During the course of immune responses B cells diversify their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR). These reactions establish highly specific and adapted humoral responses by increasing antibody affinity and by allowing the expression of a different antibody isotype with unique immunological functions. SHM diversifies the variable region Immunoglobulin heavy (IgH) and light (IgL) chain genes, producing families of related clones bearing mutated receptors that are positively selected on the basis of antigen binding affinity.
CSR diversifies the B cell repertoire by combining a single heavy chain variable region with a different constant region, switching the antibody isotype expressed (from IgM to IgG, IgE or IgA) while retaining the antigen specificity of the receptor. Both of these reactions are initiated by Activation Induced Cytidine Deaminase (AID), an enzyme that deaminates cytosines in DNA. AID-induced lesions are recognized by the DNA repair machinery and are processed in different ways to trigger mutations or double stranded DNA break intermediates during CSR.
At present in is not understood how AID-induced DNA damage results in error-prone DNA repair. We have recently conducted a genome-wide CRISPR/Cas9 knockout screen looking for genes involved in CSR. We have identified Fam72a as a key regulator in the balance between error-prone and error-free DNA repair (Rogier et al. Nature 2021). Here, we propose to further understand, at the molecular level, how Fam72a regulates the choice between error-free and error-prone DNA repair during antibody diversification. The project's goal is to generate individual knockouts in CH12 cells for a number of candidate genes, which we suspect being involved and conduct initial mechanistic studies before generating conditional knockout mouse models to study SHM and CSR in vivo.
Key words: Antibody diversification,error-prone DNA repair, CRISPR/Cas9, Molecular Biology, cancer.
RNA polymerase II (Pol II) transcription initiation is controlled by the sequential assembly of the General Transcription Factors (GTF) at promoters. TFIID is the first GTF to bind the promoter. In Metazoans, TFIID is composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). Interestingly, it has been shown that while many cells required the canonical TFIID complex, some others are less affected by depletion of some TAFs subunits. The development of the neural system seems particularly sensitive to TFIID requirement as mutations in genes coding for TFIID subunits have been associated with neurological disorders and intellectual disability in human patients.
In the mouse, Taf8 loss of function is lethal at the implantation stage. We have recently identified a homozygous splice site mutation in TAF8 in a patient with intellectual disability, developmental delay and mild microcephaly . Analysis in fibroblasts derived from this patient have surprisingly shown that while the mutant TAF8 protein was not detectable and TFIID assembly was impaired, transcription is not significantly affected.
The goal of this PhD project is to analyze the molecular consequences of the human mutation during development and in relevant cell types. To that extend, we have generated a Taf8H mouse mutant carrying the patient mutation in collaboration with the Phenomin platform.
The candidate will 1/ analyze the developmental phenotype of the homozygous mutant embryos and fetuses to study the origins of the developmental delay and microcephaly. 2/ If these mutants survive, she/he will also perform behavioral studies in collaboration with the Institut Clinique de la Souris to determine the learning abilities of the Taf8H/H mice. 3/ She/he will derive mES cells from Taf8H/H blastocysts and use in vitro differentiated neuronal cells in order to analyze TFIID assembly by immunoprecipitation associated with mass spectrometry (IP-MS) and nascent Pol II transcription. 4/ In parallel, the candidate will use CRISPR/Cas9 to generate mES lines carrying Taf mutations associated with neurological disorders in human (i.e; TAF2), in order to generate differentiated neuronal cells and analyze TFIID assembly by IP-MS and nascent Pol II transcription, in comparison to the Taf8H mutation.
Increasing evidence suggests that overnutrition in conditions such as gestational diabetes mellitus (GDM), may perpetuate altered metabolic outcomes across generations. These mechanisms involve heritable changes in gene expression that rather occur via altered epigenetic regulation. Animal studies suggest that nutritional stress – among other effects – affects hypothalamic development, predisposing offspring to obesity as the hypothalamus orchestrates a complex array of processes mediating energy intake and expenditure. Accordingly, hyperphagia and reduced satiety represent early features in obesity-prone children. However, A comprehensive analysis of the epigenetic profile in hypothalamus in the context of GDM is largely lacking thus far. We hypothesize that GDM impacts on hypothalamic gene expression via epigenetic regulation during both intrauterine and early postnatal development, predisposing offspring to an obesogenic phenotype that lasts throughout life.
To study the transmission of above epigenetic traits, we will use high fat diet-based mouse models that have been employed to study consequences of GDM in maternal as well as offspring tissues. We will then establish high resolution DNA methylation/oxidation maps of hypothalamus collected from pregnant diabetic mice and their offspring in collaboration with the team of Dr. Ali Hamiche.
Overall, we strongly believe that this collaborative set-up will provide a comprehensive understanding of the impact of overnutrition on diabetes predisposition, a major question in the diabetes field.
