Central to the process of germ cell (GC) differentiation, meiosis allows the mixing of genetic material between male (XY) and female (XX) individuals. In mammals, XX GC meiosis is initiated during foetal life, at a time when XY GC become quiescent in the G0/G1 phase of their cell-cycle. Then in females, the number of gametes (oocytes) is determined at birth, whereas in males, XY GC proliferation resumes post-natally, yielding spermatogonia stem cells (SSC), which subsequently insure the continuous production of gametes during all the life-time of the individual. The molecular mechanisms driving these gender differences in GC proliferation and commitment to meiosis are not fully understood, but the somatic environment proves to play a critical role in this process, independently of the chromosomal content of GC. In Molmechmeiosis project, we propose to identify yet uncovered cascades and to decipher the genetic and molecular interactions set up between several signalling pathways, which all appear required for somatic sexual determination and GC commitment to meiosis, namely the prostaglandin D2 (PGD2), fibroblast growth-factor 9 (FGF9), MSX homeogene, NODAL, R-spondin (RSPO1), beta-catenin (CTNNB1) and retinoic acid (RA) dependent pathways. To reach this goal, the acquisition of XX and XY GC sexual identity driven by the surrounding somatic tissues, the induction or prevention of meiosis entry, as well as the post-natal recovery of GC proliferation are being dissected in multiple mouse lines bearing either germinal or somatic gene ablations of key components relaying these pathways. These developmental processes are being investigated by means of morphological approaches such as histology, immuno-histochemistry, in situ hybridization, but also through more sophisticated, holistic, molecular approaches such as global micro-RNA profiling, transcriptome analysis using DNA microarrays and high throughput parallel sequencing of RNA (RNA-Seq), as well as through an innovative in vivo tandem-affinity purification tag-facilitated immuno-precipitation of nuclear complexes coupled to mass-spectrometry aimed at identifying RA-receptor interacting proteins. Collectively, the broad combination of both state-of the art and cutting-edge methodologies will allow us (i) elucidating the role played by PDG2, FGF9 and NODAL-dependent signalling pathways in the mitotic arrest and meiosis inhibition occurring in foetal XY GC, (ii) identifying the molecular factors downstream of MSX and RSPO1/CTNNB1 allowing XX GC to become competent for meiosis, and (iii) uncovering the genetic networks and characterizing the molecular mechanism through which CTNNB1 and RA control XY GC and SSC proliferation, as well as entry in meiosis in the post-natal testis. Overall, the combination of our respective expertises in the Molmechmeiosis project offers a unique opportunity to unravel fundamental genetic networks and molecular cascades governing the sexual fate of GC.

The central machinery by which hormones, proteins and neurotransmitters are released from exocytosis vesicles is well known. Nevertheless, we are still far from having elucidated the regulatory mechanisms that prevent random secretion events from occurring. Subtle contacts between organelles have been observed and it is increasingly established that their major roles are to harbor intensive exchanges of lipids and to control the flow of calcium through specific channels between intracellular organelles or between these organelles and the limiting membrane of the cell. Surprisingly, although the dynamics of calcium and lipids are crucial for the regulation of exocytosis and the subsequent reuptake of certain constituents of exocytosis vesicles by compensatory endocytosis, studies on the contribution of membrane contact sites in these mechanisms show default. Our proposal, based on strong preliminary evidence and novel chemobiology tools supporting the presence of three different types of contact sites between the endoplasmic reticulum, secretory granules, and plasma membrane, is to define how these are formed, what are their dynamics, and their role in the regulation of secretion. From a mechanistic perspective, we will focus on SOC-type calcium channels and the transfer of key lipids that define sites of exocytosis. Our project should provide completely new and unanticipated data to better understand the regulation of a major biological process.

