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

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.

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.

The Center for Human Immunology (CIH) supports researchers involved in translational research projects by providing access to 16 different cutting edge technologies. Currently, the CIH hosts over 60 scientific projects coming from 8 departments of the Institut Pastuer and 5 external teams. In order to respond to the growing needs of these projects in the area of single cell analysis, the CIH has introduced a significant number of single-cell/single-molecule technologies over the past 2-3 years. These new technologies, such as the Personal Genome Machine (PGM) and Ion Proton sequencers, iSCAN microarray scanner, Nanostring technology for transcriptomics profiling and real-time PCR machine BioMark, give rise to large datasets with high dimensionality. Such trend, in terms of data complexity, is also true for flow cytometry technologies (currently reaching over 20 parameters per cell). The exploration of this data is generally beyond the scope of scientists involved in translational research projects. In order to maximize the research outcomes obtained from the analysis of these rich datasets, and to ensure that the full potential of our technologies can be served to the users of the CIH, we would require a proximity bioinformatics support. A CIH-embedded bioinformatician would: 1) design and implement standard analysis pipelines for each of the data-rich technologies of the CIH; 2) provide regular ‘bioinformatics clinics’ to allow scientists the possibility to customize standard pipelines to their specific needs; 3) run trainings on the ‘R software’ platform and other data analysis tools (such as Qlucore) of interest for the CIH users. The objective would be to empower the users to run exploratory analysis by themselves, and to teach good practices in terms of data management and data analysis.    

Aim : When L. amazonensis (L.am.) amastigotes infect BMDMs, they induce multiple strategies to allow their survival and multiplication. This project aims at deciphering the transcriptional modulations induced after three days of in vitro infection of C57BL/6 BMDMs with L. amazonensis (L.am.) amastigotes, in unstimulated conditions, or conditions that induce NLRP3 inflammasome activation.

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

The Transcriptome & Epigenome Platform is dedicated to the development and use of high throughput approaches for transcriptomics and epigenomics studies. The platform is accessible to any research team from the Pasteur Institute (80% of the projects) as well as from outside. It is involved (most often as collaborator) in several projects funded by the ANR, Microbes and Brain, ERANET and by the Pasteur Institute in the framework of the PTR programs. Next Generation Sequencing (NGS) based on the Illumina technology (HiSeq 2000/2500 sequencers) is used to perform RNA-sequencing experiments for which a large amount of data is generated. After a first step involving bioinformatics, specific statistical methods must be used be analyze rigorously the data. These analyses are most often performed by the statistician(s) of the platform. They are also in charge of bibliographical survey activity.

Tuberculosis (TB) still remains a major public health problem with estimated 9 million incident cases and 1.5 million deaths in 2014 (WHO, Global Tuberculosis Report 2015). More worrisome is the emergence of multi drug resistance (MDR), or even extensively resistant (XDR) M. tuberculosis strains worldwide. The standardized treatment of pan-susceptible tuberculosis is the administration of two antibiotics (rifampicin and isoniazid) for six months, accompanied by two additional antibiotics (pyrazinamid and ethambutol) for the first two months. Although very efficacious, this treatment is very demanding due to the duration and the possible side effects. The treatment of MDR-TB is less standardized, with more toxic and poorly tolerated drugs, resulting in lower cure rates. Therefore, we need not only more molecules with antimycobacterial activity, but also, we urgently need new strategies to increase our therapeutic arsenal for treating MDR-TB. Only three new drugs, bedaquiline, delamanid and PA-824 have been tested in phase2/3 clinical trials.

In this context, the european funded project NAREB has been created. It brings together 14 partners from 8 EU Member and Associated States, and it aims to (i) screen different combinations of antibiotic drugs with nano-carriers (lipid, polymeric, biopolymeric) with and without targeting ligands, (ii) coload antibiotics in order to develop innovative therapeutic combination therapies (iii) test in vitro and in vivo the best therapeutic combinations. In particular, we will analyze more in-depth the effect of bedaquilin, new TB drugs and nano-carriers on the host/bacterial transcriptome using RNAseq.

We are interested in determining the differences in the transcriptome of select developmental stages of the malaria parasite, Plasmodium.

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, a recent paper from the Rachel Green’s lab showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected (Young et al., Cell 2015). 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) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in translation of some specific mRNA.

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 <

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

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.