Cancer cells differ from normal cells displaying a high level of proliferative capacity. That creates cancer cell vulnerabilities, which can be targeted therapeutically. One of the cellular machineries which are “hijacked” by cancer cells are the nuclear pore complexes (NPCs). NPCs constitute the sole communication gates between the nucleus and the cytoplasm and ensure cellular function and survival. Nucleoporins (Nups) are the building protein units of the NPCs and represent an “achilles heel” of cancer cells. Cancer cells increase their NPC number to adapt to proliferative demand and depletion of Nups required for NPC assembly can selectively kill cancer cells and reduce tumor growth, providing a strong proof of principle for targeting Nups in cancer disease. However, the molecular mechanisms on how rapid spatial assembly of the NPCs can be achieved in cancer cells and how exactly misregulation of Nups homeostasis can cause cancer disease remain largely unknown. Our published findings identified a novel mechanism on how spatial assembly and increased biogenesis of NPCs can be achieved in cancer cells. We plan to characterize the role of newly identified factors in Nups homeostasis in relevant cellular cancer models. Inhibition of these factors should selectively reduce viability of cancer cells. This research may create unprecedented therapeutic perspectives for cancer patients.
cancer, nuclear pore complexes, nucleoporins, nucleocytoplasmic transport, cancer-specific vulnerabilities
We have described a natural transdifferentiation (Td) event in C. elegans: the cell identity conversion of a rectal cell, named Y, into a moto-neuron, named PDA. In wild type animals, only one PDA neuron is made, through Td. However, we have identified mutant backgrounds in which 2 PDA neurons are made: 1) Notch gain-of-function mutants, in which a cell close to Y, named DA9, is transformed into a second Y cell that then transdifferentiates into a second PDA. 2) More recently we identified 2 PDAs in two other mutant backgrounds. Preliminary data suggest that the DA9 cell remain there suggesting that in these backgrounds the second PDA is made from a different cell. Furthermore, expression data of Notch pathway members suggest that it is another cell close to the Y cell that could give rise to the ectopic PDA. The objectives of this PhD project are thus to : i) Identify which cell is at the origin of the second PDA neuron; ii) Determine if this second PDA is made through a transdifferentiation process; iii) Decipher the mechanisms that normally inhibit ectopic transdifferentiation in this cell and possibly other surrounding the Y cell. To address these points, live imaging, transgenesis, genetics, signal quantification and transcriptomics approaches will be deployed. We hope to gain a better understanding of the mechanisms that make some cells naturally plastic while others are prevented from changing identity.
Key words: Cellular plasticity, transdifferentiation, Notch, C. elegans, scRNA Seq
Gene expression requires ordered and efficient processing of messenger RNA (mRNA) molecules from their sites of synthesis in the nucleus to the cytoplasm, where they are translated into proteins. Yet, we know little about the cellular processes that orchestrate the flux of mRNA in a temporal and spatial manner. Regulation of mRNA fluxes may be particularly important during cell differentiation, which depends on rapid and sometimes extreme changes in gene expression. We recently discovered that the yeast NuA4 acetyl-transferase complex, which promotes transcription through histone acetylation, is also required for nuclear export of mRNA by acetylation of Nuclear Pore Complexes (NPCs), which are multiprotein complexes communicating the nucleus with the cytoplasm. The role of NuA4 in mRNA export seems conserved in mammalian cells, because mouse embryonic stem (mES) cells lacking the NuA4 catalytic subunit accumulate mRNA in nuclear phase-separated domains called nuclear speckles. The objective of this project is to determine how the NuA4 complex regulates the flux of mRNAs from the nucleus to the cytoplasm in budding yeast and mES cells. This project will reveal novel, key roles of NuA4 acetyl-transferase, nuclear pores and nuclear speckles in the control of gene expression.
Multicellular organisms establish and maintain different transcriptional states in disparate cell types through complex and specific regulation of gene expression. This regulation is mediated by the cooperative binding of transcription factors to regulatory elements through the recognition of specific DNA sequence motifs. Additionally, the physical access of transcription factors to DNA can be modulated by epigenetic regulation, such as DNA methylation, nucleosome positioning and histone modifications. Failure to maintain this tight regulation of gene expression results in developmental defects and various diseases including cancer. The research focus on Molina’s lab is to develop computational method to integrate single-cell RNA-seq and ATAC-seq data with transcription factor binding motifs to study how transcription factors regulate gene expression.
You will work in the international team of Nacho Molina under the co-supervision of Anaïs Bardet and will benefit from our strong expertise in computational biology for the success of the project. You will analyze genomic sequencing data generated by the team and our collaborators at the IGBMC by developing novel bioinformatic and machine learning tools to gain a deeper understanding of gene regulation at the single cell level.
The ideal candidate should hold a MSc degree in Computational Biology, Bioinformatics, Data Science, Mathematics, Computer Science or similar. Bioinformatics background with experience analyzing genomic sequencing data will be required. Excellent programming skills and experience working in a Linux environment will also be required. Good knowledge of biology and interest in genomics and gene regulation will be expected. Good background in mathematics will be a plus. Ability to work in a team with both computational and experimental biologists. Good level in spoken and written English.
Key words: Bioinformatics, computational biology, machine learning, gene regulation, genomics, NGS, single-cell sequencing.
Cell morphology and dynamics relies on the actin cytoskeleton to generate forces. In animals, the core elements of this machinery are encoded by a variety of paralog genes, and modified post translationally, altering their dynamics. During embryonic development, the composition of this machinery changes dynamically. So far, the relationship between the molecular diversity and its underlying genetic code remains elusive.
C. elegans offers a unique opportunity to explore how the combinatorics of actin and other actin binding proteins can alter cytoskeletal dynamics to drive specific cell behaviors. Three actin paralogs are abundant and required in the early embryo for cortical contractility and division, and are involved in muscle at later stages. Actin paralogues display a high sequence similarity, and their participation in different networks and their respective physiological function remains unexplored. We propose first to elucidate the regulatory mechanisms that control their expression pattern, second to explore how their biochemical properties are tuned to drive specific morphogenetic processes. For example, to assess the role of actin coding sequences, we can generate CRISPR mutations or switch actin paralogs and assess expression patterns and developmental defects in embryos and adults.
