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Skeletal muscle regeneration relies on muscle stem (satellite) cells. We previously demonstrated that satellite cells efficiently and accurately repair radiation-induced DNA double-strand breaks (DSBs) via the DNA-dependent kinase DNA-PKcs. We show here that DNA-PKcs affects myogenesis independently of its role in DSB repair. Consequently, this process does not require the accumulation of DSBs and it is also independent of caspase-induced DNA damage. We report that in myogenic cells DNA-PKcs is essential for the expression of the differentiation factor Myogenin in an Akt2-dependent manner. DNA-PKcs interacts with the p300-containing complex that activates Myogenin transcription. We show also that SCID mice that are deficient in DNA-PKcs, and are used for transplantation and muscle regeneration studies, display altered myofiber composition and delayed myogenesis upon injury. These defects are exacerbated after repeated injury/regeneration events resulting in reduced muscle size. We thus identify a novel regulator of myogenic differentiation, and define a differentiation phase that does not involve the DNA damage/repair process.

Mitochondrial DNA (mtDNA) damaged by reactive oxygen species (ROS) triggers so far poorly understood processes of mtDNA maintenance that are coordinated by a complex interplay among DNA repair, DNA degradation, and DNA replication. This study was designed to identify the proteins involved in mtDNA maintenance by applying a special long-range PCR, reflecting mtDNA integrity in the minor arc. A siRNA screening of literature-based candidates was performed under conditions of enforced oxidative phosphorylation revealing the functional group of polymerases and therein polymerase ζ (POLZ) as top hits. Thus, POLZ knockdown caused mtDNA accumulation, which required the activity of the base excision repair (BER) nuclease APE1, and was followed by compensatory mtDNA replication determined by the single-cell mitochondrial in situ hybridization protocol (mTRIP). Quenching reactive oxygen species (ROS) in mitochondria unveiled an additional, ROS-independent involvement of POLZ in the formation of a typical deletion in the minor arc region. Together with data demonstrating the localization of POLZ in mitochondria, we suggest that POLZ plays a significant role in mtDNA turnover, particularly under conditions of oxidative stress.

Cockayne syndrome (CS) is a rare disease caused by mutations in ERCC6/CSB or ERCC8/CSA. We report here the clinical, genetic, and functional analyses of three unrelated patients mutated in ERCC6/CSB with a severe phenotype. After clinical examination, two patients were investigated via next generation sequencing, targeting seventeen Nucleotide Excision Repair (NER) genes. All three patients harbored a novel, c.3156dup, homozygous mutation located in exon 18 of ERCC6/CSB that affects the C-terminal region of the protein. Sanger sequencing confirmed the mutation and the parental segregation in the three families, and Western blots showed a lack of the full-length protein. NER functional impairment was shown by reduced recovery of RNA synthesis with proficient unscheduled DNA synthesis after UV-C radiations in patient-derived fibroblasts. Despite sharing the same mutation, the clinical spectrum was heterogeneous among the three patients, and only two patients displayed clinical photosensitivity. This novel ERCC6 variant in Tunisian patients suggests a founder effect and has implications for setting-up prenatal diagnosis/genetic counselling in North Africa, where this disease is largely undiagnosed. This study reveals one of the rare cases of CS clinical heterogeneity despite the same mutation. Moreover, the occurrence of an identical homozygous mutation, which either results in clinical photosensitivity or does not, strongly suggests that this classic CS symptom relies on multiple factors.

Significance Reactive species have been classically considered causative of age-related degenerative processes, but the scenario appears considerably more complex and to some extent counterintuitive than originally anticipated. The impact of reactive species in precocious ageing syndromes is revealing new clues to understand and perhaps challenge the resulting degenerative processes. Recent Advances. Our understanding of reactive species has considerably evolved, including their hormetic effect (beneficial at a certain level, harmful beyond this level), the occurrence of diverse hormetic peaks in different cell types and organisms, and the extended type of reactive species that are relevant in biological processes. Our understanding of the impact of reactive species has also expanded from the dichotomic damaging/signaling role to modulation of gene expression. Critical issues These new concepts are affecting the study of ageing and diseases where ageing is greatly accelerated. We discuss how notions arising from the study of the underlying mechanisms of a progeroid disease, Cockayne syndrome, represent a paradigm shift that may shed a new light in understanding the role of reactive species in age-related degenerative processes. Future issues. Future investigations urge to explore established and emerging notions to elucidate the multiple contributions of reactive species in degenerative processes linked to pathophysiological ageing and their possible amelioration.

