We observed that Tgfb1 is upregulated at the transcription level in cells deficient for the four and a half LIM-only protein 2 (FHL2). The upregulation of Tgfb1 leads to increased liver and kidney fibrogenesis in FHL2-/- mice. We have carried out microarray analysis with mouse embryonic fibroblasts derived from FHL2-/- embryos. We would like to have help for the analysis of our microarray data to identify the transcription factors that would be responsible for the upregulation of Tgfb1 expression in FHL2-/- cells. These transcription factors should potentially bind to the murine promoter of Tgfb1.

We search for the expressed transcript in mature normal sperm as compared to the infertil one.

We have recently identified around 500 long non-coding RNAs that follow a very precise expression pattern in undifferentiated mouse Embryonic Stem cells. In order to assess if any of those molecules are functionally relevant for the biology of ES cells we will perform a functional screening based on the CRISPR technology. We plan to use a modified version of the CRISPR-CAS9 technology, the CRISPRon system, in which (i) the guide-RNAs (gRNAs) will target the promoters of our candidate lncRNAs (at least 5 gRNAs per promoter) and (ii) an enzymatically-inactive CAS9 will be engineered to recrute 10 molecules of the potent VP64 transactivator. This will enable to upregulate the targeted lncRNA from its endogeneous locus.

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.

Grâce à la plateforme PF2 à Pasteur, nous avons réalisé une étude de microarray ( Affymetrix Genechip Mouse Gene 2.0 ST Array) avec les fibroblastes de souris. Pourriez vous SVP nous aider à analyser ces données?

Gli are transcriptional regulators involved in the Shh signalling pathway. Gli3 binding sites on DNA have been defined in the mouse genome by ChIP-chip experiments (Vokes et al., 2008). We are working on the Msx transcription factor family, and have strong evidence indicative of interactions between Msx1 and Gli3 at the protein level. This might reflect in Msx1 binding to DNA in the vicinity of Gli3 binding sites. However, Msx1 binding sites are ill-defined. One stategy to confirm this hypothesis is to compare sequences around gli3 binding sites (around 4200 such sequences in published data) and look for conserved stretches that might define Msx1 binding sites.

La PF Transcriptome et Epigenome développe des projets de séquençage à haut débit (collaboration et service) avec des équipes du Campus. Ceux-ci couvrent l'ensemble des thématiques du campus ainsi qu'une large gamme d'organismes (des virus aux mammifères). La plate-forme exerce des activités de biologie humide (construction des librairies et séquençage) et de biologie sèche (analyse bioinformatiques et statistiques). La personne mise a disposition interagira étroitement avec les autres bioinformaticiens du pôle BioMics et du Hub. Ses activités concerneront notamment: - La participation à la conception et à la mise en place des projets avec les équipes demandeuses, la prise en charge des analyses et le reporting aux utilisateurs - La mise en place d'un workflow d'analyse bioinformatique des données de transcriptome /épigénome en étroite collaboration avec le C3BI, la DSI et les autres bioinformaticiens du pole. Ce workflow permettra le contrôle qualité des données, leur prétraitement, le mapping des séquences sur les génomes/transcriptomes de réference, et le comptage des reads pour les différents éléments de l'annotation - L'adaptation du workflow d'analyse aux questions biologiques et aux organismes étudiés dans le cadre des activités de la PF - L'activité de veille technologique et bibliographique (test et validation de nouveaux outils d'analyse, updates d'outils existants...) - La mise en place et le développement d'outils d'analyse adaptés aux futurs projets de la PF: single cell RNAseq, métatranscriptome, ChIPseq, analyse des isoformes de splicing.. Ceci se fera notamment via la réalisation d'analyses dédiées avec certains utilisateurs. Les outils mis en place et validés dans ce cadre seront ensuite utilisés pour l'ensemble des projets. - L'activité de communication et de formation (participation aux réunions du consortium France Génomique,formation permanente à l' Institut Pasteur… - la participation a d autres projets du Pole BioMics (selon disponibilité) Bernd Jagla, qui était le bioinformaticien de la plateforme a rejoint le Hub au 1er janvier 2016. Rachel Legendre est mise a disposition depuis le 2 novembre 2015 et remplace Bernd Jagla. Je souhaite que Rachel Legendre soit mise à disposition de la plateforme pour une durée d'au moins 2 ans.

