Nicolas Chatron, J. Courchet team – Dec. 20, 2019
Complex chromosomal rearrangement: from precise molecular characterization to functional consequences
Human cytogenetics is a discipline aimed at studying the structure and function of the chromosomes of our species. In the early 2010’s, genome sequencing revealed chromosomal rearrangements of as yet unknown complexity, termed chromoanagenesis. While a significant proportion of tumour genomes present such anomalies, descriptions of constitutional cases are rare and the underlying mechanisms poorly understood. We report here the genome sequencing of 20 new cases of constitutional chromoanagenesis (6 balanced and 14 unbalanced), constituting the largest cohort to date. In several patients, loci of less than one kilobase appear several times in the reshuffled chromosome in what we call a “hub”. The analysis of the distribution of breakpoints of these chromoanagenesis and those in the literature showed that late chromatin replication was the main “risk factor” for chromosomal breakage. This result provides an orthogonal demonstration of the premature condensation of a chromosome hypothesis at the origin of these rearrangements and shows for the first time a common origin of constitutional and tumoral chromoanagenesis. At the same time, breakpoint distribution of simple rearrangements appears to be biased towards the center of the nucleus, opening up important avenues of research. To better understand the consequences of these complex reshuffles on the functioning of the genome, we studied the transcriptome of the 6 balanced chromoanagenesis. We have not detected any massive deregulation of the genome. Even locally, near the breakpoints, there does not appear to be any deregulation of gene expression, again raising important questions about the mechanisms of “resistance” to structural variants.
The detection of chromosomal rearrangements is (almost) no longer limited by technology (short-read , long-read, linked-read sequencing, optical mapping). Understanding the mechanisms at their origin, knowledge of their pathogenicity and more generally the mastering of genome biology are the new limits to the genotype/phenotype correlation. By studying chromoanagenesis, an exceptional fusion transcript in a hemophiliac patient and describing poorly known transposable elements (retrocopies) we are bringing important new information to the field.
Hugo Ducuing, V. Castellani Team- Dec. 20, 2019
Functional study of PlexinA1/SlitC signaling during commissural axon guidance in the spinal cord
In bilaterians, commissures are neuronal circuits interconnecting the two halves of the central nervous system (CNS). They are essential for the integration and coordination of sensory modalities, motor commands and brain processing. During embryonic development, commissural neurons extend an axon that crosses the midline of the CNS to connect its final target on the contralateral side. This navigation is highly stereotyped and controlled, at each of its stages, by multiple axon guidance signaling.
In the developing spinal cord, the axons of dorsally located commissural interneurons grow ventrally and cross the midline within a particular territory: the floor plate (FP). They navigate through the feet of the FP glial cells and acquire during their progression the ability to respond to the repulsive signals present in their local environment. This gain of sensitivity enables the repulsive signals: 1) to expel the axons out of the FP, which then move rostrally and continue their navigation towards their final targets and, 2) to prevent the axons from backcrossing the midline by setting a non-permissive barrier. During the last decades, several laboratories including ours have identified the main repulsive signaling of the FP: Semaphorin3B (Sema3B) acting via the PlexinA1 (PlxnA1) – Neuropilin-2 (Nrp2) receptor complex, SlitC via the PlxnA1 receptor and SlitN via the Roundabout1-2 (Robo1-2) receptors. However, the precise contributions and functional specificities of these different signals remain unclear.
During my thesis, I first participated in a study that aimed at understanding whether the generation of functional specificities of repulsive signaling could result from spatial and temporal dynamics at the growth cone (GC) surface specific to each guidance receptor. In particular, I developed a FRAP paradigm (fluorescence recovery after photobleaching) on native embryonic spinal cords, and performed experiments on GC electroporated with guidance receptors fused to the pHluorin, a pH-sensitive GFP allowing to follow the membrane pool of receptors. These experiments allowed me to characterize the dynamics of PlxnA1 and Robo1 receptors during the crossing of the FP and to show that the exocytosis of PlxnA1 is significantly stronger than that of Robo1. I also imaged in super-resolution the membrane and total pools of different receptors in GCs navigating the FP. We found a complementary distribution of Robo1 and PlxnA1 at the GC surface. More generally, these results revealed a unique sequence of membrane insertion events of the receptors in the GCs, which could allow them to functionally discriminate the different repulsive signals they are exposed to during their FP navigation.
