The aim of our project is to improve our understanding of the mechanisms regulating the expression of protein coding genes. We initially focused our attention on the transcription/repair factor TFIIH and studied its role in both mechanisms. Mutations in this complex yield rare autosomal recessive disorders, such as Trichothiodystrophy (TTD) and Xeroderma Pigmentosum (XP, sometimes associated to Cockayne syndrome, XP/CS). These diseases, also found in patients bearing mutations in other repair factors (the nucleotide excision repair NER factors XPC, XPA, RPA, XPG, XPF, CSB), were initially defined as DNA repair syndromes. However, the clinical complexity of the patients cannot be explained solely on the basis of a DNA repair defect and may also involve transcription deficiencies. In line with this hypothesis, we recently observed that mutations in TFIIH as well as in NER factors disrupt the transactivation mediated by hormonal nuclear receptors (NR), revealing an unexpected function of the NER factors during transcription. Apart from NER factors, other genetic disorders result from mutations in other transcriptional partners of TFIIH, such as the Mediator. Indeed, mutations in one of the Mediator subunits lead to different diseases, such as Opitz-Kaveggia syndrome (related in particular to the MED12/R961W mutation), cerebellar atrophy (found in patients bearing the MED17/L371P mutation) or nonsyndromic intellectual disability (associated to MED23/R617Q mutation). These observations prompt us to study the molecular mechanism of the Mediator-associated disorders that will help us to better understand how gene expression is regulated at the transcription initiation level. We would like in the next years: - To better understand at a molecular level the roles played during transcription by TFIIH, the Mediator and more particularly the NER factors. - To unveil how mutations in either TFIIH or one of its partners (such as the NER factors and the Mediator) disrupt the transcription and lead to various syndromes. Several experiments will be performed to define at a molecular level the role of the different factors of interest. In this exciting study with potential impact on human health, in addition to conventional molecular biology technics, different mouse models will be monitored. Moreover, and since limited number of mouse model are available at the present time, innovative approaches will be initiated to generate human Induced Pluripotent Stem (hiPS) cells from a number of mutated cell lines isolated from patients. This will help us to analyze in a specific cellular context the incidence of numerous mutations in either TFIIH, the NER factors or the Mediator, leading to a better understanding of the heterogeneity of the phenotypes depending on the mutations. In fine, this work will allow us to define the aetiology of several transcription diseases resulting from mutations in TFIIH and some of its partners. With a long-term perspective in mind, the information obtained from these studies will help to correct or to attenuate the phenotypes of the patients.

Adaptation to fluctuating conditions is usually associated with a trade-off between growth and defense, a dynamic process that fine-tunes resources allocation for either plant growth or defense. This balance is tightly governed by plant hormones, which trigger adapted responses to the environment through transcriptional regulations impacting metabolic fluxes. Among phytohormones, gibberellins (GA) control major aspects of plant growth and development throughout the lifecycle. GA promote growth by stimulating the degradation of DELLA growth repressing proteins (DELLAs), a family of transcriptional regulators. In many cases it has been shown that changes in the environment restrain plant growth as a result of reduced GA content, which in turn enhances the function of the DELLAs. Interestingly, whereas the growth of multiple della mutants is less affected by adverse conditions, growth restraint conferred by DELLAs enhances survival to stress. Thus DELLA-dependent growth restraint is advantageous to flowering plants and permits flexible and appropriate modulation of growth in response to changes in natural environments. Strikingly, while much effort has been invested in the identification of signaling pathways by which GA/DELLA modulate plant growth, little is known about the regulatory networks that control stress tolerance. By using a combination of targeted and untargeted omics analyses, the DELLAdef project aims at identifying the DELLA-dependent defense pathways activated in response to drought, an important question in the actual context of global warming. To reach this goal, the project is structured in 5 highly complementary tasks: (1) decipher the physiological role of GA in the adaptive responses to drought stress; (2) map the spatiotemporal distribution of GA in response to drought; 3) obtain GA-dependent metabolomics profiles in a kinetic drought stress assay; 4) identify the gene networks; 5) reveal the DELLA-dependent defense networks associated with plant tolerance to drought stress by generating gene-metabolite correlation heatmap.

Eukaryotic cells are compartmentalized in distinct organelles, each of them hosting specialized functions. They are not isolated from each other but physically and transiently connected, through the formation of membrane contact sites (MCS). The endoplasmic reticulum (ER) is the most connected organelle, owing to the presence along its surface of receptors of the VAMP-associated protein (VAP) family. These receptors VAP-A, VAP-B and the recently identified MOSPD2, by recognizing a short linear peptide motif called FFAT, are critical for the formation and the function of ER-organelle contact sites. Yet our knowledge on MCSs reaches its limit. We do not know how these contacts are regulated. The aim of this project is to unveil a new mechanism regulating the formation of MCS and establish the repertoire of constitutive and regulatable complexes bridging the ER with other organelles.

