During vertebrate development, embryonic cells proliferate, differentiate, and reorganize to form functional tissues and organs in a stereotypical manner. How tissue morphogenesis and differentiation are coordinated to ensure this developmental robustness remains poorly understood. A population of stem cells located in the caudal part of vertebrate embryos is essential for building the body axis. These progenitors, known as neuro-mesodermal competent cells or NMCs, self-renew in their niche or exit to form the neural tube (future central nervous system) or paraxial mesoderm tissues (future musculoskeletal system). In the quail embryo, NMCs show strong intercellular heterogeneity in expression levels of Sox2 and Bra, two transcription factors involved in neural and mesodermal specification respectively. Our recent results indicate that contrary to established models, progenitors first change their biophysical properties under the effect of these transcription factors to migrate to their destination tissue, where a specific mechanical environment will subsequently enable them to progress in their differentiation process. By coupling morphogenesis and differentiation, this phenomenon provides a better explanation for the robustness of embryonic development. We propose to explore this new concept in bird and human NMCs, bringing together expertise in quail transgenesis, live imaging, biophysics, mathematical modeling, transcriptomics, and organoids.

Early stages of ribosome synthesis in the nucleolus require the recruitment of a plethora of assembly and maturation factors (AMFs), which interact co-transcriptionally with the nascent RNA polymerase I (RNAPI) transcripts. Among these, small nucleolar ribonucleoprotein particles (snoRNPs) are recruited to deposit nucleotide chemical modifications (methylations and pseudouridylations), which are important for ribosomal RNA (rRNA) folding and ribosome function in translation. How eukaryotic cells spatially and temporally organize the early steps of ribosome assembly remains poorly understood. Many nucleolar proteins contain so-called intrinsically disordered regions (IDRs), whose functions in the activity of the proteins and in nucleolar organization remain debated. Our project focuses on an abundant IDR called the KKE/D domain, present exclusively in RNAPI, in all snoRNPs and in some RNA helicases involved in snoRNP dynamics. Our preliminary data show that KKE/D domains fulfill a dual function in rRNA modification and nucleolar (re-)organization depending on RNAPI activity. Indeed, mutant cells lacking all KKE/D domains of snoRNPs show a global defect in rRNA modifications in exponentially growing cells, and display a novel, striking nucleolar compaction defect after the post-diauxic shift when glucose becomes limiting. This defect is associated with a delay in growth recovery upon refeeding. The aim of the DUKKED project is to explore the dual role of the KKE/D domains in the chronology of rRNA modifications and nucleolar organization. We will decipher, using mostly S. cerevisiae as a model system, the molecular events allowing snoRNP recruitment to the unfolded pre-rRNA during RNAPI transcription. We will assess the global importance of KKE/D domains for nucleolar organization, rDNA transcription by RNAPI, pre-rRNA processing, translation and cell fitness, both in exponentially growing cells and upon inhibition of rRNA synthesis. The function of the KKE/D domains will also be investigated in metazoan cells when experimentally possible to highlight the evolutionary conservation of our findings. We will more specifically answer three questions: What is the role of the KKE/D domains in the chronology of rRNA nucleotide modifications during active ribosome production? Using innovative RiboMethSeq, HydraPsiSeq and CRAC technologies, we will demonstrate that KKE/D domains promote the early deposition of nucleotide modifications and explore the chronology of modifications in early rRNA precursors and nascent transcripts (WP 1). How do KKE/D domains maintain the compartmentalization of the nucleolus? Using Co-IPs, Turbo-ID and advanced approaches in microscopy, we will better characterize the interactome of KKE/D domains and their importance for nucleolar organization in exponentially growing cells. In addition, we will decipher the molecular mechanisms allowing KKE/D domains to promote nucleolar compaction and the sequestration of early-acting AMFs upon inhibition of RNAPI transcription. Using quantitative microscopy approaches, we will characterize the physical properties of KKE/D domain condensates formed in vivo and in vitro (WP 2). What is the function of KKE/D domains in cell physiology and stress adaptation? We will investigate the relevance of KKE/D domains for all physiological mechanisms related to ribosome biogenesis and translation, namely rDNA transcription by RNAPI, pre-rRNA modification and processing, and translation. Through time course experiments following refeeding of post-diauxic wild-type and KKE/D deletion mutant cells, we will identify the function(s) of KKE/D domains in rRNA modification, nucleolar re-organization or both that is/are required for stress adaptation (WP 3). Altogether, our study should allow to unravel the crucial function of KKE/D domains in continuously adapting the availability of snoRNPs and other AMFs, as well as nucleolar organization, to RNAPI activity.