My thesis work then focused on the functional specificity of one of these signaling: PlxnA1/SlitC. Following a previous in vitro study of our team showing that the PlxnA1 Y1815F mutation allows specifically abrogating SlitC signaling but not that of Sema3B, we generated a mouse model carrying this mutation. I first analyzed the guidance phenotypes of commissural axons in this model, and observed defects providing evidence that PlxnA1/SlitC signaling specifically prevents axons from performing U-turns while crossing the midline. By reintroducing PlxnA1Y1815F fused to pHluorin into the spinal cord of PlxnA1-/- embryos, I could show that the mutated receptor is unable to rescue this phenotype of U-turns. I then became interested in the impacts of the Y1815F mutation on PlxnA1. First, I observed that PlxnA1Y1815F is well expressed by commissural neurons but its temporal pattern of membrane addressing shows some differences when compared to that of the native receptor. Moreover, I found that the surface expression of PlxnA1Y1815F is altered, both in vivo, when the receptor is re-expressed in the embryonic spinal cord, and in vitro, in an N2a cell model and in cultured commissural neurons. Finally, I investigated whether the Y1815F mutation alters the spectrum of PlxnA1 interactions by comparing the binding partners of the native and mutated receptors by a mass spectrometry approach. The analyzes established a first list of candidates whose interactions with PlxnA1 were disrupted by the Y1815F mutation. Additional experiments are in progress to validate these alterations.
Collectively, my results bring new information on the mechanisms governing commissural axon navigation and provide in this context the first dissection of ligand-specific functional contributions mediated by a guidance receptor shared by several ligands.
Jessica Bouvière, B. Chazaud team – Sept. 30, 2019
Role of selenoprotein P and glutathione peroxidase 3 in macrophage phenotype and skeletal muscle regeneration
Supervisor: Rémi Mounier
Tissue repair after skeletal muscle injury is supported by various cells and notably by macrophages that orchestrate both the inflammatory response, the resolution of inflammation and the recovery phase allowing tissue recovery. After injury, pro-inflammatory macrophages activate myogenic cell proliferation and phagocyte debris. At the time of resolution of inflammation, macrophages switch their phenotype to acquire an anti-inflammatory state, which stimulates the fusion of myogenic cells into multinucleated myofibers. The effectors by which anti-inflammatory macrophages sustain their pro-myogenic effect are poorly known. Thanks to a transcriptomic/secretomic screen comparing the secretome of pro- and anti- inflammatory human macrophages, we identified two anti-oxidant molecules that are two selenoproteins: selenoprotein P (SEPP1) and glutathion peroxidase 3 (GPX3), which functions remain poorly characterized in both muscle biology and inflammation. Macrophage-derived GPX3 and SEPP1 are involved in the function of anti-inflammatory macrophages in the stimulation of the late steps of myogenesis. In vivo, these two selenoproteins are necessary for the homeostasis of skeletal muscle. Moreover, using a skeletal muscle injury model of animals lacking selenoprotein within macrophages (LysMCre/+ Sepp1fl/fl model) we found that the absence of SEPP1 impaired the transition from a pro to an anti-inflammatory phenotype during skeletal muscle regeneration.
Thus, we have demonstrated for the first time the role of selenoproteins secreted by macrophages in tissue repair establishing a link between antioxidant molecules and inflammation resolution.