Together this work will allow us to better understand the genetic and molecular “actin code” as well as better grasp the mechanisms related to Nonmuscle actinopathies- a human rare disease we are studying in the lab.
Actin, C. elegans embryogenesis, Nonmuscle actinopathies
mRNA decay and its regulation have emerged as important players in the control of gene expression. In eukaryotes, this pathway initiates with the shortening of the poly(A) tail mediated by protein assemblies harboring ribonucleolytic activities, among which the CCR4-NOT complex. We have identified a new subunit of this assembly complex: CNOT11. This conserved protein binds to the N-terminal region of the CNOT1 protein that scaffolds the CCR4-NOT complex, making CNOT11 likely to contribute to the function of the CCR4-NOT complex in controlling gene expression (1). We have also identified GGNPB2 (Gametogenetin Binding Protein 2) as a direct partner of the CNOT11 protein. Interestingly, GGNBP2 is strongly expressed in the testis and its inactivation leads to male sterility in knockout out mouse models (2). This ANR-funded project aims to decipher the physiological roles of the CNOT11 module of the CCR4-NOT complex, as well as that of its new partner GGNBP2. This will be achieved through the phenotypic analysis of mice lacking CNOT11 or GGNBP2 either in germ cells or in their supporting cells, as previously done (3). Given the current knowledge, our main hypothesis is that these factors control stability and/or translation of specific transcripts during the production of male gametes. We propose to identify these mRNAs.
(1) Mauxion et al., (2013) RNA Biol. 10 :267 ;
(2) Liu et al., (2017) Am. J. Pathol. 187 :2508 ;
(3) Teletin et al., (2017) Curr. Top. Dev. Biol. 125: 191.
Key words: mRNA decay, gene regulation (RNA-seq, single cell), FACS, cell differentiation,
We are deciphering the physiological role played by retinoic acid (ATRA) nuclear receptors (RAR), by using a large variety of techniques ranging from biochemistry, through mouse genetics, to molecular biology and imaging (1). We use the seminiferous epithelium (SE) of the testis as a model system because ATRA is required for both its differentiation and maintenance (2). Our previous work indicates that RARA isotype is required in Sertoli cells, the supporting somatic cells of the SE, to allow proper spermiation i.e., the release of spermatozoa from the SE (3). However, the genetic cascade controlled by RARA is not yet defined. Evidence indicate that Sertoli cells cyclically change their functions in a coordinated manner with germ cell differentiation to support the entire process of spermatogenesis. We hypothesized that RARA exerts complementary roles in Sertoli cells by alternatively repressing genes during the stages of SE cycle preceding spermiation, when unliganded, and activating genes for spermiation and during the following stages of SE cycle, when ATRA-liganded. The thesis project is aimed at deciphering the molecular mechanisms by which RARA is able to alternatively repress and activate transcription in Sertoli cells.
(1) Mark et al., (2006) Annu. Rev. Pharmacol. Toxicol. 46:451;
(2) Teletin et al., (2017) Curr. Top. Dev. Biol. 125:191;
(3) Vernet et al., (2006) EMBO J. 25:5816
Key words: nuclear receptors, gene regulation (RNA-seq, single cell), epigenetics (ChiP-seq, ATAC-seq), cell differentiation, cell morphogenesis, stem cell niche, mouse genetics, bioinformatics
The regulation of gene expression is particularly complex in eukaryotes as cells need to adapt to changing conditions and / or differentiate. This regulation occurs largely by changes in the rates of transcription or degradation of messenger RNAs (mRNAs) that adjust the synthesis of proteins to quantities needed by cells.
Our work on the degradation of mRNAs led to the characterization of several protein complexes involved in critical steps of this process. Among them, the yeast and human decapping complexes, composed of the catalytically active Dcp2 protein and its mandatory Dcp1 cofactor. Decapping is indeed a key irreversible step in the constitutive and regulated turnover of mRNAs, preventing translation of the transcript and irremediably targeting it to degradation. Thus, decapping must be tightly regulated. The decapping process has been shown to target several families of transcripts, including mRNAs, non-coding RNAs, or defective transcripts suggesting that the decapping is active in multiple contexts. In particular, decapping must be tightly coordinated with translation. Consistently, previous analyses have identified several decapping regulators whose functions are only partly understood.
Recent data from the team provide new insights into the activities of some decapping regulators. Their biological roles will be investigated through in vivo analyses (characterization of mutants, analyses of reporter genes...) and biochemical assays (activity tests, protein-protein interaction studies...). This project will provide a molecular and functional understanding of some decapping regulators and of theirs roles at the interface between degradation of mRNAs and translation. These analyses contribute to elucidate the physiological role of decapping, including its links to genetic diseases and cancer.
Key words: mRNA, cap, regulation of gene expression, RNA decay, translation, diseases
TFIIH is a basal transcription factor involved in the expression of class II genes. The complex contains nine subunits including the CDK7 kinase which is involved in the phosphorylation of RNA Polymerase II. Recently, chemical inhibition of CDK7 has been shown to be very effective in targeting cancer cells due to the specific involvement of CDK7 in expression of super-enhancer dependent genes such as the oncogene cMYC. Melanoma cells are broadly divided in proliferative cells showing high protein level of the oncogene MITF and invasive melanoma cells that show low level of MITF. Our team has shown that targeting CDK7 was highly effective against proliferative melanoma cells expressing MITF. However, we also observed that prolonged exposure to CDK7 inhibition caused a transcriptional reprograming of proliferative melanoma cells to invasive cells poorly expressing MITF and showing significant resistance to targeted therapies. By seeking to unveil the molecular details of this resistance, we showed that MITF represses the expression of the transcription factor GATA6, involved in drug resistance, by binding to an intronic region of the gene. A repressive function for MITF has been proposed but has never been studied in detail. Our goal is now to use our model of MITF repression of GATA6 to unveil the molecular basis of this mechanism and to broaden this knowledge to the role of GATA6 in melanoma aggressiveness.