Protein homeostasis or proteostasis is the concept that integrated biological pathways within cells maintain proteins in the correct concentration, folding, and subcellular location. A key feature of protein homeostasis in all organisms is involvement of proteasome components and the expression of ubiquitous chaperone proteins often referred to as protein quality control (PQC) systems. The transcription factor Hsf1 drives the expression of PQC components in eukaryotes and is considered as the guardian of protein homeostasis from yeast to human. Ribosome biogenesis in rapidly growing cells produces thousands of ribosomes per minute in eukaryotes and is critical for sustaining high rates of growth (mass accumulation) and proliferation. At the same time, though, ribosome assembly poses a constant threat to cellular protein homeostasis, since it requires the coordinated and large-scale assembly of tens of thousands of Ribosomal Proteins (RPs), which are highly prone to aggregation. Indeed, due to their particular features, such as a highly basic amino acid composition and enrichment in disorder promoting residues, the vast majority of RPs are mostly unstructured in their unbound state, which may promote non‐specific interactions resulting in insolubility. This implies that perturbations of ribosome assembly could lead to a rapid and dramatic increase in unassembled RPs, i.e. “orphan” RPs, which could seriously disrupt protein homeostasis. My recent discovery of a novel regulatory system in yeast, the Ribosome Assembly STress Response (RASTR), that rapidly adjusts chaperone and RP production in response to ribosome biogenesis disruption, sheds new light on regulation of protein homeostasis in eukaryotes. Our published and unpublished data indicate that any perturbation of ribosome assembly causes a rapid increase in unassembled RPs forming nuclear aggregates, leading to a conserved homeostatic response impacting transcription, translation and cell cycle regulation through an unknown dynamic aggregation mechanism. Furthermore, our findings suggest that RASTR represents the earliest transcriptional response to several different types of stress (temperature, starvation) to which cells must adapt in order to survive. The aim of this project is to identify the components and the mechanisms that make up this system operating at the intersection between proteostasis and ribosome biogenesis, driven by dynamic protein condensation. This project will address numerous questions as fundamental as the physiological consequences on proteostasis of the frenetic pace of ribosome assembly in rapidly growing cells. This project is a fundamental research project that is relevant to virtually all organisms. The RASTR studies promise to revolutionize our understanding of a fundamental interplay between ribosome biogenesis and proteostasis networks by the characterization of a new branch of stress response in eukaryotes dedicated to balance the proteotoxic burden of ribosome biogenesis with proteostasis network capacity. Importantly, we expect to characterize a new nuclear membrane-less compartment whose formation is driven by opposite electrostatic charge densities, namely the positive charges of “orphan” RPs and negative charges of glutamic acid motifs enriched in proteins found in insoluble fraction during RASTR. Given the high evolutionary conservation of RP features (small, highly charged, intrinsically disordered domains enriched) in virtually all kingdoms of life, such a discovery will represent ground-breaking advances in the stress regulation field. Lastly, RASTR project should generate results interesting for a broad audience, beyond the limits of our research community, and will directly contribute to medical research as highlighted by citation of our first study (Albert et al, 2019, Elife) describing RASTR in very recent publications related to cancer development, neurodegenerative disorders and recently ribosomopathies.