Colline Sanchez, V. Jacquemond team – Sept. 27, 2019
Investigating sarcoplasmic reticulum function during skeletal muscle excitation-contraction coupling using fluorescent biosensors
Excitation-contraction (EC) coupling in skeletal muscle corresponds to the sequence of events through which muscle fiber contraction is triggered in response to plasma membrane electrical activity. EC coupling takes place at the triads; these are nanoscopic domains in which the transverse invaginations (t-tubules) of the surface membrane are in close apposition with two adjacent terminal cisternae of the sarcoplasmic reticulum (SR) membrane. More precisely, EC coupling starts with action potentials fired at the endplate, propagating throughout the surface membrane and in depth into the muscle fiber through the t-tubules network. When reaching the triadic region, action potentials activate the voltage-sensing protein Cav1.1. In turns, Cav1.1 directly opens up the type 1 ryanodine receptor (RYR1) in the immediately adjacent SR membrane, through intermolecular conformational coupling. This triggers RYR1-mediated SR Ca2+ release which produces an increase in cytosolic Ca2+ triggering contraction.
Current understanding of the mechanisms involved in the control and regulation of RYR1 channels function is still limited. One reason is related to the fact that detection of RYR1 channel activity in intact muscle fibers is only achieved with indirect methods. Also, whether the SR membrane voltage experiences changes during muscle activity has so far never been experimentally assessed. Yet, deeper knowledge of these processes is essential for our understanding of muscle function in normal and disease conditions.
In this context, the general aim of my PhD project was to design and use fluorescent protein biosensors specifically localized at the SR membrane of differentiated muscle fibers, by fusing them to an appropriate targeting sequence. Thanks to a combination of single cell physiology and biophysics techniques based on electrophysiology and biosensor fluorescence detection, we were able to study the SR activity during muscle fiber function. Specifically, my PhD work focused on two major issues: SR membrane voltage and SR calcium signaling during EC coupling.
The first aim of my work was to characterize SR membrane voltage changes during muscle fiber activity. For this, we used voltage sensitive FRET-biosensors of the Mermaid family. Results show that the SR trans-membrane voltage experiences no substantial change during EC coupling. This provides the first experimental evidence, in physiological conditions, for the existence of ion counter-fluxes that balance the charge deficit associated with RYR1-mediated SR Ca2+ release. Indeed, this process is essential for maintaining the SR Ca2+ flux upon RYR1 channels opening and thus critically important for EC coupling efficiency.
The second objective of my work aimed at detecting the changes in Ca2+ concentration occurring in the immediate vicinity of the RYR1 Ca2+ release channels during muscle fiber activation. For this, we took advantage of one member of the recent generation of genetically encoded Ca2+ biosensors: GCaMP6f. The SR-targeted biosensor provides a unique access to the individual activity of RYR1 channels populations within distinct triads of a same muscle fiber. Beyond allowing a detailed characterization of the biosensor properties in this preparation, results highlight the remarkable uniformity of SR Ca2+ release activation from one triad to another, during EC coupling. These results open up stimulating perspectives for the investigation of disease conditions associated with defective behavior of RYR1 channels.
Keywords: skeletal muscle; excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptor; fluorescent biosensors; membrane voltage; intracellular calcium.
Thibaut Desgeorges, B. Chazaud team – May 22, 2019
Crosstalk of Glucocorticoid Receptor and AMP-activated protein kinase in macrophages during skeletal muscle regeneration
Supervisors: Rémi Mounier & Bénédicte Chazaud
Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). Inflammation, and notably macrophages, plays important roles during this process. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Then, macrophages switch their phenotype toward an anti-inflammatory restorative profile and promote muscle stem differentiation, fusion and myofiber growth. This sequence of macrophage profile is essential for an efficient skeletal muscle regeneration. The lab has shown that this phenotype switch is dependent of AMP kinase (AMPK)α1, a major energetic sensor in the cell controlling cellular metabolism. Besides, glucocorticoids have been used for decades for their anti-inflammatory effects on inflammation. Their actions are mediated by the Glucocorticoid Receptor which induces or represses gene expression by direct or indirect DNA-binding. As AMPKα1 and glucocorticoids induce similar anti-inflammatory effects on macrophages, we hypothesized that these 2 pathways could be interconnected in macrophages to allow the resolution of inflammation and muscle repair. Data from an in vitro model of skeletal muscle injury using bone marrow derived macrophages showed that: i) glucocorticoids induce AMPK phosphorylation; ii) AMPKα1 is required for the functional acquisition of the anti- inflammatory phenotype induced by glucocorticoids. Indeed, AMPKα1-deficient macrophages did not switch their phenotype and did not sustain myogenesis. In vivo experiments using LysMCre/+;AMPKα1fl/fl mice in which AMPKα1 is depleted only in myeloid cells, showed that macrophagic AMPK drove the beneficial effects of glucocorticoids during skeletal muscle regeneration. Inversely, in the absence of AMPK in macrophages, glucocorticoids induced a delayed muscle regeneration and modifications in myofiber maturation, assessed by the alteration of myosin heavy chain expression. Altogether, these data show that glucocorticoids need AMPKα1 in macrophages for the resolution of inflammation and an efficient skeletal muscle regeneration.