Macrophages represent our first line of defense against internal and external challenges. In addition, it has become clear that these cells also put in contact distant tissues, hence providing ideal sensors of the internal state. Given the strong evolutionary conservation observed in innate immunity, we are dissecting in vivo the role and mode of action of the macrophages in neural development and function using the Drosophila model system. Our preliminary findings indicate that the fly macrophages are necessary for brain development, in line with the association observed between defective immune cells and severe neural diseases in humans. We have also recently shown that the Drosophila immune cells are heterogeneous and display unique epigenetic signatures. The major aim of the project will be to analyze the molecular and epigenetic pathways underlying the role of fly immune cells in brain development and function.
The project involves a variety of approaches, from genetics and molecular biology to high throughput assays (single cell and bulk transcriptomics and epigenomics) and bioinformatics, imaging and cell biology.
Key words: hematopoiesis, neural development, stem cells, Drosophila, single cell analyses, high throughput assays, genetics, epigenetics, imaging
At the most fundamental level, eukaryotic DNA is packaged into chromatin by making nearly two turns around an octamer of histone proteins, forming nucleosomes. Several multi-protein, chromatin-modifying complexes contain at their core an ATPase subunit that remodels chromatin: replacement of core histones with histone variants that influence DNA accessibility; sliding, addition or eviction of nucleosomes from the chromatin fibre to modulate the compaction of DNA or expose certain regions. Deregulation of this process can severely impact gene expression patterns and genome integrity. Their importance is such that chromatin remodelling protein mutations are strongly associated to several diseases, including cancer. Unfortunately, we currently do not understand well enough the molecular details of their mode of action to be able to translate this into improved cancer treatment. The mammalian SWI/SNF complex comprises a set of evolutionary conserved ‘core and enzymatic’ subunits, but also ‘auxiliary’ subunits present only in mammalians, thought to reflect increasing biological complexity. Although the topology and 3D structure of the SWI/SNF complex are becoming clearer, the structure, the molecular details of interaction and the function of these auxiliary yet important subunits are poorly defined at best. Despite some evidence that certain auxiliary subunits are involved in DNA damage repair (DDR), our knowledge of their actual function in this process is very limited. Intriguingly, several of these proteins are predicted to be intrinsically disordered (ID), suggesting they may undergo liquid‐liquid phase separation (LLPS). By using a combination of cryo-EM, x-ray crystallography, biochemistry and in cell experiments, this project aims to define the structure and function of auxiliary subunits of the mammalian SWI/SNF complex, determine how they interact with the core, with the nucleosome and better understand their roles at sites of DNA damage and rationalize the disease associated mutations.
Key words: Chromatin remodeling, Epigenetic, Cryo-EM, X-ray crystallography
The bioactive vitamin D (1,25D3) is a key regulator of calcium homeostasis by controlling calcium absorption, reabsorption and resorption in intestine, kidney and bones, respectively. 1,25D3 activities are mediated by VDR, a member of the nuclear receptor (NR) superfamilly.
Hypercalcemia induced by high 1,25D3 circulating levels (CMH) is a hallmark of several rare refractory pediatric disorders of various etiology. Current treatments are poorly efficient and greatly impact the development of children. Therefore, there is an urgent need to develop new therapies.
Recently, we identified promising drug candidates targeting selectively VDR (Publication 1 and our unpublished data). Moreover, we established several genome-wide technologies to characterize the genomic landscape and the binding of nuclear proteins to DNA (Publication 2), as well as single cell transcriptomics (Publication 3), to gain insight into the mechanisms underlying NR activities in vivo.
The objective of this project is to perform in-depth characterization of the effects of the new drugs in relevant CMH mouse models and identify their underlying mechanisms in vitamin D target tissues using cutting-edge technics. Moreover, thanks to our collaboration with clinicians, the effect of these compounds will be determined in patient-derived primary cells. The results gained will allow to identify new therapeutic options for hypercalcemic disorders and improve our knowledge on cell-specific NR activities.
3 relevant publications:
1. Rovito, D. et al. Cytosolic sequestration of the vitamin D receptor as a therapeutic option for vitamin D-induced hypercalcemia. Nature Communications, 2020. 11(1): p. 6249.
2. Rovito D, et al. Myofiber transcriptional repertoire driven by collaboration of glucocorticoid receptor, Nrf1 and Myod. Nuclei Acid Research, 2021. 7;49(8):4472-4492
3. AbuElMaaty, et al. Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions. Science Advances, 2021. 7 : eabg5982.
Key words: Vitamin D, calcium, rare disease, high-throughput sequencing, single cell transcriptomics, mouse models
The skin is the body's primary barrier against physical insults and microbial pathogens, representing a unique environment in which various immune cells interact with skin cells in physiological and pathological contexts. The lab is interested in deciphering how the dysregulated cytokine signalings derived from epithelial cells coordinate innate and adaptive immune responses in the skin, which are crucially implicated in inflammatory pathogenesis of diseases such as atopic dermatitis (AD), and the progression of AD to asthma (atopic march). Interestingly, recent studies from the lab also uncovered inflammatory pathways shared by skin cancers.