Cell state dictates some characteristic cytoskeletal architectures and its reciprocal also holds true. Actin architectures, while driving cell morphology, mechanics or gene expression profile, feedback into cell state. For example, a decrease of actin nucleation can change cell commitment during C. elegans embryo left/right symmetry breaking, revealing an interplay between actin equilibrium and cell fate. The aim of this proposal is to reveal, how the nucleation of actin architectures is temporally and spatially controlled in the different founder cells of C. elegans early embryo and how it affects their fate. We will characterize the specific actin architectures of each blastomere at the single cell level to define actin cytoskeleton identities throughout the early lineage. First using in vivo quantification of endogenously expressed GFP-tagged actin binding proteins, which will enable us to map molecular content and dynamics throughout the lineage. Second, using an in vitro biochemical approach, to probe actin related molecular content in a controlled environment. To do so we are developing a novel device in order to produce single cell extracts and use this content for actin in vitro polymerization assays using micropatterns. Finally, we will proceed in a series of perturbation experiments either affecting actin dynamics and quantifying how it affects cell states or vice versa, in order to assess the interplay between actin and cell identity. This project will provide fundamentally new insights into actin biochemistry as it focuses on the acquisition of cytoskeleton specific identities arising in a natural situation while cells are undergoing commitment changes. It will be of upmost importance to verify how actin dynamics feedback into early embryo development. The main objectives of this project are to reveal in a living model system, how the nucleation of actin architectures can be temporally and spatially controlled in the different cell types found in a developing embryo and how it affects cell states. The proposal has 3 main work packages: WP 1. Identify actin cytoskeletal content variation throughout the early lineage pattern. Here we will focus on the developmental control of the actin proteome, by a quantification of ABPs levels and inheritance through the lineage of the early embryo. The main work is to develop an automated single cell image quantification toolbox to integrate 4D imaging and define the proper references. WP 2. Use cell extracts to test in vitro polymerization capacities of single cells. Here we will probe molecular content by using single cell extracts for actin in vitro nucleation assays using micropatterns. Some technological development will be performed in order to combine extract preparation, actin in vitro polymerization and visualization on the same device. It will allow us to quantify the phenotypes linked to the different actin proteomes. WP 3. Link actin organization, cell state and developmental control. In this final WP, we will assess the interplay and dependency of cell states versus cytoskeletal specific states, via perturbation of either the actin nucleation capacity and observing the impact on cell identity or perturbation of the cell identity and observation of actin nucleation changes. I propose an interdisciplinary project combining top down and bottom up approaches, relying on quantitative experiments in single cells of C. elegans early embryo. The results of this proposal will lead to a precise characterization of cell specific actin related content and actin nucleation capacities, it will also reveal particular requirements linked to cell identity acquisition in the early developing C. elegans embryo.

While cells of a multicellular organism are genetically homogeneous, their function, structures and behaviour vary. Many of these differences arise from distinct gene expression programs. Environmental cues are integrated and mediate epigenetic changes that regulate cell activity, differentiation and development. Cells sense their microenvironment not only through soluble signals but also through mechanical cues. Tissue-level elasticity responsiveness is of an important physiological and pathological significance. Indeed, stiffening of the extracellular matrix promotes invasiveness behaviour of cancer cells and prime Mesenchymal Stem Cells (MSCs) differentiation programs. Here, in this proposal, I intend to study how physical constraints control epigenetic marking and set MSCs fate decision. I plan first to determine how mechanical cues influence transcription programs using RNA-seq and assess their effects on regulatory elements activity using ChIP-seq of histone marks. Next, I propose to study how physical cues contribute in shaping topologic domains in the nucleus using HiC and Lamin-B ChIP-seq. Recent works have suggested an important function of the chromatin association to the lamina in defining chromatin topological domains. In this context, the response to matrix plasticity appears as a powerful tool to study fundamental mechanism of the chromatin architecture’s organisation and to address the central question of how changes of the environment, of the cytoskeleton and of the nuclear envelope impact on the epigenetic landscape and gene expression. Indeed, lamins A/C expression levels range with the matrix stiffness and cytoskeletal rearrangements that represent physiological conditions controlling the nuclear membrane properties. Finally, using chemical compounds and targeted gene deletion, I intend to dissect how epigenetic changes, chromatin reorganisation and gene expression programs occur in response to mechanotransduction and impact MSCs differentiation.