Keywords: muscle, regeneration, macrophages, AMPK, glucocorticoids
Elena Cerutti, A. GIGLIA-MARI team – May 10, 2019
Nucleotide Excision Repair at the crossroad with transcription
The integrity of DNA is continuously challenged by a variety of endogenous and exogenous agents (e.g. ultraviolet light, cigarette smoke, environmental pollution, oxidative damage, etc.) that cause DNA lesions which interfere with proper cellular functions. Nucleotide Excision Repair (NER) mechanism removes helix-distorting DNA adducts such as UV-induced lesions and it exists in two distinct sub-pathways depending where DNA lesions are located within the genome. One of these sub pathways is directly linked to the DNA transcription by RNA Polymerase 2 (TCR). In the first part of this work, we demonstrated that a fully proficient NER mechanism is also necessary for repair of ribosomal DNA, transcribed by RNA polymerase 1 and accounting for the 60 % of the total cellular transcription. Furthermore, we identified and clarified the mechanism of two proteins responsible for the UV-dependent nucleolar repositioning of RNAP1 and rDNA observed during repair. In the second part of this work, we studied the molecular function of the XAB2 protein during NER repair and we demonstrated its involvement in the TCR process. In addition, we also shown the presence of XAB2 in a pre-mRNA splicing complex. Finally, we described the impact of XAB2 on RNAP2 mobility during the first steps of TCR repair, thus suggesting a role of XAB2 in the lesion recognition process.
Linda Gsaier, B. Chazaud team – October 22, 2018
Role of cell-autonomous regulation of metabolism on muscle stem cell fate and skeletal muscle homeostasis
Supervisor: Rémi Mounier
During muscle regeneration following injury, muscle stem cells, also called satellite cells, leave their quiescent state and activate. MuSCs are capable of both differentiating to repair muscle tissue after an injury and self-renewing to replenish the pool of stem cells. The regulation of their fate is modulated by several signaling pathways such as Wnt, Notch or TGFß pathway. However, there are few data concerning the involvement of metabolism in the fate of satellite cells. Yet it has been shown that the activation of satellite cells is closely related to cellular metabolism, which one of the main players is AMPK protein kinase. This heterotrimeric complex, composed of three subunits α, ß et 𝜸, is responsible for the balance between energy consumption and energy production within the cell. With the modulation of mTORC1, AMPK 1 has also been shown to be responsible for cell growth and proliferation of myogenic precursors. Using different mouse models, primary lines and sorted satellite cells, we determined the role that each isoform, AMPKα1 and AMPKα2, could play within the cell, on myogenesis and on the homeostasis of the regenerated muscle. First, we demonstrated that AMPKα1-LDH signaling pathway regulates the satellite cells self-renewal by controlling metabolism. Indeed, at the time of cell fate choice between commitment into terminal differentiation versus self-renewal, the AMPK 1 pathway induces a decrease in LDH activity, allowing cells to adopt an oxidative phosphorylation metabolism responding to their energy needs. In a second time, we demonstrated that the AMPKα2 isoform, expressed during myogenesis only after the induction of muscle cell differentiation, was responsible for a modulation of the muscular regeneration and that its absence induced a lack of differentiation and a delay in maturation of the new formed myofibers. Our work allowed us to confirm the central role of AMPK protein kinase in the regulation, by the modulation of metabolism, of muscle stem cell fate in a context of skeletal muscle regeneration in a mouse model.