Aim : In vitro infection of innate immune cells by L. amazonensis (L.am.) seems to be associated to an increase in resistance to cell death of infected cells. This project aims at deciphering the impact of in vitro L. amazonensis (L.am.) amastigotes infection on cell death processes including apoptosis, autophagy, pyroptosis and necroptosis in different host cells, i.e. Bone-marrow- derived macrophages (BMDMs) and dendritic cells (BMDCs) after one day of infection. Material : RNA samples were obtained from control and infected BMDMs (BALB/c mice) or BMDCs (BALB/c, C57BL/6 and DBA/2 mice) after their sorting by Fluorescence Activated- Cell sorting. For BALB/c BMDCs, amastigotes were also added to cells in presence of immune serum to trigger their opsonization. Samples were analysed a few years ago at the « Plateforme Transcriptome et Epigénome » with the Affymetrix technology. RNAs from BMDMs and BMDCs were analysed with the Affymetrix Mouse430_2 GeneChips and the Affymetrix Mouse Gene ST 1.0 arrays respectively.  

We are mapping the interactions between argonaute proteins and their target RNAs at nucleotide resolution to reveals preferential binding sites on RNAs

assess the faisability of the chip analysis to our samples from laser capture microdissection of the mouse intestine, of diverse quality and preparation levels

Detection of nucleic acids at the single cell level using microscopy has now reach high throughput levels which promise exciting discoveries concerning the functionning of the genomes. The HSC3D project funded by the CITECH in the Pasteur institute aims at multiplexing nucleic acid detection by Fluorescence In Situ Hybridisation (FISH). This necessitates libraries in the range of tens of thousands of  100mer DNA oligonucleotides which will be synthesised by digital lithography. Several constrains apply to these oligonucleotides which therefore require heavy duty bioinformatics for their design .

16S OTU assignment and statistical analysis of zebrafish microbiota in different experimental conditions.

Screening for NSm function in infected mammalian cells by proteomic analysis.

Mycobacterium abscessus is the fast growing mycobacterial species the most frequently associated with lung infection, characterized by severe and very inflammatory cases, after a slow and chronic infectious process. The Cystic Fibrosis patients are particularly susceptible to this bacteria. M. abscessus can exist in a smooth (S) or rough (R) shape depending respectively on the presence or absence of glycopeptidolipids (GPL) associated with the wall of the bacillus. These GPL are involved in motility and biofilm formation, two important features of bacterial colonization. A correlation between the morphotype R, capable of producing morphological structures called "cording" visible in microscopy, and virulence was established. Natural switching from a phenotype S to R allow the transition from a colonizing status to an invasive and pathogenic form. Our project is focusing on deciphering specific mechanisms and analysis of benefits from this transition S/R in vivo. On the basis of transcriptomic data and RNA-Seq previously obtained and/or by screening a transpositional library of colonies deficient in the cording formation, several genes were selected and their corresponding mutants were generated. Since are analyzed : i) the (glyco)lipid composition of their wall; ii) their respective virulence in zebrafish and mouse models and iii) the involvement/recruitment of macrophages and neutrophils and their role in inflammation in response to infection. Our aims are to identify and describe new virulence determinants specific to the establishment of chronic stages of M. abscessus and identification of molecular locks of the S/R switch to understand how an environmental mycobacterium evolves into a pathogen in the host.

After the behavioural characterisation of the Shank3 KO cohort, we extracted and dissected different region of the brain: cortex, hippocampus, striatum, cerebellum.

These regions were selected according to the expression level of Shank3 (Peça et al. 2011). Using QUIAGEN miRNAeasy with DNAse, miRNA-enriched RNA was extracted from the brain regions in order to perform RNAseq.

We will ask 3 questions:

• What is the pattern of Shank3 isoforms in different brain regions?

• What are the genes/pathways differentially expressed in wild-type and Shank3 knock-out mice?

• What are the genes/pathways associated with the severity of the self grooming behaviour ?

Beside data driven eperiments, we will test candidate genes/pathways such as glutamatergic receptors and <

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

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, senescence and cancer, 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.

Gene ontology analysis of RNAseq data from uninfected and Leishmania-infected mouse macrophages.  Scientific context During the course of cutaneous or visceral disease in humans or experimental animal models, the resolution of leishmanial infections or the control of parasite growth is dependent on appropriate innate and adaptive immune responses developed by the parasitized host. Leishmania largely evades and subverts these responses by its intracellular life style inside the mammalian host, where the parasites develop into amastigotes mainly within macrophages (BMDMs). We have focused our interest in the BMDM inflammasome and the way Leishmania amastigotes interfere or subvert BMDM inflammatory responses. Our recent data are in favor of an absence of stimulation, even a down-regulation of the inflammasome in BMDMs harboring replicating amastigotes at the gene and protein expression levels. To go further on this, we have performed RNAseq experiments on uninfected and infected BMDMs. This project was done at the “Transcriptome and Epigenome” platform and in close collaboration with the C3BI for normalization and statistical analysis procedures. Objective In the present proposal we will perform a deep analysis of the repartition of modulated genes between the different conditions using these RNAseq data. Using C3Bi expertise we will classify known functions of modulated genes into GO aspects i.e. molecular function, cellular component and biological process, visualize gene annotations and perform statistical analyses for the distribution of the annotated genes over the GO hierarchy for the different gene lists analyzed. Hopefully, these analyses will bring us a better understanding of the mechanisms underlying the subversion of BMDM functions in the innate and adaptive immune response to Leishmania infection which is a prerequisite to design novel anti-parasitic intervention strategies targeting the infected host cell rather than the parasite.