The objective of this project is to delineate immune pathways through which skin keratinocytes program the immune response in skin microenvironment via their production of cytokines to control the T cell response. One particular interest is on the T follicular helper (Tfh) cell responses, which is implicated both in AD and atopic march, and how such responses are controlled by skin dendritic cells (DCs) and what is the pathogenic role. The study will integrate in vivo studies using elegant genetic tools (newly generated conditional knockout, reporter, cell-depleting mice), in combination with immunological approaches (flow cytometry, adoptive cell transfer, cell sorting, ex vivo cell co-culture; in vitro cell differentiation), and molecular biology tools particularly single cell RNA-Sequencing and bioinformatic analyses. Results are expected to reveal missing cellular and molecular pieces of skin immunity, and to identify potential targets for developing preventative/therapeutic strategies for skin inflammatory diseases.
Key words: Immunity; Inflammation; Skin; T cell; Th2; Tfh; Dendritic cell; Epithelial cell; Cytokine; Mice; Human; Immunology; single cell RNA sequencing
Title: Functional analysis of a novel domain specifically occurring in plant mitochondrial ribosomes
Description: about 200 word
Mitochondria are the powerhouses of eukaryote cells. These endosymbiotic organelles are semi-autonomous since they have retained a genome and a fully functional gene expression machinery. In plants, the mitochondrial ribosome has recently been characterized by our team, i.e. through the determination of its structure by cryo-EM. This showed that plant mitoribosomes contain many specific proteins, 10 of which are pentatricopeptide repeat (PPR) proteins. Moreover, these mitoribosomes have a distinctive architecture with the occurrence of novel domains. The most remarkable one, localized on the head of the ribosome small subunit is composed of a 400 nt long extension of the 18S rRNA bound with two PPR proteins. The function of this novel domain, called hereafter the elongated head domain (EHD), is completely unknown.
The proposed work aims at determining the function of the EHD by two complementary reverse genetic approaches in Arabidopsis thaliana. The first one is based on a CRISPR/Cas genome editing system and targets rPPR6, a PPR protein that shapes the EHD. The second one is based on an innovative system using TALEN directed to mitochondria (mitoTALENs) and targeting the 18S rRNA EHD-coding sequence to cause a deletion of the extension. These two kind of mutants will be used as tools to investigate whether the EHD plays a role in translation related processes such as initiation or regulation, or whether its function is independent of translation and could serve e.g. as a hub for mRNA maturation. In the longer term, this work will contribute to unravel the diversity of translation machineries across eukaryotes.
Key words: Ribosome; translation; PPR proteins; mitochondria; Genome editing; TALEN
C19ORF12/Nazo are evolutionarily conserved proteins that have been identified independently by unbiased genetic approaches for their roles in a neurodegenerative disease in humans and in antiviral immunity in the model organism drosophila. These mysterious proteins do not contain structural domains that could hint to a possible function and seem to co-localize with mitochondria and endoplasmic reticulum membranes. A number of observations further suggest that they are involved in vesicle trafficking. The goal of this application is to unite the findings made in flies and humans and to provide explanations for the connection of C19ORF12/Nazo proteins to seemingly unrelated physiological functions. We will combine experiments in vivo, taking advantage of the fly model, and in tissue culture cells, including skin fibroblasts from patients, to establish the link between C19ORF12/Nazo proteins and vesicle trafficking in order to understand how they impact the biology of neurons and resistance to viral infection in flies and possibly also in humans. State of the art cell biology techniques, coupled to structure/function studies, will contribute to the understanding of the regulation and the dynamics of these enigmatic proteins.
Key words: conserved protein, flies, human, neuron biology, viral infection, vesicle trafficking
The androgen receptor (AR) is a ligand dependant transcription factor that regulates genes expression under the action of androgens such as testosterone in both physiological (male phenotype, muscle mass regulation…) and pathological contexts (prostate cancer). This nuclear receptor is composed of three structural domains: a ligand binding domain (LBD), a DNA binding domain (DBD) and a N-terminal domain (NTD) which is intrinsically disordered but plays a major role in the regulation of the receptor through recruitment of co-regulatory complexes and post-translational modifications. The receptor maturation and proper folding requires the action of a range of chaperones and co-chaperones which also play a poorly understood role in the repression of AR in absence of ligand.
The thesis goal is to decipher the structural basis of AR maturation and regulation by chaperone proteins by providing an atomic description of interactions between AR and its chaperones in order to better understand how certain pathological AR variants escape such regulatory mechanisms and to propose new strategies to modulate AR activity.
Approach: This integrative structural biology project will combine biophysical methods and NMR spectroscopy for the characterisation of transient interactions that regulate AR activity and cryoEM for structure determination of identified stable complexes. The student will benefit from the expertise of the team in structural characterisation of the androgen receptor. He/she will also have access to the state of the arts infrastructures and facilities of the IGBMC.
Key words: Structural biology, NMR, cryoEM, androgen receptor, chaperones, transcription regulation
Gene expression employs several steps in all kingdoms of life: i) mRNA is transcribed from DNA by RNA polymerase (RNAP); ii) mRNA is translated to protein by the ribosome; and iii) in eukaryotes, the mRNA is further processed before protein synthesis. The machineries executing gene expression have been mostly studied in isolation in the past but we know many of them are organized in large, supramolecular assemblies where their activities are coordinated and where new functions emerge not predictable from the individual components. We aim to close this gap. For example, we study the expressome, a supramolecular assembly line where RNAP is coupled to the ribosome (see Webster et al., Science 2020 for recent result from the lab). In collaboration with Valérie Lamour, we study how DNA topology influences transcription and how RNAP is coupled to DNA topoisomerases. Finally, we are interested how transcription in eukaryotes is coupled to mRNA processing.