Keywords: muscle, regeneration, metabolism, myogenesis, AMPK
Sonia El Mouridi, T. BOULIN team – Oct. 18, 2018
Régulation de l’excitabilité musculaire par le canal potassique EGL-23 et la voie de signalisation LIN-12/Notch chez le nématode C. elegans.
Philippe Tardy, T. BOULIN team – Sept. 18, 2018
Caractérisation du trafic cellulaire du canal potassique à deux domaines P UNC-58 par la protéine UNC-44 chez le nématode C. elegans.
Alexis Weinreb, J-L BESSEREAU team – Sept. 11, 2018
Impact de l’activité postsynaptique sur le développement et le maintien de la jonction neuromusculaire de C.elegans.
Diane Lebrun, C. Marcelle team – June 8, 2018
Caractérisation et généralisation de l’implication de la voie NOTCH cytoplasmique au cours des processus de transition épithélio-mésenchymateuse chez l’embryon de poulet
La transition épithélio-mésenchymateuse (EMT) est un processus incontournable dans de nombreux contextes normaux et pathologiques, tels que gastrulation, organogenèse, fibroses et cancers. Cette transformation de cellule épithéliale en cellule mésenchymateuse est indissociable de l’acquisition de propriétés migratoires et est généralement associée à un changement de destin cellulaire. Différentes voies moléculaires sont impliquées selon le contexte de l’EMT concernée. Récemment, notre laboratoire a mis en évidence que la voie Notch cytoplasmique contrôle l’EMT des cellules de la lèvre dorso-médiale du somite (DML). Les crêtes neurales exprimant DLL1 activent « en passant » le récepteur NOTCH, liberant ainsi le domaine intra-cytoplasmique de NOTCH (NICD). Dans le cytoplasme, NICD inhibe la kinase GSK3ß, conduisant à la stabilisation de SNAIL, un gène maître de la transition épithélio-mésenchymateuse. Il en résulte une libération de la βcaténine des jonctions adhérentes qui, après translocation dans le noyau, active la transcription des gènes de la myogénèse (Myf5). Ainsi, l’activation de la voie Notch cytoplasmique permet une induction concomitante de l’EMT et de la myogénèse. La fonction cytoplasmique de Notch reste controversée et le mécanisme par lequel NICD inhibe GSK3ß reste obscur.
Au cours de ma thèse j’ai cherché à élucider le mécanisme par lequel NICD inhibe l’activité kinase de GSK3ß. J’ai confirmé l’interaction de GSK3ß et de NICD en démontrant leur interaction via CoIP. Après avoir démontré l’implication de la sérine-thréonie kinase AKT dans la myogenèse des cellules de la DML, j’ai mis en évidence, via CoIP et électroporation, que l’inhibition GSK3ß par NICD est très certainement médiée par AKT, connue pour être impliquée dans l’EMT et inhiber GSK3ß par phosphorylation. En comparant le NICD1 de poulet et les 4 NICD de souris, j’ai montré que l’expression exogène de ces 5 molécules induit l’EMT et la différenciation myogénique de manière similaire. J’ai aussi montré que parmi des différents domaines de NICD, le domaine RAM, connu pour se lier à l’ADN (via RBPJ), est nécessaire et suffisant à l’inhibition de GSK3ß.
Un second axe de ma thèse a été de tester l’implication de la voie Notch cytoplasmique dans d’autres contextes d’EMT. Pour ce faire, j’ai mis en évidence que cette voie est impliquée dans les autres lèvres du dermomyotome mais aussi dans les crêtes neurales qui délaminent du toit du tube neural. J’ai en particulier mis en évidence une co-activation des voies Wnt et Notch, une inhibition de la kinase GSK3ß par NICD cytoplasmique ainsi qu’une inhibition de la différenciation en présence d’une ß-caténine mutée, retenue à la membrane, ou en présence d’une molécule SNAIL2 dominant-négative.