In order to study a differential regulation of RNA and protein expression level during Rift valley fever virus (RVFV) infection in a murine model, we want to compare transcriptomic and proteomic analysis.  

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.

We have identified a candidate gene associated with increased resistance to a pathogen. This gene is poorly annotated in public databases. To get insight into its function we are focusing on genes that are co-expressed with this candidate gene in various datasets, including microarray collection on various mouse tissues or in a given tissue across inbred strains of mice. Indeed, coexpression is one of the central idea in gene expression analysis. The 'Guilt by association' principle states that gene coexpression might indicate shared regulatory mechanisms and roles in related biological processes. We have established lists of genes that are co-expressed in various tissues, and in collection of individuals with different genotypes. We are willing to get graphical representation giving a compact overview of genes that are coexpressed with our candidate gene.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carries bacteria belonging to the Borrelia burgdorferi sensu lato complex. Our study was focused on peri-urban forests of Île-de-France. These forests are frequented by many visitors and the risk of exposure to tick bites is high. One of them, the Sénart forest, is located 30 km south of Paris (in the Île-de-France region) and has a large number of visitors (3 million per year in the late 1990s). This forest has the characteristics of being partly invaded by chipmunks (Tamias sibiricus). The chipmunk has been introduced from Eurasia, particularly Siberia, China and Korea. The first individuals were released by their owners at the western end of the Sénart forest, in the 1970s. The northeastern part of the forest was colonized recently. Our current study aims to evaluate the evolution of the infection of Ixodes ricinus by Borrelia burgdorferi sl. by comparing the results obtained during 3 years and to determine the consequences of the proliferation of this non-native rodent species, Tamias sibiricus, on the risk of transmission of Lyme borreliosis. For this purpose, we analyzed the rate of infection and the density of infected ticks during 2008, 2009 and 2011 in several locations of the Sénart forest. These results were compared to those obtained for ticks collected in 2009 in two other peri-urban forests of Île-de-France (Rambouillet and Notre-Dame) that have not yet been colonized by these rodents. The density of nymphs, adults as well as the infected density of nymphs and adults were compared according to several factors: location of tick collection in the forest,  presence or absence of chipmunks, type of vegetation, temperature and humidity.

I am interested in gut-brain axis and specifically how a bacterial metabolite, MDP, can be sensed directly by neurons.

Notre but est de trouver un test statistique capable de dire si deux matrices de nombres sont différents. Ces matrices correspondent à l’utilisation de fragments de gènes (V ou J) pour créer un gène ré-arrangé (V-J) codant pour un anticorps de spécificité particulière. Ici nous avons représenté pour chaque gène V la famille de gène J utilisé (parmi J1, J2, J3, J4). Le nombre correspondant au nombre d’occurrences que nous avons trouvé après séquençage du répertoire d’anticorps spécifiques à partir de souris immunisées contre l’un de ces antigènes. Les matrices ont été réalisées pour les deux chaines codant la spécificité d’un anticorps: la chaine lourde (VH) ou la chaine légère (VL). Le but est de comparer les deux matrices VH l’une avec l’autre, et les deux matrices VL l’une avec l’autre.

The present work is to systematically investigate the role of TLRs and NODs in the host defense during C. trachomatis infection using KO animals. The inflammatory cytokines and bacterial burden will be measured using Bio-Plex ELISA kit. C3BI will provide help in the data analysis.

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.

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.