Cellular senescence has causative links with ageing and age-related diseases, however, it remains unclear if progeroid factors cause senescence in normal cells. Here, we show that depletion of CSB, a protein mutated in progeroid Cockayne syndrome (CS), is the earliest known trigger of p21-dependent replicative senescence. CSB depletion promotes overexpression of the HTRA3 protease resulting in mitochondrial impairments, which are causally linked to CS pathological phenotypes. The CSB promoter is downregulated by histone H3 hypoacetylation during DNA damage-response. Mechanistically, CSB binds to the p21 promoter thereby downregulating its transcription and blocking replicative senescence in a p53-independent manner. This activity of CSB is independent of its role in the repair of UV-induced DNA damage. HTRA3 accumulation and senescence are partially rescued upon reduction of oxidative/nitrosative stress. These findings establish a CSB/p21 axis that acts as a barrier to replicative senescence, and link a progeroid factor with the process of regular ageing in human., Senescence of metabolically active cells is a process linked to ageing. Here the authors reveal that CSB is required to block replicative senescence, and epigenetic control of CSB downregulation triggers proliferative arrest in a p21-dependent manner.

Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.

Cockayne syndrome (CS) and UV-sensitivity syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and chromatin remodelling proteins CSA or CSB, but only CS patients display a progeroid and neurodegenerative phenotype. As epigenetic modifications constitute a well-established hallmark of ageing, we characterized genome-wide DNA methylation (DNAm) of fibroblasts from CS versus UVSS patients and healthy donors. The analysis of differentially methylated positions and regions revealed a CS-specific epigenetic signature, enriched in developmental transcription factors, transmembrane transporters, and cell adhesion factors. The CS-specific signature compared to DNAm changes in other progeroid diseases and regular ageing, identifyied commonalities and differences in epigenetic remodelling. CS shares DNAm changes with normal ageing more than other progeroid diseases do, and according to the methylation clock CS samples show up to 13-fold accelerated ageing. Thus, CS is characterized by a specific epigenomic signature that partially overlaps with and exacerbates DNAm changes occurring in physiological aging. Our results unveil new genes and pathways that are potentially relevant for the progeroid/degenerative CS phenotype.

Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.

Mitochondrial DNA (mtDNA), which is essential for mitochondrial and cell function, is replicated and transcribed in the organelle by proteins that are entirely coded in the nucleus. Replication of mtDNA is challenged not only by threats related to the replication machinery and orchestration of DNA synthesis, but also by factors linked to the peculiarity of this genome. Indeed the architecture, organization, copy number, and location of mtDNA, which are markedly distinct from the nuclear genome, require ad hoc and complex regulation to ensure coordinated replication. As a consequence sub-optimal mtDNA replication, which results from compromised regulation of these factors, is generally associated with mitochondrial dysfunction and disease. Mitochondrial DNA replication should be considered in the context of the organelle and the whole cell, and not just a single genome or a single replication event. Major threats to mtDNA replication are linked to its dependence on both mitochondrial and nuclear factors, which require exquisite coordination of these crucial subcellular compartments. Moreover, regulation of replication events deals with a dynamic population of multiple mtDNA molecules rather than with a fixed number of genome copies, as it is the case for nuclear DNA. Importantly, the mechanistic aspects of mtDNA replication are still debated. We describe here major challenges for human mtDNA replication, the mechanistic aspects of the process that are to a large extent original, and their consequences on disease.