Le dernier axe de ma thèse a consisté à élucider le mécanisme de régulation de l’induction de l’EMT et de la myogenèse via l’activation de NICD. Il a été mis en évidence que toutes les cellules de la DML peuvent être activées via DLL1 et que la surexpression massive de NICD dans la DML provoque une différenciation massive et une déplétion du groupe de cellules progénitrices. Afin de déterminer si la régulation de cette initiation se fait avant ou après induction de NICD, j’ai créé un plasmide permettant de répondre à cette question et afin de visualiser son expression in vivo, j’ai initié une collaboration avec une équipe de l’ILM afin de créer un microscope vertical SPIM biphoton permettant l’observation d’embryon de poulets vivants. Ce projet est encore en cours.
Ainsi, mon travail de thèse a permis de i) préciser le mécanisme par lequel NOTCH et GSK3ß interagissent physiquement au niveau cytoplasmique lors de l’induction de l’EMT et de la myogenèse des cellules de la DML et j’ai ajouté un nouvel acteur à ce complexe, AKT qui permettrait de médier l’inhibition de GSK3ß par NICD via phosphorylation, de ii) démontrer que cette voie Notch cytoplasmique est présente dans au moins deux autres EMT embryonnaires, celles de la lèvre ventro-latérale du somite et celle des crêtes neurales, et iii) de mettre en place de nouveaux outils d’imagerie in vivo pour explorer les aspects dynamiques de ces mécanismes.
Isabella Scionti, L. Schaeffer team – Nov. 20, 2017
Epigenetic regulation of skeletal muscle differentiation
Isabella Scionti- Directeur de thèse Laurent SCHAEFFER- equipe Schaeffer
LSD1 and PHF2 are lysine de-methylases that can de-methylate both histone proteins, influencing gene expressionand non-histone proteins, affecting their activity or stability. Functional approaches using Lsd1or Phf2 inactivation in mouse have demonstrated the involvement of these enzymes in the engagement of progenitor cells into differentiation.
One of the best-characterized examples of how progenitor cells multiply and differentiate to form functional organ is myogenesis. It is initiated by the specific timing expression of the specific regulatory genes; among these factors, MYOD is a key regulator of the engagement into differentiation of muscle progenitor cells. Although the action of MYOD during muscle differentiation has been extensively studied, still little is known about the chromatin remodeling events associated with the activation of MyoDexpression. Among the regulatory regions of MyoDexpression, the Core Enhancer region (CE), which transcribes for a non-coding enhancer RNA (CEeRNA), has been demonstrated to control the initiation of MyoDexpression during myoblast commitment.
We identified LSD1 and PHF2 as key activators of the MyoDCE. In vitroand in vivoablation of LSD1 or inhibition of LSD1 enzymatic activity impaired the recruitment of RNA PolII on the CE, resulting in a failed expression of the CEeRNA. According to our results, forced expression of the CEeRNA efficiently rescue MyoDexpression and myoblast fusion in the absence of LSD1. Moreover PHF2 interacts with LSD1 regulating its protein stability. Indeed in vitroablation of PHF2 results in a massive LSD1 degradation and thus absence of CEeRNA expression. However, all the histone modifications occurring on the CE region upon activation cannot be directly attributed to LSD1 or PHF2 enzymatic activity.
These results raise the question of the identity of LSD1 and PHF2 partners, which co-participate to CEeRNA expression and thus to the engagement of myoblast cells into differentiation.
Adeline Mergoud dit Lamarche, J-L BESSEREAU team – July 13, 2016
Étude des modifications subcellulaire associées au vieillissement musculaire chez Caenorhabditis elegans– Rôle du facteur de transcription UNC-120/SRF.
Viviane Lainé, J-L BESSEREAU team – June 23, 2016
Conséquences fonctionnelles de la suractivation des récepteurs de l’acétylcholine et des canaux calciques de type L sur l’homéostasie des cellules musculaires striées de Caenorhabditis elegans.