Students interested to tackle challenging problems and using state-of-the-art structural biology approaches, will be able to choose from a number of projects and study the allosteric crosstalk between RNAP and other key enzymes involved in bacterial and eukaryotic gene expression.
Interested candidates will use single particle cryo-EM, the ideal method to gain mechanistic insights of large, dynamic complexes, to obtain high-resolution reconstructions. Biochemical, and in-vivo approaches will complement your results. Your work will unravel how synergism and crosstalk of distinct macromolecular machines in the context of a supramolecular assembly regulates the conversion of genotype to phenotype.
Key words: RNA polymerase, ribosome, molecular machines, gene expression, cryo-electron microscopy, structural biology
Eukaryotic ribosomes are complex molecular machines whose assembly is tightly controlled to ensure faithful protein biosynthesis. Ribosome assembly is initiated in the nucleolus by the transcription and processing of ribosomal RNAs, which together with ribosomal proteins form pre-60S ribosomal particles. The pre-60S particles mature by transiently interacting with various assembly factors during cytosolic export. The nearly 5000 residues long maturation factor Rea1 is vital for the export of the pre-60S particles. Rea1 belongs to the AAA+ protein family and harnesses the energy of ATP hydrolysis to mechanically remove assembly factors from pre-60S particles. Despite its key importance for ribosome maturation, the Rea1 structure and mechanism are poorly understood. We will carry out genetic manipulations in yeast to tag proteins for subsequent Rea1-pre-60S purification. The PhD student will also be trained in state-of-the-art cryo-electron microscopy to structurally characterize these complexes. There are already preliminary structural data on a Rea1-pre-60S complex providing a solid starting ground for the PhD project. These structural investigations will be complemented by in-vitro as well in-vivo activity assays carried out in collaboration with external partners. He/she will also learn to work in a highly international environment and to develop project management skills. Furthermore, presentation skills will be acquired due to regular progress meetings and the attendance of international conferences.
Key words: Rea1, molecular machines, ribosome maturation, AAA+ proteins, cryo-electron microscopy, structural biology
Description: Transcription initiation is a major regulatory step in eukaryotic gene expression. The DNA in eukaryotic cells is organized into chromatin, a highly conserved polymer that controls many crucial functions of the genome. The accessibility of DNA to the transcription machinery is regulated by the degree of chromatin compaction. Co-activators have critical roles in regulating DNA accessibility in the chromatin to facilitate RNA polymerase II transcription initiation. One of the major chromatin modifications is acetylation of specific lysine residues of nucleosomal histones. Human ATAC and SAGA complexes are two related multi-subunit histone acetyltransferase (HAT) complexes. Both complexes harbour the same HAT enzyme, GCN5 or PCAF and were reported to interact with the TATA-box Binding Protein TBP. The metazoan specific ATAC complex contains ten subunits and plays a vital role in cellular homeostasis. The aims of the project are to determine the 3-D structure of the human ATAC complex by cryo-electron microscopy, to understand how ATAC interacts with nucleosomes, post-translational modifications and general transcription factors and the functional importance of the different structural elements and subunits of ATAC genome-wide in chromatin modifications and gene regulation in human cells.
Key words: eukaryotic transcription, transcription initiation, human transcriptional co-activators, cryo-electron microscopy
The ability to accurately predict protein structures from their amino-acid sequence is a long sought-after goal that promises to profoundly influence life sciences and medicine by vastly accelerate efforts to advance drug discovery. Innovative technologies such as artificial intelligence (AI) and machine learning are making important inroads in the fields of biotechnology, biomedical research, pharmaceutical drug discovery, and life sciences.
The recent spectacular advances in protein structure prediction afforded by state-of-the-art AI machine learning methods coupled with evolutionary analysis (doi: 10.1038/s41586-021-03819-2; doi: 10.1038/s41586-021-03828-1) have opened the way to obtaining unprecedented in silico structural information on protein-protein complexes (doi: 10.1126/science.abm4805). These new developments will have an important impact on the development of future novel therapeutics and drug discoveries once being thoroughly assessed both in silico and experimentally to harness their full potential.
This thesis project is focused on employing the recently developed AlphaFold2 (doi: 10.1038/s41586-021-03819-2; doi: https://doi.org/10.1101/2021.10.04.463034) software to predict protein-protein interactions of nuclear hormone receptors within regulatory complexes. There exists few structures of such complexes aside from several low resolution cryo-EM structures of the androgen receptor-coregulator (doi.org/10.1016/j.molcel.2020.06.031) and estrogen receptor-coregulator (doi: 10.1016/j.molcel.2015.01.025) complexes, as well as somewhat higher resolution cryo-EM structures of the glucocorticoid receptor in complex with chaperone proteins (doi: 10.1038/s41586-021-04252-1 & doi: 10.1038/s41586-021-04236-1). These protein complexes are important targets for the development of new therapeutic compounds targeting diseases such as cancer, autoimmune, inflammatory, allergic, and lymphoproliferative diseases. While structures of the individual proteins are generally well established, their 3 dimensional assemblies are open to much debate making
these low-resolution structures of limited use for the development of new therapeutic compounds and even less useful for the development of structure-based personalized medicine.
Our project will use the revolutionary AlphaFold2 software to obtain structural predictions for nuclear receptor complexes of therapeutic importance and to establish a database where this structural information is integrated with sequence, disease and therapeutic compounds, thereby establishing the first such database that can be used in a personalized medicine procedure.
