the Fc. The molecular and cellular mechanisms underlying the activation of Fc-dependent effector functions of antibodies, including bNAbs, are incompletely understood. Our aim is to analyze the interactions between HIV-1 specific antibodies, infected cells, and the immune system

Our research team recently identified an Aedes aegypti mosquito population that is partially resistant to dengue virus infection. We also have candidate genes that potentially innerly this phenotype. In this project, we would like to test wether specific SNPs are associated with virus infection in those mosquitoes. Sequencing data has already been generated with the Omics platform and handled by C3BI.

Adaptive immune cells play important role in pathogenesis of multiple sclerosis by infiltrating the central nervous system, thereby contributing to demyelination and maintenance of an inflammatory microenvironment. In particular, autoreactive T cells, CD4+ T helper and CD8+ T cells exert a detrimental effect, while CD4+ regulatory T cells (Treg) have impaired suppressive function. Interferon b (IFNb), a commonly prescribed treatment for relapsing-remitting multiple sclerosis (RR-MS) decreases relapse rates and brain disability progression. However, around 30% of patients are or become non-responsive to treatment. Our aim is to determine the immune signatures associated with clinical efficacy of IFNb treatment in RRMS patients

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient T cell response. We aim at characterizing the molecular determinants underlying this efficient antiviral response. We have previously shown that CD4+ T cells from HIV controllers express T cell receptors (TCRs) of particularly high affinity for Gag peptides/ MHC II complexes. Furthermore, HIV controllers shared Gag-specific TCR clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control. To extend these studies, we propose to characterize the TCR repertoire and the transcriptional pattern of HIV-specific CD4+ T cells at the single cell level. We will compare HIV-specific CD4+ T cells obtained from HIV controllers included in the ANRS CO21 CODEX cohort to those from patients receiving antiretroviral therapy.

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Defenses against ROS, e.g. peroxidase activity, are crucial for Leptospira virulence and the adaptive response to ROS is controlled by PerR regulators. We aim at studying how small non-coding RNA participate in the adaptive response to oxidative stress in pathogen Leptospira.

The transcriptome signature during rabies infection will be analyzed both in animal model (mouse) and in human, at the central nervous system level, to identify the main canonical pathways involved during the infection.

Rabies virus (RABV) is a causative agent of lethal neurological disease, a member of Lyssavirus genus, belonging to Rhabdoviridae family in the order Mononegavirales. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that it contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase). The MTase domain is suggested to mediate the transfer of methyl molecules on mRNA cap structure and or at the ribose 2’OH of RNA residues. The MTase domain is assumed to be acquired for: 1) mRNA translation into viral proteins and, 2) protection of viral RNA from detection by host cytoplasmic sensors: RIG like receptors (RLR) such as RIG-I and MDA-5. However, the RABV MTase domain role in pathogenicity and immune evasion is not known. The objective of this work is to characterize the MTase domain and identify the critical residues involved in this function. To this aim, mutations of this domain are introduced by site directed mutagenesis, and recombinant viruses were recovered and characterized. We therefore evaluated the ability of MTase mutants to modify viral RNAs. We used RiboMethSeq analysis to identify eventual internal 2’O-methylated residues in RABV mRNAs {Ringeard et al., 2018}. This project was already started and developed in collaboration with biomics platform by Rachel Legendre (n°B885).

Rabies virus (RABV) is a causative agent of lethal neurological disease. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that L contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase) mediating the transfer of methyl molecule on mRNA cap structure. The detection of viral presence in a host cell is mediated by pattern recognition receptors (PRRs) that sense viral pathogen-associated molecular patterns (PAMPs). The cytosolic receptors include RIG-I-like receptors (RLRs). Three RLRs are known: RIG-I, MDA5 and LGP2. Upon ligand recognition, MDA5 and RIG-I activate signaling pathways leading to the production of pro-inflammatory cytokines, including type-I interferon (IFN), and the establishment of an antiviral state in the host. We have recently performed RLR siRNA knockdown experiments suggesting that upon RABV infection RIG-I is the major RLR implicated in RABV RNA sensing and IFN induction. However, the precise nucleic acid sequence of RABV RNA sensed by RLRs remains unknown. In order to determine RABV RNAs signatures recognized by RIG-I and MDA5, in collaboration with A. Komarova (IP), we have applied a novel ribonucleoproteomic approach that consists in affinity purification of tagged RLRs followed by extraction of associated RNA molecules. This approach has recently provided comparative views on viral RLR ligands in the context of infection with Measles, Chikungunya and Dengue viruses. We are now ready to perform NSG analysis of RIG-I- and MDA5- specific RNA ligands purified from RABV infected cells.