Skeletal muscle stem cells or satellite cells are responsible for muscle regeneration in the adult. Although satellite cells are highly resistant to stress, and display greater capacity to repair molecular damage than the committed progeny, their regenerative potential declines with age. During ageing, satellite cells switch to a state of permanent cell cycle arrest or senescence which prevents their activation. A recent study reveals that the senescence of satellite cell relies on defective autophagy, the quality control mechanism that degrades damaged proteins and organelles. Molecular damage is generated by oxidative stress that also promotes epigenetic changes that activate the expression of master genes, in a double-hit mechanism that ensures senescence. Importantly, genetic, and pharmacological correction of defective autophagy reverses satellite cell senescence and restores muscle regeneration in geriatric mice, with perspectives of modulating age-related functional decline of muscle. This study provides new clues to understand stem cell and organismal ageing.

During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process., Summary: Distinct energy metabolism pathways act during mouse skeletal muscle stem cell commitment and differentiation in different physiological states.

// Rahel Stefanie Wiehe 1 , Boris Gole 1, 2 , Laurent Chatre 3, 4 , Paul Walther 5 , Enrico Calzia 6 , Miria Ricchetti 3, 4 and Lisa Wiesmuller 1 1 Department of Obstetrics and Gynecology, Ulm University, Ulm, 89075, Germany 2 Present address: Centre for Human Molecular Genetics and Pharmacogenomics, Medical Faculty, University of Maribor, Maribor, SI-2000, Slovenia 3 Department of Developmental and Stem Cell Biology, Institute Pasteur, Stem Cells and Development, 75724 Cedex 15, Paris, France 4 Team Stability of Nuclear and Mitochondrial DNA, Unit of Stem Cells and Development, CNRS UMR 3738, 75724 Cedex 15, Paris, France 5 Central Facility for Electron Microscopy, Ulm University, Ulm, 89081, Germany 6 Institute of Anesthesiological Pathophysiology and Process Engineering, Ulm University Hospital, Ulm, 89081, Germany Correspondence to: Lisa Wiesmuller, email: lisa.wiesmueller@uni-ulm.de Keywords: endonuclease G; oxidative damage; base excision repair; mitochondrial DNA degradation Received: January 12, 2018 Accepted: March 12, 2018 Published: April 06, 2018 ABSTRACT Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG’s nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.

International audience; Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

International audience; Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life threatening condition associated with multiple organ failure. Survivors may display long-term disability with muscle weakness that remains poorly understood. Recent data suggest that long-term myopathy in sepsis survivors is due to failure of skeletal muscle stem cells (satellite cells) to regenerate the muscle. Satellite cells impairment in the acute phase of sepsis is linked to unusual mitochondrial dysfunctions, characterized by a dramatic reduction of the mitochondrial mass and hyperactivity of residual organelles. Survivors maintain the impairment of satellite cells, including alterations of the mitochondrial DNA (mtDNA), in the long-term. This condition can be rescued by treatment with mesenchymal stem cells (MSCs) that restore mtDNA alterations and mitochondrial function in satellite cells, and in fine their regenerative potential. Injection of MSCs in turn increases the force of isolated muscle fibers and of the whole animal, and improves the survival rate. These effects occur in the context of reduced inflammation markers that also raised during sepsis. Targeting muscle stem cells mitochondria, in a context of reduced inflammation, may represent a valuable strategy to reduce morbidity and long-term impairment of the muscle upon sepsis.

Aims: Storkhead box 1 (STOX1) is a winged-helix transcription factor that is implicated in the genetic forms of a high-prevalence human gestational disease, pre-eclampsia. STOX1 overexpression confers pre-eclampsia-like transcriptomic features to trophoblastic cell lines and pre-eclampsia symptoms to pregnant mice. The aim of this work was to evaluate the impact of STOX1 on free radical equilibrium and mitochondrial function, both in vitro and in vivo. Results: Transcriptome analysis of STOX1-transgenic versus nontransgenic placentas at 16.5 days of gestation revealed alterations of mitochondria-related pathways. Placentas overexpressing STOX1 displayed altered mitochondrial mass and were severely biased toward protein nitration, indicating nitroso-redox imbalance in vivo. Trophoblast cells overexpressing STOX1 displayed an increased mitochondrial activity at 20% O2 and in hypoxia, despite reduction of the mitochondrial mass in the former. STOX1 overexpression is, therefore, associated with hyperactive mitochondria, resulting in increased free radical production. Moreover, nitric oxide (NO) production pathways were activated, resulting in peroxynitrite formation. At low oxygen pressure, STOX1 overexpression switched the free radical balance from reactive oxygen species (ROS) to reactive nitrogen species (RNS) in the placenta as well as in a trophoblast cell line. Innovation: In pre-eclamptic placentas, NO interacts with ROS and generates peroxynitrite and nitrated proteins as end products. This process will deprive the maternal organism of NO, a crucial vasodilator molecule. Conclusion: Our data posit STOX1 as a genetic switch in the ROS/RNS balance and suggest an explanation for elevated blood pressure in pre-eclampsia. Antioxid. Redox Signal. 21, 819–834.