ADP-ribosylation signaling by PARP1 is a key early event of the DNA damage response. While recent works have elucidated the role of PARP1 auto-ADP-ribosylation, less is known about the impact of the ADP-ribosylation of histones, which are the second main target of this signaling pathway after PARP1 itself. In this project, we aim to uncover the functions of histone ADP-ribosylation in three key steps of the DNA repair process known to be regulated by this signaling pathway: i) modulation of the chromatin architecture, ii) recruitment of repair factors and iii) control of the timely release of PARP1 from the sites of damage. To study these different components of the repair mechanism, we set up an interdisciplinary consortium of 5 research teams with complementary expertise in the fields of ADP-ribosylation signaling, chromatin architecture and repair protein dynamics. The chromatin architecture will be characterized both in vitro and in living cells using cutting edge microscopy methods that will give us access to the chromatin structure and dynamics at the single nucleosome level. These tools will be applied in cells lacking specific components of the ADP-ribosylation signaling pathway to be able to delineate the impact of histone ADP-ribosylation on the chromatin architecture. We will also investigate two alternative mechanisms by which histone ADP-ribosylation could promote the recruitment of repair factors at sites of damage. First, by combining advanced proteomics assays with live cell microscopy, we intend to identify factors displaying binding specificity to ADP-ribosylated histones. Second, we will study whether histone ADP-ribosylation could promote the accumulation of repair proteins to DNA lesions via liquid-liquid phase separation mechanisms, focusing on the two key actors 53BP1 and BRCA2. Using complementary assays to characterize the dynamics of these two proteins at the population and single-molecule levels in living cells, we will be able to distinguish between accumulation mechanisms driven by specific binding to the damaged chromatin, and confinement within liquid droplets surrounding the lesions. Then, we will analyze the dynamics of 53BP1 and BRCA2 at DNA lesions in cells lacking histone ADP-ribosylation compared to control cells to establish the specific impact of this histone mark on the recruitment mechanism. Finally, we will monitor PARP1 recruitment kinetics and turnover at sites of damage to define how histone ADP-ribosylation could contribute to the rapid dissipation of PARP1 from the DNA lesions. Altogether, building-up on the interdisciplinary expertise of the consortium established for this project and the use of high-end quantitative methods, this project will provide a precise description of the roles of histone ADP-ribosylation in the context of the DNA damage response. We anticipate that these findings will deepen our understanding of the complex histone-based epigenetic landscape by dissecting the functions of one of its least characterized components.

Most biological activities follow a nearly-24 hours rhythm entrained to the daily environmental Light/Dark cycle referred to as circadian rhythm. Circadian rhythms are driven by an internal clock. This clock regulates locomotor activity quantitatively, as an ON/OFF switch of movements. Our consortium of two experts, a specialist of circadian activity (E.Cau) and an expert of locomotor circuits (C.Wyart) will analyze whether the circadian system also regulates qualitative aspects of locomotor activity (type of movements, chain of movements), using a diurnal vertebrate: the zebrafish larva. Especially, we have recently described two different locomotor styles, referred to as ‘cruising’ (involving forward locomotion) and ‘wandering’ (where the larvae turn a lot more) that we postulate could prevail during day and night respectively. Ablation of the pineal gland impairs circadian rhythms in a number of species. We have obtained strong indications that the pineal gland control quantitative rhythms of locomotion. Based on these, we will study the roles of the pineal gland during the establishment of circadian rhythms of locomotor activity as well as their regulation by light, together with a possible role in controlling qualitative aspects of locomotor activity (aim1). How can the pineal gland control locomotion? One of the main functions of the pineal gland is the nocturnal secretion of the neurohormone melatonin. However, in lower vertebrates and fetal mammals, the pineal gland also sends axonal projections whose functions have never been addressed. Our unpublished data shows the existence of a neuronal projection from the pineal gland to a high motor center: the posterior tuberculum. The posterior tuberculum corresponds to the mammalian substantia nigra, a dopaminergic center involved in the etiology of Parkinson Disease. The posterior tuberculum is directly implicated in locomotion. It projects onto the MLR (Mesencephalic Locomotor Region), an area which we recently showed controls movements. We will perform a complete characterization of the sites of projection of the larval pineal gland with specific focus on other locomotor centers onto which the pineal gland is known to project at adult stages. We will address the respective roles of melatonin (aim2) and of projection to synaptic targets (aim3) in mediating the effects of the pineal gland on quantitative and qualitative (type of movements, chain of movements) control of locomotor rhythms. At the root circadian organization, the molecular clock, a transcriptional regulatory loop, regulates circadian rhythms in every cell of the body. We will use an innovative live-imaging strategy to report for this molecular clock. This tool will help us assess if melatonin controls the phase of the clock in melatonin responding cells located in the locomotor circuit (aim2) as well as whether neural activity in the pineal gland modifies the phase of the molecular clock through synaptic contact within its neuronal target cells (aim3). Our research program will contribute to understanding how the pineal gland controls the activity of a dopaminergic locomotor center involved in the etiology of Parkinson disease and restless leg syndrome, two diseases that associate motor and circadian disturbances. Our study performed in a diurnal vertebrate will inspire future mammalian studies. More generally, highlighting a qualitative effect of the circadian system on locomotion represents a major conceptual advance in the circadian field, opening the possibility that different locomotor sub-circuits are activated depending on the time in the day/night cycle. Finally, our program will provide a conceptual framework to understand how circadian rhythms control a “simple” behavior.