The implication of nucleic acid sensing pathways in human pathologies, ranging from viral infections, malignancies to autoimmune disorders, has recently emerged. The Stimulator of interferon genes (STING) adaptor protein plays a pivotal role in the triggering of pathological nucleic acids-associated inflammatory responses. In this respect, dysregulation of STING-associated signaling is at the heart of several human pathologies. Consequently, a lot of effort has been put into the identification of pharmacological means to contend with STING activation, leading to the discovery of several promising compounds. Notwithstanding, it is as of today difficult to predict their impact in specific pathologies notably due to the poor current knowledge concerning tissue/cell-type-specific nucleic acid detection, potential cross talk between nucleic acid sensing pathways, and yet to be characterized functions of nucleic acid receptors. Furthermore, targeted delivery of these compounds to specific microenvironments where inflammation is deleterious, is a difficult task. To overcome these hurdles while investigating nucleic acid-dependent inflammation from new perspectives, we propose to combine emerging technologies, including nanotechnologies, and deep-learning approaches. To this aim, we propose to build a consortium that will allow strong interactions between biologists, chemists, artificial intelligence (AI) specialists, clinicians and pharmaceutical companies. Such a consortium will group competencies that will serve as a framework for the development a novel and ambitious training program for early stage researchers with multi-, inter- and trans-disciplinary skills. Therefore, with the support of the MRSEI, and in the frame of the Build-INovATE project, we ambition to build a strong pan-European network – the INovATE network – between both academic and non-academic partners, and to apply for the MSCA Innovative Training Network (ITN) funding scheme. INovATE will provide an unprecedented opportunity for exchange of knowledge and skills, while allowing the identification of novel therapeutic targets and biomarkers, instrumental for harnessing innate immune pathways to the benefit of patients. INovATE will thus allow break-through discoveries and contribute to the emergence of both new avenues to tackle inflammation-related pathologies and of a new challenge-oriented young research community.

MicroRNAs ("miRNAs") are important gene regulators: they recognize specific target mRNAs through sequence complementarity. Their expression level depends on their transcription and maturation (which are well known), as well as on their stability. Several observations suggest that miRNA stability is actively regulated, in particular in neurons, but the mechanism of such regulated degradation has long remained mysterious. A phenomenon of miRNA stability control by long RNAs which exhibit sequence complementarity to the miRNA (hence: similar to miRNA targets) has recently been revealed: TDMD ("target-directed microRNA degradation"). TDMD redefines the expression pattern of miRNAs, thereby controlling neuronal and behavioral phenotypes. While the biochemistry of TDMD is starting to be understood, many aspects of that process remain mysterious: for the moment, only two cellular RNAs, and a few viral RNAs, are known to induce TDMD; rules distinguishing TDMD inducers from classical miRNA targets are imprecisely described; and it seems that TDMD is neuron-specific, while its known molecular effectors are ubiquitous. Combining our expertise (high-throughput molecular biology, advanced computational analyses), we want to characterize that phenomenon more precisely. First, using the few known rules for RNA/RNA pairing that trigger TDMD, and complementing them with phylogenetic analyses, we established a list of candidate TDMD inducers in mouse cortical neurons, whose activity we want to verify experimentally. We will extend that work (predictions and experimental validation) to Drosophila, and we will perform a comprehensive prediction of TDMD inducers in mammals (that resource will be made available freely on the Internet). Second, we will clarify the recognition rules between miRNAs and TDMD inducers, with a high-throughput CRISPR screen followed by a machine-learning analysis. Our approach will use a randomized sequence library, allowing us to interrogate the TDMD inducing activity of dozens of millions of distinct sequences, for 8 different miRNAs, exhaustively exploring every possible pairing geometry and nucleotide identity. Finally, we will determine whether TDMD is more active in neurons, as has been apparent so far (or: whether this is a mere coincidence, on the few known examples). If neuronal tropism is confirmed, we will search for neuronal factors responsible for such a specificity, by a complementation experiment in non-neuronal cells. That work will therefore explore an emerging field, it will be articulated with the most recent international discoveries. It will also allow to better understand cross-dependency relationships in gene expression, making our list of predicted mammalian TDMD inducers accessible to all. Such a mechanistic understanding of inter-relations in the transcriptome is essential in order to make sense out of "omics" datasets which are currently descriptive and correlative. In particular, the development of personalized medicine can only become fertile if one can understand causal links among gene expression levels, and give some biological sense to "big data" which is currently piling up.

Global chromatin organization is highly conserved across cell types and species. Indeed, most somatic cells accumulate compact heterochromatin at the nuclear periphery while the less condensed euchromatin is internally localized in the nucleus. This organization highly depends on the association of heterochromatin with the nuclear lamina, which in turn participates in gene repression. Changes in association of specific genomic loci with the nuclear lamina are correlated with differentiation, suggesting a role for nuclear lamina association in the establishment of cell identity. In rupture with the classical view that the nuclear lamina stands as the main determinant of heterochromatin accumulation at the nuclear periphery, I propose that it rather results from an equilibrium between attraction at the nuclear lamina and repulsion from the adjacent nuclear compartment: the nuclear pores. I hypothesize that changes in nuclear pore density may therefore be an important mechanism allowing regulation of this equilibrium during cellular transitions. I will use oncogene-induced senescence in human fibroblasts and differentiation of mouse embryonic stem cells as two models of cellular transitions associated with changes in nuclear pore density to ask (1) how heterochromatin is repelled from nuclear pores, (2) how nuclear pore-dependent global heterochromatin reorganization affects gene activation in senescence and (3) how nuclear pores affect association of specific genomic loci with the nuclear lamina during differentiation. I will use synthetic biology approaches to modify the position of nuclear pore components or genomic loci and interrogate the relationship between nuclear positioning and genome regulation at the single cell level using mainly microscopy analysis. aNChOr will provide a mechanistic analysis of global scale chromatin organization by nuclear pores and how changes in the equilibrium between the nuclear lamina and the nuclear pores can affect cellular fate.

Heterochromatin is a closed and mostly transcriptionally repressed state of chromatin, which is critical for gene silencing, cell differentiation and genome maintenance. Although how heterochromatin is maintained through cell proliferations is well understood, how heterochromatin is formed in the first place is still poorly understood. This is largely because there are very few biological models in which we can separately study the process of heterochromatin establishment from its maintenance. The programmed DNA elimination in the ciliated protozoan Tetrahymena can be synchronously induced in laboratory in a large scale and heterochromatin is established de novo during this process. Therefore, Tetrahymena DNA elimination serves as a unique model for heterochromatin initiation and spreading processes that can be studied genetically and biochemically. The goal of this proposal is to understand the heterochromatin establishment process using this DNA elimination as a model. Our previous studies demonstrated that heterochromatin establishment on eliminated sequences in Tetrahymena is dependent on an RNA interference mechanism. In this proposal, I aim to understand: 1) how small RNAs interact with chromatin to recruit the initial set of heterochromatin components; 2) how the initial set of heterochromatin components induces production of the secondary small RNAs that leads to propagation of heterochromatin; and 3) how the spreading of heterochromatin is stopped precisely at the borders of eliminated sequences. I am confident that this proposed study will provide several novel insights for the basic molecular mechanisms of heterochromatin establishment. In addition, because heterochromatin is suggested to be important for genome integrity, development, aging and cancer progression, I believe the results obtained by the proposed project will eventually aid in improving human health.