A founding event in the origin of eukaryotes is the acquisition of an extraordinary organelle, the mitochondrion, which contains its own genome. Being linked to energy metabolism, oxidative stress, cell signalling, and cell death, the mitochondrion to a certain extent controls life and death in eukaryotic cells. The large metabolic diversity and living strategies of the Kingdom Fungi make their mitochondria of particular evolutionary interest. The review focuses first on the characteristics of mitochondria in the Kingdom Fungi, then on their implications in the organism survival, pathogenicity and resistance, and finally on proposing unconventional strategies to investigate the biology of fungal mitochondria, unveiling the possibility that mitochondria play as the Achilles' heel of this kingdom.

Contains fulltext : 202265.pdf (Publisher’s version ) (Closed access) Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Nuclear (nDNA) and mitochondrial DNA (mtDNA) communication is essential for cell function, but it remains unclear whether the replication of these genomes is linked. We inspected human cells with a novel fluorescence in situ hybridization protocol (mitochondrial Transcription and Replication Imaging Protocol) that identifies mitochondrial structures engaged in initiation of mtDNA replication and unique transcript profiles, and reconstruct the temporal series of mitochondrial and nuclear events in single cells during the cell cycle. We show that mtDNA transcription and initiation of replication are prevalently coordinated with the cell cycle, preceding nuclear DNA synthesis, and being reactivated towards the end of S-phase. This coordination is achieved by modulating the fraction of mitochondrial structures that intiate mtDNA synthesis and/or contain transcript at a given time. Thus, although replication of the mitochondrial genome is active through the entire cell cycle, but in a limited fraction of mitochondrial structures, peaks of these activities are synchronized with nDNA synthesis. After release from blockage of mtDNA replication with either nocodazole or double thymidine treatment, prevalent mtDNA and nDNA synthesis occurred simultaneously, indicating that mitochondrial coordination with the nuclear phase can be adjusted in response to physiological alterations. These findings will help redefine other nuclear–mitochondrial links in cell function.

Mitochondria autonomously replicate and transcribe their own genome, which is present in multiple copies in the organelle. Transcription and replication of the mitochondrial DNA (mtDNA), which are defined here as mtDNA processing, are essential for mitochondrial function. The extent, efficiency, and coordination of mtDNA processing are key parameters of the mitochondrial state in living cells. Recently, single-cell analysis of mtDNA processing revealed a large and dynamic heterogeneity of mitochondrial populations in single cells, which is linked to mitochondrial function and is altered during disease. This was achieved using mitochondrial Transcription and Replication Imaging Protocol (mTRIP), a modified fluorescence in situ hybridization (FISH) approach that simultaneously reveals the mitochondrial RNA content and mtDNA engaged in initiation of replication at the single-cell level. mTRIP can also be coupled to immunofluorescence or MitoTracker, resulting in the additional labeling of proteins or active mitochondria, respectively. Therefore, mTRIP detects quantitative and qualitative alterations of the dynamics of mtDNA processing in human cells that respond to physiological changes or result from diseases. In addition, we show here that mTRIP is a rather sensitive tool for detecting mitochondrial alterations that may lead to loss of cell viability, and is thereby a useful tool for monitoring sublethal cytotoxicity for instance during chronic drug treatment.

The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo . These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ −/− mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ −/− MEFs, associated with high levels of the tumor-suppressor p16 INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ −/− MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ −/− MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.