To develop a robust protocol based on AlphaFold2 for obtaining structural insight on nuclear receptors (NRs) co-regulatory complexes, we will first use our protocol to predict the recent experimental structures determined at reasonably high resolution (doi: 10.1038/s41586-021-04252-1 & doi: 10.1038/s41586-021-04236-1) thereby validating our procedure. Subsequently, for the lower resolution structures, comparison with in silico prediction will be used both to further validate the computational methodology and to obtain complementary biological insight.
This protocol will then be applied to obtain structural information on complexes for which no prior structural information is available, focusing on experimentally established interactomes of the NRs. The ultimate goal will be to link this 3D structural information on protein-protein interactions of functional importance with nuclear receptor sequences, and mutations observed in clinical studies, using as first targets, breast cancer (estrogen receptor alpha) or bladder cancer (PPPARgamma receptor). Being able to include a 3 dimensional structure of the mutant complex will allow to study the effects of mutations on the assembly of functional complexes. Personalized clinical decisions can eventually be made after integrating an individual patient’s genomic data at the molecular level within our computational pipeline and rank the efficacy of binding of particular drugs to variant proteins.
Transfer RNA (tRNAs), are critical regulators of cell homeostasis in their role of adaptors in mRNA translation. By dictating the efficiency and accuracy of translation, cellular tRNAs abundance controls the quantity and the quality of the cellular proteomes. As proteomes are as diverse as the cell types, tRNAs repertoires need to extensively vary in distinct cell types. tRNAs diversity comes from the fact that tRNAs isoacceptors (that carry the same amino-acid) are encoded by multiple genes, many of them being identical in sequence. This provides ample opportunity for regulation of tRNA abundance in different cellular contexts. Genetic defects impairing tRNAs function are leading to neurological diseases (up to 79%) through unknown mechanisms. As tRNAs ensure optimal ribosome speed during mammalian brain development, dynamic tRNA abundance likely constitutes a mechanism to control neuronal development. We therefore propose to probe the functional impact of tRNAs pools during mammalian brain development.
The overarching goal of this project is to define how changes in tRNA pool regulate cell fate acquisition during mammalian corticogenesis. Our hypothesis is that the fine-tuned balance between cellular self-renewal and differentiation in the developing cortex requires tight regulation of cell-type specific tRNA pools.
To understand whether tRNAs pool is shaped during cortical development to allow generation of fully functional neurons, we will analyze the consequences of perturbing tRNA transcription (by manipulating the expression of gene specifically involved in tRNAs transcription) in vivo on neurogenesis and neuronal migration. We have also identified variants in those genes (Brf1 and GTF3C1) in patients with neurodevelopmental disorders. The intern will be in charge in defining the consequence of perturbing global tRNA content at the cell population level and with time during cortical development, as well as assessing the pathogenicity of the identified variants. To do so, the student will combine in vivo mouse genome editing, genome wide tRNA and ribosome footprint sequencing and immunochemistry.
tRNA, cortical development, neurodevelopmental disorders, neurogenetics
Inherited myopathies are severe genetic diseases with strong muscle weakness impacting the autonomy and quality of life of patients. No therapy exists for primary myopathies. There is thus an urgent unmet need to validate novel therapeutic strategies in laboratory models to foster preclinical and clinical development of cures.
We previously identified several therapeutic targets for myopathy sub-classes, which can be modulated through gene therapy, RNA interference or pharmacology. In the current project, the PhD candidate will develop novel methodological approaches to modulate known and novel therapeutic targets, using adeno-associated virus, drugs, and potentially CRISPR-assisted genome editing. The efficacy of the therapies will be validated in cell and animal models, in particular in available mouse models. The treated animals will be phenotyped at different levels: motor function, muscle force, histology and myofiber organization, molecular pathways, through quantitative analysis including omics approaches.
This project is expected to have a major medical impact by providing a methodology that will be translated to human through pre-clinical development.
Key words: myopathy, gene therapy, virus transduction, animal model
Amyotrophic Lateral Sclerosis (ALS) is the third most common neurodegenerative disease worldwide. This devastating disease is characterized by degeneration of lower and upper motor neurons leading to muscle wasting, resulting in paralysis and death of patients in few years.
The most common genetic cause of ALS was identified as an expansion of GGGGCC repeats located within the C9ORF72 gene. This mutation leads to DNA epigenetic changes resulting in decreased expression of the C9ORF72 protein. Recent results of our group indicate that C9ORF72 regulates autophagy (Sellier et al., 2016; Boivin et al., 2020), a catabolic process essential for neuronal cell viability. Autophagy is based on the formation of a vesicle engulfing the material to eliminate, which is then directed to lysosomes for degradation. Importantly, autophagy is essential to degrade protein inclusions and altered organelles, which are too big for the proteasome. Among the organelles directed tor autophagy, altered mitochondria should be swiftly and efficiently degraded as their dysfunctions lead to oxidative stress and induce neuronal cell degeneration.
Interestingly, we recently uncovered that C9ORF72 regulates autophagy of altered mitochondria. Thus, the objectives of this PhD project will be to better characterize this mechanism through a wide range of molecular, cellular and biochemical approaches (cell culture, transfection, siRNA and CRISPR-Cas9, immunofluorescence, immunoblotting and immunoprecipitation, mass spectrometry and RNA-Seq, etc.), as well as analysis of our mouse models of ALS (locomotor and behavior phenotyping, tissue analysis immunoblotting, quantitative RT-PCR and immunohistochemistry, etc.).