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

We are analyzing a Listeria protein secreted in the supernatant. From cells transfected with this protein, after crosslinking, we have isolated RNA and found the protein in the RNA fraction. In addition, the protein binds several splicing factors in RNA dependent manner. Furthemore, the protein localizes to the nucleus. In this project, we have isolated the protein from the supernatant and from bacterial extract after growth in BHI. We have extracted the RNA from the supernatant fraction containing the protein and from the bacterial extract containing the protein. We want to alalyze the RNA in these different fractions.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB), is the deadliest disease due to a single infectious agent. Despite considerable efforts to fight the disease, TB remains a major public health problem. Even more worrying for the future, multidrug resistant (MDR) strains of MTB are continually emerging and about 10% of people with MDR-TB have extensively drug-resistant TB (XDR-TB). Drug-sensitive TB can be cured by a 6-month treatment using 4 antibiotics, but MDR-TB and extensively drug-resistant XDR-TB require treatment for up to 2 years with more toxic and costly second- and third-line drugs. Toxicity of these drugs is well described; it includes hepatotoxicity, liver injury, skin reactions, gastrointestinal and neurological disorders. However how MTB drugs influence the host response to MTB infection has been poorly addressed. The main goal of project is to understand how drugs interact with the host in order to improve the treatment.

Our team studies interactions between arboviruses and Aedes aegypti mosquitoes. We recently generated RNAseq data to analyse the response of the mosquito to virus infection. We would now like to analyse that data using Gene Ontology to find pathways or processes that are activated upon infection.

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

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.

In a previous project (collaboration with PF2 and C3BI) , we determined the transcriptome landscape of the pathogen Leptospira interrogans (Zhukova A, Fernandes LG, Hugon P, Pappas CJ, Sismeiro O, Coppée JY, Becavin C, Malabat C, Eshghi A, Zhang JJ, Yang FX, Picardeau M. Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans.Front Cell Infect Microbiol. 2017 Jan 19;7:10.). In the present project, we performed RNA-seq with the PF2 on the saprophyte Leptospira biflexa and a mutant of a gene encoding a hfq-like protein. Hfq is a chaperone usually involved in the stability of sRNA in other bacteria and we would like to : 1) identify the sRNA ((intergenic regions, antisense RNA, etc) of L. biflexa wt strain 2) compare the level of expression of wt and hfq-like mutant strains.

The RpoS/σS sigma subunit of RNA polymerase is the master regulator of the general stress response in many Gram-negative bacteria. σS also contributes to virulence and biofilm formation of the human pathogen Salmonella enterica serovar Typhimurium. We have used RNA-sequencing to unravel the σS-dependent transcriptome in S. Typhimurium ATCC14028. These studies have revealed a major effect of σS on the remodelling of metabolism and membrane functions, and have highlighted the importance of down-regulation of gene expression by σS. More recently, we have used a mass spectrometry-based proteomics approach to explore the wide σS-dependent proteome of ATCC14028. Our proteomics data, and complementary assays, have unravelled the large impact of σS on the Salmonella proteome in stationary phase. As expected, the majority of genes up-regulated by σS at the protein level were also up-regulated at the transcript level. In sharp contrast, our data have revealed a large number of genes down-regulated at the protein level, but not at the transcript level, suggesting that post-transcriptional regulation plays a larger role in σS gene regulation than previously recognized. In this project, we will determine the impact on genome expression of a single amino-acid substitution in RpoS which confers specific phenotypes.

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.

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.

There is a wide variation in both animal and human risk and outcome of infection, generally encompassing asymptomatic, to more severe and sometimes lethal cases. Dengue virus, a re-emerging arbovirus is a great examples of this, as it lead to a very wide spectrum of disease severity. Genetic variation associated with intrahost populations has been postulated to influence viral fitness and disease pathogenesis. This project aims to characterize the complexity of viral populations in samples from patients affected with different degree of dengue disease severity.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

Despite the worldwide importance of malaria due to Plasmodium vivax, there is currently almost no data on the molecular responses of the Anopheles mosquito vectors to this parasite species. Understa

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

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. This neglected but emerging infectious disease has a worldwide distribution and affects people from developing countries, m

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

A number of mammalian cell types are susceptible to Salmonella Typhimurium infection, including epithelial cells, fibroblasts and macrophages. It has recently been demonstrated that Salmonella display

Cocaine is the most widely used illicit stimulant in Europe1, with a recent increase in use in the French general population. Cocaine addiction (CocAdd) is recognised as a public health priority world

Microbes are prone to rapid changes and they can either exploit or countervail their variation in a context-dependent manner. To this purpose, both genetic diversity and non-genetic phenotypic variati

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

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

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen spec

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the ord

The aim of this RNA-seq project is to characterize three transcriptional regulators of Streptococcus agalactiae, the main etiological agent of bacterial meningitis in neonate, and one transcriptional