Skeletal muscle stem cells in the adult display heterogeneity that has been functionally linked to their behavior, self-renewal capacity, and resistance to stress in hostile environments. Behavioral heterogeneity emerges also during developmental myogenesis. Muscle stem cell diversity may be functionally linked to the changing needs of skeletal muscle regeneration. Intriguingly, dramatic reduction of stem cell diversity, the “clonal drift”, that implies loss of stem cells and related expansion of clonally related stem cells has been reported for tissue replacement in several adult tissues and suggested in the zebrafish embryo. A recent study shows clonal drift of muscle stem cells in the zebrafish embryo caused by inhibition of the cell cycle and directed by the homeobox protein Meox1. Although stem cell quiescence is associated with inhibition of the transition phase G0/G1 of the cell cycle, Meox1 triggers the muscle stem cell fate by an arrest in G2 phase. Why efficient muscle growth in the zebrafish embryo requires sacrificing stem cell heterogeneity in favor of a small number of dominant clones has not been elucidated. The significance of G2-halted stem cells, which are generally associated with robust regeneration capacity, is also intriguing. These processes are relevant for understanding organ growth and the mechanisms that govern stem cell quiescence.

Mitochondria autonomously replicate and transcribe their own genome, which is present in multiple copies in the organelle. Transcription and replication of the mitochondrial DNA (mtDNA), which are defined here as mtDNA processing, are essential for mitochondrial function. The extent, efficiency, and coordination of mtDNA processing are key parameters of the mitochondrial state in living cells. Recently, single-cell analysis of mtDNA processing revealed a large and dynamic heterogeneity of mitochondrial populations in single cells, which is linked to mitochondrial function and is altered during disease. This was achieved using mitochondrial Transcription and Replication Imaging Protocol (mTRIP), a modified fluorescence in situ hybridization (FISH) approach that simultaneously reveals the mitochondrial RNA content and mtDNA engaged in initiation of replication at the single-cell level. mTRIP can also be coupled to immunofluorescence or MitoTracker, resulting in the additional labeling of proteins or active mitochondria, respectively. Therefore, mTRIP detects quantitative and qualitative alterations of the dynamics of mtDNA processing in human cells that respond to physiological changes or result from diseases. In addition, we show here that mTRIP is a rather sensitive tool for detecting mitochondrial alterations that may lead to loss of cell viability, and is thereby a useful tool for monitoring sublethal cytotoxicity for instance during chronic drug treatment.

UV-sensitive syndrome (UV(S)S) and Cockayne syndrome (CS) are human disorders caused by CSA or CSB gene mutations; both conditions cause defective transcription-coupled repair and photosensitivity. Patients with CS also display neurological and developmental abnormalities and dramatic premature aging, and their cells are hypersensitive to oxidative stress. We report CSA/CSB-dependent depletion of the mitochondrial DNA polymerase-γ catalytic subunit (POLG1), due to HTRA3 serine protease accumulation in CS, but not in UV(s)S or control fibroblasts. Inhibition of serine proteases restored physiological POLG1 levels in either CS fibroblasts and in CSB-silenced cells. Moreover, patient-derived CS cells displayed greater nitroso-redox imbalance than UV(S)S cells. Scavengers of reactive oxygen species and peroxynitrite normalized HTRA3 and POLG1 levels in CS cells, and notably, increased mitochondrial oxidative phosphorylation, which was altered in CS cells. These data reveal critical deregulation of proteases potentially linked to progeroid phenotypes in CS, and our results suggest rescue strategies as a therapeutic option.

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity., Sepsis patients often develop muscle atrophy that can last for years. Here the authors show in a mouse model that sepsis causes long-term impairment of the satellite cells, affecting mitochondrial function and energy metabolism, and that injection of mesenchymal stem cells restores satellite cell metabolism and muscle regeneration.

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.