Overall, this proposal will help contribute to better understanding of the cause mechanisms of neuronal degeneration in ALS patients, in order to define therapeutic strategies for this devastating disease.
Key words: Neuronal disease, Genetic diseases, Mitochondria, Autophagy
Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma (HCC) world-wide. A key feature of HBV replication is the synthesis of the covalently closed circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation and cccDNA targets are only poorly defined. One major challenge to address this urgent question is the absence of robust models for the study of the cccDNA biology. To overcome this roadblock, we established a simple cell-based HBV cccDNA reporter assay. Using this model combined with a targeted loss-of-function screen encoding the human DNA damage response machinery, we identified YBX1 as a novel cccDNA host factor and candidate antiviral target. The aim of the PhD project will be to (1) validate and explore candidates identified during the primary screen as antiviral targets in authentic HBV infection models; (2) to identify novel cccDNA-related host factors by high-throughput loss-of-function screens based on CRISPR/Cas9 and shRNA perturbation. The PhD candidate will apply established cutting-edge technologies in molecular and cell biology including functional genomics combined with patient-derived models for clinical translation. Based on our approach and previous results, we expect that this project will pave the way to the discovery of novel therapeutic strategies for HBV cure – an urgent global unmet medical need.
KEY WORDS: antivirals, HBV cccDNA, genetic screen, HBV cure
Liver fibrosis is the main risk factor for liver cancer – a key unmet medical need with rising incidence world-wide. Using single-cell RNA-seq we previously identified a novel cell type of EPCAM+ liver progenitor cells. This stem-cell like cell population expresses molecular regulators relevant for liver regeneration and disease. We hypothesize that liver injury alters the composition and cellular fate of this EPCAM+ population contributing to perturbed liver regeneration and fibrosis progression to cancer. We aim to understand the role of EPCAM+ cells for liver disease progression to cancer and explore identified cellular circuits as therapeutic targets. The PhD candidate will conduct single-nuclei sorting and RNA-seq from snap frozen liver tissues of mice with diet-induced fibrosis and patients with fibrotic liver disease. Differential gene expression will be correlated with transcriptional signatures of disease-relevant signaling pathways using established bioinformatics tools. Selected disease drivers and pathways will be validated using perturbation studies (RNAi, CRISP-Cas9, small molecules) in liver progenitor cell lines and mice with liver-specific KO. The project will unravel novel regulators of liver regeneration and of fibro- and hepatocarcinogenesis. We expect that the understanding of perturbed liver homeostasis and regeneration contributes to novel therapeutic concepts to prevent and treat fibrosis-induced liver cancer.
KEY WORDS: liver, regeneration, fibrosis, cancer, signaling, liver microenvironment
Chronic liver disease progressing to hepatocellular carcinoma (HCC) is a major public health burden with limited therapeutic options. HCC is a leading cause of cancer-related death with rising incidence world-wide. Over the last decade our team successfully applied human liver chimeric mouse models based on the engraftment patient-derived human hepatocytes or patient-derived xenografts (PDX) into immune-deficient mice to advance the knowledge of chronic hepatitis B and C virus infection, fibrotic liver disease and HCC (Zhuang et al. Nature Comm 2021, Aizarani et al. Nature 2019, Mailly et al. Nature Biotech 2015). However, the absence of a human immune system in these models limits the full modeling of hepatocarcinogenesis and precludes the study of therapeutic approaches targeting the human immune system (e.g. immuno-oncology).
Funded by the RHU program DELIVER of the French Research Agency (ANR), this project aims to develop a fully humanized mouse for liver disease and HCC containing a human immune system based on the use of human bone marrow-derived mesenchymal stem cells (hBMSC). The PhD candidate will develop solid expertise in human stem cell biology, organoids, cell and molecular biology and following detailed training animal experimentation together with the institute’s animal platform. This program will significantly advance the understanding of the human immune system in liver disease biology and hepatocarcinognesis and contribute to discover urgently needed novel therapeutic strategies for HCC.
KEY WORDS: cancer, humanized mouse model, liver disease, human mesenchymal stem cells, immuno-oncology
Chronic liver disease progressing to cancer such as hepatocellular carcinoma (HCC) is a major public health burden with limited therapeutic options. HCC is a leading cause of cancer-related death with rising incidence world-wide. Therapeutic discovery has been hampered by the lack of suitable models recapitulating patient liver disease and cancer for clinical translation. Recently, our team established a 3D patient-derived liver disease model based on both cancer cell lines and patient-derived spheroids (Crouchet et al. Nature Comm 2021). Combined with scRNASeq and perturbation studies this model enabled to uncover novel compounds for treatment of advanced liver disease and cancer prevention. Funded by the RHU program DELIVER of the French Research Agency (ANR), this project aims to use our established patient tissue pipeline to develop next generation patient-derived liver disease models for the investigation of liver disease and cancer biology and drug development. Within this project, the PhD candidate will develop patient-derived spheroids and organoids combined with microfluidics, precision cut slices, aiming to build a “liver-on-a-chip model” from human patient tissues in collaboration with the Strasbourg University Hospitals. The liver-on-a-chip model will be applied to investigate the effect of therapeutic compounds using clinically relevant read outs in collaboration with Pharma. The PhD candidate will develop skills in cell and molecular biology, scRNASeq and related technologies, bioinformatics and build expertise in translational medicine and drug development.
KEY WORDS: Drug discovery, drug development, fibrotic liver disease, liver cancer, patient-derived models.