Nuclear Receptors are ligand-activated transcription factors involved in the regulation of critical cellular processes such as regulation of cell growth and differentiation, or the regulation of metabolism. NRs form a gene superfamily that is widely conserved in Metazoans. Many members of the NR superfamily bind major hormones, such as steroids, thyroid hormones, or retinoids. They occupy a special position in gene regulation by providing a direct link between the ligand and the target gene whose expression they regulate. There are also many nuclear receptors for which no known ligand has been identified and are called “orphan NRs”. Some Phylogenetic analyses have shown that NRs arose very early in the metazoan lineage long before the divergence of protostomes and deuterostomes and diversified through complex series of gene duplications and gene losses The history of this gene family is relatively well understood in terms of molecular evolution, however the situation is more elusive at the functional level. It is difficult to trace back the history of the three main functional features of NR genes: their DNA binding activity, their ability to interact with co-regulators and their ligand binding abilities. The later feature has been the most controversial with two competing models published in the literature: the “orphan early” who suggests that NRs were originally orphan receptors that gained ligand binding ability during evolution, and the “ligand exploitation” model which in contrast suggests that liganded receptors occurred early during evolution. The main reasons why it is still difficult to reconcile these two views is that the factors governing the evolution of ligand specificity are not well known for NRs. In this project, in the context of the evolution of NR’s functions, we propose to use an original model system, the cephalochordate amphioxus (Branchiostoma lanceolatum) to tackle the question of the functional evolution of nuclear receptors. This model organism is widely regarded as the best available stand-in for the chordate ancestor, and is characterized by an overall body plan and a genome that are vertebrate-like, but simpler. By using the unique advantages of the amphioxus model and by concentrating our efforts onto 3 receptor types that offer distinct situations we propose to combine structural, functional and developmental approaches to better identify the main trends governing the evolution of NR ligand binding and the evolution of the ligand/receptor couple. The three receptor types we have chosen represent three different possibilities of evolution of a ligand/receptor couple: - TR, with a unique divergent receptor in amphioxus (AmphiTR) related to duplicated vertebrate copies (TRa and TRb) - NR1H, with a massive lineage specific expansion in amphioxus (it contains 10 NR1H receptors for only 2 in vertebrates) - NR7, a completely new subfamily of receptors probably secondarily lost in vertebrates but present in different protostome and deuterostome invertebrates. The use of these three examples of NRs in amphioxus offer unique opportunities to study the consequences of gene duplications in terms of the evolution of the ligand binding capacity. In addition, since two of the studied receptors are major pharmacological targets, we anticipate that a better knowledge of the evolutionary plasticity of the ligand binding pocket will bring new information that could be exploited for drug design. We will characterize these events at the structural and functional levels by combining 3D structure determination and testing the activities of the receptors (ligand binding, transactivation). Because it will be important to relate these findings with the biological role played by the receptor we will associate to these approaches a developmental analysis of the receptors for which the amphioxus is also an excellent model system.

Eukaryotic transcription is a fundamental mechanism of gene expression regulation with important implications in human health. The nucleosome - the key component of chromatin - is a barrier for the numerous cellular processes that require accessibility of distinct protein factors to their cognate DNA sequences, in particular the RNA polymerase. To overcome the nucleosome barrier, the cell uses different strategies including histone modifications, chromatin remodelers and histone variants. Chromatin-modifying proteins regulate transcription by modulating the DNA accessibility of a target gene to the RNA polymerase, for example through the deacetylation / acetylation of histone protein tails within the nucleosome, thus inducing a transition from a repressive to an activated state. Associated to these regulatory proteins are ATP-dependent chromatin-remodelling enzymes such as the SWI/SNF or the Mi2/NuRD complex that induce nucleosome sliding or histone replacement by histone variants characteristic of transcriptionally active genes. Understanding the molecular mechanisms by which chromatin modifying and remodelling complexes modulate transcription is crucial for both fundamental reasons and therapeutic applications in cancer and numerous other diseases such as skin or immune diseases. The aim of this innovative project is to reconstitute recombinant human nucleosomes and use their core structure as a platform for assembling regulatory complexes with chromatin-modifying and -remodelling enzymes, with a focus on (i) the NuRD complex containing histone deacetylase and remodelling activities, and (ii) the acetyltransferases of different families such as GCN5, HAT1 and the nuclear receptor co-activator SRC1. The structure-function relationship of these complexes - prepared either as subunits or domains interacting with the nucleosome or as full complexes whenever these can be isolated in homogeneous form - will be studied by using an integrative biology approach including molecular biology, biochemistry, biophysics, bio-informatics, and using in parallel crystallography and cryo-electron microscopy (cryo-EM). In order to achieve a detailed interpretation at the molecular level, single particle cryo-EM structures will be determined in the 10-6 Å resolution range by advanced image processing of data recorded on a newly installed state-of-the-art high-resolution electron microscope. The structures will be analysed in combination with the crystal structures of the nucleosome and of complexes determined along this project, integrating known and newly obtained functional data. Expertise in the relevant techniques is well-established in the two partner groups thus forming an interdisciplinary team, supported by in-house facilities and technical platforms for imaging, for structural biology and genomics, and for bioinformatics. The study of chromatin-related transcription regulation and de-regulation will on the long-term contribute to the development of novel therapeutic applications.