In their report “Reverse transcriptase motifs in the catalytic subunit of telomerase” ([25 Apr., p. 561][1]), Joachim Lingner et al . demonstrate that such motifs are present in the catalytic subunit of the telomerase that they purified and identified in Euplotes aediculatus . They show that similar motifs exist in a homologous yeast gene and that alteration of these motifs by classical yeast genetics affects telomere elongation. After mentioning that telomerases are frequently called “specialized reverse transcriptases,” they stress that three main features distinguish them from other reverse transcriptases: (i) telomerase uses only a small portion of its RNA subunit as a template; (ii) during processive synthesis of telomeric repeats, the substrate translocates from one end of the template to the other by an as-yet-unknown mechanism; and (iii) the telomerase protein is stably associated with its RNA subunit (a feature also found in retrotransposon reverse transcriptase). On the other hand, we have recently shown that, under specific in vitro conditions, human immunodeficiency virus–type 1 (HIV-1) reverse transcriptase uses a portion of its template RNA to perform a reiterative synthesis; the substrate then translocates from one end of the template motif to the other, and the enzyme maintains a stable association with its RNA ([1][2]). On specific template sequences, it is therefore sufficient to modify the cationic environment (from manganese chloride to magnesium chloride in the reaction buffer) in order to switch the HIV-1 reverse transcriptase mode of synthesis from a regular one to a telomerase-like reiterative synthesis (as described in figure 9 of the report by Lingner et al .). Thus, HIV-1 reverse transcriptase can display an appreciable telomerase-like activity, indicating that these enzymes are biochemically closely related. 1. [↵][3]1. M. Ricchetti, 2. H. Buc , Biochemistry 35, 14970 (1996). [OpenUrl][4][CrossRef][5][PubMed][6][Web of Science][7] [1]: /lookup/doi/10.1126/science.276.5312.561 [2]: #ref-1 [3]: #xref-ref-1-1 "View reference 1 in text" [4]: {openurl}?query=rft.jtitle%253DBiochemistry%26rft.volume%253D35%26rft.spage%253D14970%26rft_id%253Dinfo%253Adoi%252F10.1021%252Fbi961274v%26rft_id%253Dinfo%253Apmid%252F8942663%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [5]: /lookup/external-ref?access_num=10.1021/bi961274v&link_type=DOI [6]: /lookup/external-ref?access_num=8942663&link_type=MED&atom=%2Fsci%2F277%2F5328%2F883.7.atom [7]: /lookup/external-ref?access_num=A1996VV23700038&link_type=ISI

International audience; The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Amplification of oligonucleotide repeats is a major cause of variability and instability of genomes. This phenomenon is probably due to an aberration in the copying process of polymerases. We show here that in the presence of MnCl2, mismatch formation commits HIV-1 reverse transcriptase to a new mode of DNA synthesis which generates repetitive products. This activity is distinct from terminal transferase since it requires specific DNA motifs in the template. This mechanism, which is processive, also works on homologous RNA templates where it generates reiterative products more than 150 nucleotides long. The corresponding mechanism, which involves extensive primer misalignment, is strikingly similar to that postulated for telomerases.

Le sepsis est une infection associee a un syndrome de reponse inflammatoire systemique conduisant a des dommages tissulaires et des defaillances d’organes pouvant entrainer une mort rapide [1] . L’une des consequences du sepsis peut etre l’atrophie musculaire, pouvant avoir de nombreux impacts cliniques [1] . En situation normale, le muscle squelettique a la capacite de regenerer ad-integrum apres une blessure, grâce aux cellules souches : les cellules satellites [2] . Nous avons montre qu’apres un choc septique, les cellules souches musculaires ne sont plus capables d’assurer de nombreuses fonctions comme leur activation, la division ou la differentiation. Ces effets perdurent dans le temps et l’effet negatif du sepsis a l’echelle cellulaire se fait ressentir meme 3 mois apres le choc septique. Nous avons egalement montre que ces defauts proviennent, en partie des mitochondries, dont la masse s’effondre et l’activite augmente entrainant une deterioration de l’etat metabolique. L’injection de cellules souches mesenchymateuses s’est egalement revelee interessante pour contrer ces dysfonctions, entrainant une diminution systemique de l’inflammation et une restauration quasi complete de tous les parametres des cellules souches musculaires. Cette injection de cellules souches mesenchymateuses entraine egalement une amelioration fonctionnelle du muscle avec un reel gain de force a l’echelle de la fibre, contrant ainsi la perte de masse musculaire et de la force engendree par le sepsis.