The RIG-I like receptors (RLRs) play a major role in sensing viral infection to initiate and modulate antiviral immunity. These are three RNA helicases entitled RIGI, MDA5 and LGP2 that interact with particular signatures of viral RNA molecules in cytoplasm of infected cell triggering a downstream signalling cascade resulting in the expression of type-I IFNs, proinflammatory cytokines and divers set of antiviral genes. We have recently generated stable cell lines expressing tagged RLRs. By applying affinity chromatography purification of RLR ribonucleoprotein complexes followed by RNA extraction, we observed that short-length RNAs were enriched on RLRs. We have previously sequenced small RNAs of RLRs (PF2 Pasteur) and now need to adjust our new bioinformatic analysis to these RNAseq data with the help of C3BI.

Left-right asymmetry of the heart is essential for establishing the double blood circulation. Impairment of left-right embryo patterning is associated with severe congenital heart defects. During embryonic development, the heart initially forms as a straight tube. While it elongates, it acquires the shape of a rightward helix, a process referred to as heart looping. Previous work suggests that dynamic signaling in the heart precursor cells are essential for heart looping completion, but so far only a few genes have been identified as asymmetrically expressed. In order to investigate the dynamic spatiotemporal patterning of heart precursors, and screen for novel players in asymmetric heart morphogenesis, we aim to perform several transcriptomic analyses.

We propose to identify peptides derived from the S protein that can modulate the HLA-E restricted activity of NK cells. Through in-silico analyses, we have already identified in the S protein of SARS-Cov1, SARS-Cov2 and MERS-Cov regions coding for peptides with HLA-E binding motifs. We will perform functional and structural analyses of the binding of these peptides to MHC-E. We will study MHC-E restricted responses of NK cells in blood and tissues during SARS-Cov2 infection in non human primates. From longitudinally collected samples from blood and tissues before and after the SARS-CoV2 infection of the animals, we will analyse the phenotype and transcriptome profile of NK cells and compare to viral loads and disease progression of the animals.

This project aims at characterizing the transcriptional signature of skeletal muscle during regeneration.

In recent years the concept that only adaptive immunity mediates memory responses has been challenged by several examples of innate immune memory, as shown for macrophages and NK cells. NK cells are innate lymphoid cells, with well-established roles in targeting cancer and virally infected cells, and in addition, are crucial players in antibacterial immunity. Similarly to what has been shown in response to virus, our work is revealing that NK cells maintain a memory of bacterial infection, as seen by a faster and more tailored response to subsequent infections. In response to viral infection, it has been shown that a certain subpopulation of NK cells become “memory” cells, which can expand and contract according to necessity. Similar studies in search of a memory population to bacterial infection have not been shown. Indeed, a major setback in such experiments is the lack of established markers for memory cells. Without a known receptor-ligand interaction, like what has been described for viruses, specific memory subpopulations are very difficult to identify by classical approaches. Single cell transcriptomics represents a novel approach that has been used to discover multiple immune subtypes and will provide a method to identify new sub-populations of memory cells within the NK cell pool.

SARS-CoV-2 depends on the activation or inactivation of specific cellular pathways for efficient replication. The project screened small molecule compounds that affected epigenetic regulation of such pathway. The project aims to characterize these specific pathways, so identify specific steps important in the viral replication cycle and with this to describe the mode of action of these compounds.

HPV RNA-Seq, a novel and unique molecular test that allows detection of pre-cancerous lesions of the cervix from cervical samples.

Establishment of a left-right asymmetry is essential for the function of the heart, which is to ensure a double blood circulation. This asymmetry is initiated during an embryonic process, in which the heart tube acquires a loop shape. The molecular cascade at the origin of left-right asymmetry is well established and involved in the severe heart defects of the heterotaxy syndrome. However, another 20% of heart malformations display cardiac chamber misalignment and their origin is poorly understood. Using a mutant mouse model and expertise in heart morphogenesis, we want to provide novel insight into malformations displaying an abnormal alignment of ventricles.

