Simon Roubille, team Lomonte – 8 november 2022
Implication of the SETDB1-HUSH-MORC2 axis in the control of herpes simplex virus 1 latency
The human body is colonized by numerous commensal microorganisms forming the human microbiota. The human microbiota is now recognized as an essential player in the health and physiology of its host. Efforts to describe the human microbiota have focused on its bacterial component, but it is becoming increasingly clear that many viruses also belong to the human microbiota. The viral component of the human microbiome therefore introduces a more recently accepted notion, that of the human virome, which corresponds to all viruses detected in humans. Among these viruses, we find the Herpesvirus family and in particular the herpes simplex virus 1 (HSV-1). This virus persists in the host organism in latent form within the trigeminal ganglia innervating the face. This latency is characterized by the absence of virus replication at the time of the initial infection, which results in transcriptional repression of the viral genome under the pressure of the intrinsic antiviral response. However, reactivation episodes are very frequent. This reactivation leads to variable clinical manifestations in the peripheral and central nervous system, with increasing degrees of severity ranging from simple epithelial damage to the face such as fever blisters, to keratitis that can lead to blindness, but also to fulminant encephalitis or even be a risk factor for Alzheimer’s disease.
The establishment of HSV-1 latency is controlled by PML nuclear bodies (PML NBs) but their exact involvement remains unclear. One of the major characteristics of the latency of the virus is the interaction between the viral genome and PML NBs forming structures called viral DNA-containing PML-NBs (vDCP NBs). The use of an infection model of primary human fibroblasts, which mimics the formation of vDCP NBs, combined with an immuno-FISH approach, allowed to show that vDCP NBs contain the SETDB1-HUSH-MORC2 entity. ChIP- qPCR experiments demonstrated the involvement of this axis in the latency of the HSV-1 genome via the deposition of the chromatin mark : trimethylation of lysine 9 of histone 3 (H3K9me3). Depletion experiments of the different components of this entity showed its involvement in the maintenance of HSV-1 latency, including in neurons derived from human induced pluripotent stem cells (huiPSDN). Finally, this study revealed the SETDB1-HUSH-MORC2 axis as a restriction factor of HSV-1 at the epigenetic level.
Constance Kleijwegt, P. Lomonte team – July 1st, 2021
Role of PML nuclear bodies and of the histone chaperone complex HIRA in the regulation of chromatin dynamics
Within the nucleus of eucaryotic cells, DNA is wrapped around histone proteins in order to form a structure called chromatin. This organization allows the compaction of a genome of 2 meters in a nucleus of around ten micrometers. It also allows the regulation of gene expression. Indeed, chromatin carries a source of information called epigenetic information and the modulation of its structure has important consequences on the transcriptional program. The histone chaperone complex HIRA is involved in the deposition of the histone variant H3.3 in euchromatin regions and in regions devoid of nucleosomes, in order to ensure chromatin and epigenetic integrity. Under normal conditions, the HIRA complex localizes homogeneously in the cell nucleus; on the other hand, under certain stressful conditions, such as during inflammation, the entry of cells into senescence or, as I showed in my thesis, during the induction of double-strand DNA breaks, the HIRA complex accumulates with H3.3 in spherical nuclear membrane-less organelles called PML nuclear bodies. The functional impact of such accumulation is still poorly understood. During my thesis, I sought to understand how and why the HIRA complex localizes in these organelles. In particular, I sought to analyse the functional impact of this localization on the deposition of H3.3 in particular regions of the chromatin.
First, I investigated the mechanisms and role of HIRA relocalization during inflammation response to type I interferon (IFN-I) treatment, a pro-inflammatory protein. My work has shown that the HIRA complex relocalizes within PML nuclear bodies in a manner dependent on interactions between SUMO modifications and a SUMO-interacting motif (SIM). In addition, I have shown that the HIRA complex and PML nuclear bodies are important in the regulation of the expression of IFN-I-stimulated genes (ISGs), by participating in the incorporation of the histone variant H3 .3 in these gene loci. Their action could allow better control of the inflammatory response in order to ensure its homeostasis. Secondly, I was interested in the relocalization of HIRA within PML nuclear bodies induced by double-strand breaks, never described before. I have shown that this relocalization is independent of IFN-I signaling and probably depends on signaling in response to DNA damage. Recruitment of HIRA is also dependent on SIM-SUMO interactions. Interestingly, the HIRA complex in PML nuclear bodies juxtaposes near the sites of damage, and this could serve to incorporate new histones after repair and thus ensure the reestablishment of the chromatin structure.
Nathalie Couturier, V. Gache team – June 09, 2020
Identification of new regulators of myonuclei positioning during skeletal muscle fibers development
Skeletal muscle fibers are built from fusion of myoblasts allowing the formation of myotubes. Those syncytia contain hundreds of nuclei that undergo many movements, simultaneously with myotubes maturation process, until reaching a final peripheric localization in mature fibers (myofibers). Disorganization of nuclei disposal is always associated with myofibers misfunctioning (i.e.: sarcopenia, centronuclear myopathy). Peripheral nuclei positioning in mature fiber appears to be essential for their functionality. Understanding the process of nuclei positioning turned up as key point.
Involvement of molecular motors are known in spacing myonuclei in the first steps of muscle differentiation, however the role of microtubules associated proteins (Maps) is less described. Consequently, identify new Maps involved in nuclei positioning during muscle formation may 1) provide a better understanding of muscle differentiation; 2) bring to light new targets potentially involved in pathologies presenting nuclei mispositioning.
In this context, my thesis work consisted in identifying proteins linked to microtubules, directly or indirectly, and involved in the regulation of myonuclei positioning throughout skeletal muscle fibers differentiation. Proteomes associated to microtubules and differentially expressed during muscle differentiation were identified using a mass-spectrometry analysis. Based on this identification, 239 proteins from the total cellular extracts of myotubes and myofibers; and 240 proteins identified in proteomes linked to microtubules were selected. A siRNA screen was performed on primary myotubes in order to decipher the involvement of those selected targets in myonuclei positioning during the first steps of muscle differentiation. This siRNA screen allowed the identification of three proteins which roles on myonuclei positioning appeared to be crucial.
Unexpectedly, we identified a cytoplasmic role of the mitotic protein NuMA1 in muscle biology field. This protein progressively accumulates into the cytoplasm where it stabilizes microtubule network in differentiating myofibers. This cytoplasmic fraction of NuMA1 proteins, in association with the dynein, regulates microtubule network architecture and myonuclei spacing. Its role is also crucial in maintaining peripherized myonuclei in mature myofibers and in neuromuscular junction regions.
Keywords: Skeletal muscle development, nuclei movements, microtubules, MAPs, primary muscle cells, sarcopenia, centronuclear myopathy, NuMA1
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.
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
Isabella Scionti, L. Schaeffer team – Nov. 20, 2017
Epigenetic regulation of skeletal muscle differentiation
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.