International audience; The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

Mitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial Transcription and Replication Imaging Protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: i) replication initiation active and transcript-positive (Ia-Tp), ii) replication initiation silent and transcript-positive (Is-Tp), and iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations which provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.

The specialized DNA polymerase μ (pol μ) intervenes in the repair mechanism non-homologous end-joining (NHEJ) as an end-processing factor but its role has not been fully elucidated. Pol μ has been shown to participate in DNA synthesis at junctions in vitro, including on unpaired substrates, and to promote annealing. However, the phenotypes observed in vivo poorly recapitulate the functions of pol μ reported in vitro. We analysed the repair of DNA double-strand breaks (DSBs) in a cellular context using improved NHEJ substrates. These substrates do not replicate in mammalian cells, thereby result in clonal repair events, which allows the measure of the efficiency of repair. We validated this paradigm by comparing the repair of NHEJ substrates to the repair reported for chromosome DSBs in mouse cells. Molecular analysis and, in most cases sequencing of more than 1500 repair events on a variety of NHEJ substrates in wild type and pol μ(-/-) mouse embryonic fibroblasts shows that, unexpectedly, the absence of pol μ decreases the efficiency of joining of all types of DSBs, including those that do not undergo end-processing. Importantly, by reducing the efficiency of accurate events, lack of pol μ also affects the overall fidelity of the repair process. We also show that, although pol μ does not help protect DNA ends from resection, the efficiency of repair of resected ends is reduced in the absence of pol μ. Interestingly, the DNA synthesis activity of pol μ, including on non-aligned substrates, appears negligible at least in a cellular context. Our data point to a critical role for pol μ as a global repair player that increases the efficiency and the fidelity of NHEJ.

Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli . Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.

Efficient and faithful repair of DNA double-strand breaks (DSBs) is critical for genome stability. To understand whether cells carrying a functional repair apparatus are able to efficiently heal two distant chromosome ends and whether this DNA lesion might result in genome rearrangements, we induced DSBs in genetically modified mouse embryonic stem cells carrying two I- Sce I sites in cis separated by a distance of 9 kbp. We show that in this context non-homologous end-joining (NHEJ) can repair using standard DNA pairing of the broken ends, but it also joins 3′ non-complementary overhangs that require unusual joining intermediates. The repair efficiency of this lesion appears to be dramatically low and the extent of genome alterations was high in striking contrast with the spectra of repair events reported for two collinear DSBs in other experimental systems. The dramatic decline in accuracy suggests that significant constraints operate in the repair process of these distant DSBs, which may also control the low efficiency of this process. These findings provide important insights into the mechanism of repair by NHEJ and how this process may protect the genome from large rearrangements.

Double strand break (DSB) repair plays an important role in chromosome evolution. We have investigated the fate of DSBs as a function of their location along the yeast chromosome XI, in a system where no conventional homologous recombination can occur. We report that the relative frequency of non-homologous endjoining (NHEJ), which is the exclusive mode of DSB repair in the internal chromosomal portion, decreases gradually towards the telomere, keeping the absolute frequency nearly constant, and that other repair mechanisms, which generally involve the loss of the distal chromosomal fragment, appear in subtelomeric regions. Distance of the DSB from chromosome ends plays a critical role in the global frequency of these repair mechanisms. Direct telomere additions are rare, and other events such as break-induced replication, plasmid incorporation, and gene conversion, involve acquisition of heterologous sequences. Therefore, in subtelomeric regions, cell survival to DSBs is higher and alternative modes of repair allow new genomic combinations to be generated. Furthermore, subtelomeric rearrangements depend on the recombination process, which, unexpectedly, also promotes the joining of heterologous sequences. Finally, we report that the Rad52 protein increases the efficiency of NHEJ.

Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.