Our project aims to study the consequences of the systemic inflammation on muscle stem cells (MuSCs). To address this question, we use two in vivo mouse models associated with high systemic inflammation, an acute viral respiratory infection, with the influenza virus, and a solid tumor growth. Both paradigms are associated with muscle catabolism, named cachexia, which is intensively studied in the cancer field. However, most studies are focusing on the muscle fibres, the contractile apparatus of the muscle. Our project is mainly focused on the study of the MuSCs, cells which give rise to skeletal muscle. We aim to characterize the molecular and functional modifications occurring in MuSCs when exposed to high inflammatory stress. RNAseq was performed for both paradigms to gain information on gene expression.

This projects aims at performing transcriptomics on mosquitoes infected with arboviruses such as Dengue or Zika virus.

Hematopietic stem cells (HSCs) are essential for supplying blood and immune cells of the vertebrate body. They are essentially formed during the embryonic time-period, from the wall of vessels and in particular the dorsal aorta. Their survival and self-renewal capabilities are depending on environmental conditions provided by hematopoietic niches among which the bone marrow microenvironment, in adult mammals. During embryonic development, HSC precursors home in transient niches; the main organ being the fetal liver. Currently, functional characteristics of developmental hematopoietic niches - including spatially-resolved biochemical and biomechanical specificities -, are lacking. This information is mandatory not only for fundamental aspects but also for the ultimate goal of producing in vitro HSCs with regeneration potential, for the purpose of regenerative medicine. The aim of our project is to characterize HSCs and niche cells at the single cell and spatial transcriptomic levels so as to identify spatially-restricted molecular components essential for HSC survival and stemness maintenance. To do so, we will use the zebrafish embryo that offers unique advantages in comparison to other vertebrates, in particular for investigating single cell HSC-niche interactions at the level of entire hematopoietic organs.

Bacteria use two-component systems (TCS) to sense and adapt to their environment. The aim of this project is to characterize the regulatory network of each of the 20 TCS in the neonatal pathogen Streptococcus agalactiae.

We aim to characterise the transcriptome of OMM12 mice under different experimental conditions

Nod2 is expressed on brain neurons, however the downstream effects of its activation by its ligand MDP are not know. We used RNAseq strategy to answer this question

Measles virus (MeV) impairs the mitochondrial network. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA). We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of pro-inflammatory cytokines. The expression of human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded mitochondrial DNA substrates, resulting in the catabolism of edited DNA. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5’TpCpG context. Using cell lines deprived of mtDNA, we have demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that MeV induces mitochondrial network fragmentation and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

The fliC locus codes for the FliC protein, the flagellin molecule that constitutes the flagellar filament of motile bacteria, like Y. pseudotuberculosis. Until now, it was commonly accepted that transcription of this locus was completely repressed in the non-motile Yersinia pestis (a recent clone of Y. pseudotuberculosis, which still harbors flagellar genes), but by transcriptomics analysis we observed that it was actually active. Even more, we showed that deletion of the fliC locus in Y. pestis was associated to an attenuation of virulence in the mouse model of bubonic plague. However, for the time being we have no proof that FliC protein is indeed synthesized. Recently, a more detailed analysis of the transcriptomics data showed that a great part of the transcripts detected was actually coming from transcription of the fliC locus in the ‘anti-sense’. Therefore, we would like to explore the hypothesis that the actual molecule playing a role in the phenotype observed is an anti-sense ARN with regulatory functions, instead of the genic product of the fliC ORF, i.e. the protein FliC.

Chronic inflammatory diseases (CID) are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. Genome-wide association studies (GWAS) demonstrated that Th17 cells play a pivotal role in the pathogenesis of these diseases and several loci involved in the IL-23/IL-17 are associated with the CID (IL23R, IL12B, IL6R, IL1R2, RORC, RUNX3, TYK2, JAK2, CARD9). IL-23 is important for the expansion and the functional activity of the Th17 cell subset and several studies have suggested that IL-23 may also regulate the function of IL-17-producing innate immune cells, which express the IL-23 receptor (IL-23R). The overall goal of this project is to improve our understanding of the so-called “IL-23/IL-17 axis” in the pathophysiology of CID and in particular to understand the effects of IL-23 on distinct cell populations and how is the expression of the IL-23 receptor regulated.