Adult neurogenesis is the process by which adult neural stem cells (NSCs) produce new neuronal and glial cells throughout an animal life. Studies in vertebrates have unveiled the crucial importance of this phenomenon for neural tissue homeostasis and proper brain function. Fundamentally, this process is a balance between maintaining a quiescent NSC pool and recruiting them into the neurogenesis cascade. Using the adult zebrafish anterior brain (telencephalon) as a model, we aim at deciphering the molecular mechanisms governing this balance. We identified a microRNA, miR-9, as a prominent quiescence enforcer. Unexpectedly, miR-9 concentrates into the nucleus of quiescent adult NSCs, together with Argonaute proteins (notably Ago2), effector proteins of microRNAs. This nuclear enrichment of Ago/miR-9 is not observed in embryonic or juvenile fish, being thus a signature of deep adult NSC quiescence. It is also observed in mouse NSCs. We wish to use nuclear miR-9/Ago2 as molecular entry points into the molecular mechanisms controlling this adult-specific deep NSC quiescence state. Within this frame, the present project aims to identify Ago2-bound nuclear targets. Through fractionation experiments, we could detect Ago proteins both in the nuclear soluble and in the chromatin-associated fractions of adult zebrafish NSCs. Thus, we built genetic tools to recover the nuclear Ago2-bound transcripts and genomic loci, ie. using CLIP-seq and DamID approaches, respectively. The enriched transcript sequences will be screened for potential miRNA-binding motifs. Putative genomic targets will be screened, in addition, for specific motifs and for their coincidence with defined functional regions (eg. coding or regulatory). The data will be cross-matched with the transcriptome and proteome of miR-9-positive versus –negative adult NSCs. Together, these data should help elaborate hypotheses on the molecular mode(s) of action of nuclear miR-9/AgAgo2 when controlling adult NSC quiescence.

Non human primates are an important reservoir for zoonotic disease. Here we analyze in Cameroon how human activities in the forest influence contact with non human primates to better understand processes of emergence.

The human gene ANOS1, responsible for the X-chromosome-linked form of Kallmann syndrome (a developmental disease affecting the olfactory system), has been identified in 1991 by positional cloning. It is located on the X chromosome short arm (at Xp22.3), close to the STS gene and close to the boundary of the pseudoautosomal region (common to the X and Y sex chromosomes). Since then, orthologous genes have been identified in all animal species (including invertebrates), except in the mouse, rat, and other rodents (an orthologue in the naked mole-rat Heterocephalus glaber is however present in the GeneBank data base). The orthologous STS in the mouse has been identified in the mouse, and is located in the XY pseudoautosomal region in this species. The sequence of the mouse STS is unusually GC-rich and has markedly diverged from the human orthologous sequence, even though the amino acid sequence of the protein is highly conserved. The ANOS1 orthologous genes have not yet been identified in the mouse/rat, although we have been able to detect the encoded protein anosmin-1 in these species, with antibodies directed against the human protein (the orthologous proteins in the mouse and rat have the expected size in western blot analysis, i.e. about 690 amino-acids).

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation

Tissue resident stromal cells form the scaffold of all organs. In addition, they provide signals for proper positioning, survival and interaction of a number of other cell types, such as immune cells.

To study the neuronal mechanisms underlying the generation of distinct memories, it is necessary to perform experiments in which the sensory elements of the environment are under the precise control o

Our goal is to have a bioinformatic tool that performs the 2 x 2 comparison of matrices of numbers in matrix batches. Each matrix corresponds to the use of gene fragments (V or J) to create a re-arran

The development of the mammary gland occurs in five distinct phases: embryogenesis, puberty, pregnancy, lactation, and involution. Due to its extraordinary regenerative capacity, the mammary epitheliu

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

Stromal cells are essential during organ morphogenesis and for the maintenance of tissue homeostasis. In addition, increasing evidence indicates that stromal cells play a role in certain type of chron

The project aims at developing (1) a database dedicated to the storage and statistical analysis of Collaborative Cross based data and (2) implement genetic analysis tools based on rqtl2 for the identi

Disruption of GABAergic inhibitory circuits is one of the common alteration responsible for several psychiatric developmental disorders. Gephyrin (GPHN) is the common and main molecular organizer of i

Complex chronic diseases are caused by the accumulation of genetic, microbial and lifestyle factors. The number and complexity of such factors makes prediction of pathogenesis and therapy particularly

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation

Dengue virus (DENV) induces strong T and B cell responses upon infection. However, there is currently neither vaccine nor specific treatment against DENV, which is spreading worldwide causing 400 mill

This projects aims at characterizing at the single cell level the stromal microenvironment of the skeletal muscle, to get a better insight into the heterogeneity and function of different cell populat

The therapeutic anti-IgE antibody Omalizumab is used for the treatment of severe asthma, and is known to trigger anaphylaxis in some patients. Since Omalizumab is a humanized IgG1, so we hypothesized

Gene-modified T cells expressing a chimeric antigen receptor (CAR) targeting the CD19 molecule have demonstrated promising clinical efficacy in the treatment of B cell malignancies. However, the frequ

The global aim of the project is to characterize and explore the role of dermal macrophage populations in the control of vascular integrity in health and diseases. Currently, we are limited by the la

Cellular senescence is a stable cell cycle arrest that can be triggered by various biological stresses. Importantly, senescent cells remain metabolically active and secrete numerous molecules, such a