Dissecting the molecular defects in rare genetic disorders like Cockayne syndrome (CS), in which ageing is dramatically accelerated, is critical to develop treatments, which are missing to date, and elucidate dysfunctions that are possibly implicated in physiological ageing. CS also displays a large spectrum of clinical severity which does not rely on simple genotype/phenotype correlation. This project is based on a working model established in the lab that identified CS-specific depletion of the mitochondrial DNA polymerase POLG1 leading to mitochondrial dysfunction, as a possible cause of CS progeroid defects. POLG1 depletion required overexpression of the HTRA3 protease, which was trigged by increased oxidative/nitrosative stress. Scavenging both reactive species, rescued these defects and opened the path to a treatment for CS. This PhD work: i) Contributed to the discovery that the CS-defective pathway is recapitulated in replicative senescence of normal cells, a process linked to regular aging. ii) Identified the mechanism of HTRA3-dependent POLG1 degradation in CS and senescent cells with implications for POLG1 homeostasis in normal cells. iii) Developed multiple isogenic cellular models (skin fibroblasts, induced pluripotent stem cells and cerebral organoids) with CRISPR-Cas9 that are essential for mechanistic studies and to address genotype/phenotype correlations, in the absence of a reliable mouse model for CS. Taken together, these studies provide novel insights into the mechanisms leading to defects in progeroid CS and their links with physiological ageing. They also establish unique CS models for studying CS pathogenesis.; La compréhension des altérations moléculaires du syndrome de Cockayne (CS), une maladie génétique rare dans laquelle le vieillissement est accéléré, est essentielle pour développer des traitements, et élucider les dysfonctionnements impliqués dans le vieillissement physiologique. Le CS présente un large spectre de sévérité clinique qui ne repose pas sur une simple corrélation génotype/phénotype. Ce projet est basé sur une altération spécifique aux cellules CS, démontrée dans le laboratoire, qui implique la déplétion de l'ADN polymérase mitochondriale POLG1 conduisant à un dysfonctionnement de l’organelle. Cette altération est due à la surexpression de la protéase HTRA3, qui est déclenchée par un stress oxydatif/nitrosatif accru. La réduction de ces espèces réactives a permis de corriger ces altérations et a ouvert la voie à un traitement pour le CS. Ce travail de thèse i) a contribué à la découverte que la voie défectueuse du CS est récapitulée au cours de la sénescence réplicative des cellules normales, un processus lié au vieillissement physiologique, ii) a identifié le mécanisme HTRA3-dépendant de dégradation de POLG1 dans les cellules CS et dans les cellules sénescentes, iii) a développé de multiples modèles cellulaires isogéniques (fibroblastes, cellules souches pluripotentes induites et organoïdes cérébraux) avec CRISPR-Cas9, permettant des études mécanistiques et de corrélation génotype/phénotype. Ces études fournissent de nouvelles informations sur les mécanismes conduisant aux altérations progeroïdes dans le CS, sur leurs liens avec le vieillissement physiologique, et établissent des modèles expérimentaux uniques pour l'étude de la pathogenèse de cette maladie.

DNA double-strand breaks (DSBs) are dangerous DNA damages and a risk factor for genome stability. The maintenance of genome integrity is crucial for adult stem cells that are responsible for regeneration of damaged tissues and tissue homeostasis throughout life. Muscle regeneration in the adult relies on muscle stem cells (satellite cells, SCs) that have a remarkable DSB repair activity, but the underlying mechanism is not known. The aims of the present PhD project were to investigate how muscle differentiation is affected when DSB repair is impaired, and which are the mechanism(s) and the consequences on muscle regeneration. During this study, a novel possibility has arisen, namely that DSB repair factors affects myogenesis independently of their DNA repair activity, suggesting a novel function, not previously anticipated, of these factors. The present study has addressed the role of DNA-dependent protein kinase (DNA-PK), a crucial factor in non-homologous end-joining (NHEJ) repair of DSBs, in muscle differentiation in the mouse. Studies have targeted SC activation and muscle regeneration in vitro and in vivo and also addressed the regulation of this kinase. In parallel the more “canonical” role of DNA-PK, and thereby of NHEJ, has been investigated in SCs via radiation-induced DNA damage. The role of ATM, a kinase that orchestrates cellular responses to DSBs in muscle regeneration has also been addressed. These results support the emerging notion of multifunctional repair proteins in a variety of physiological processes beyond the repair process itself, on which I have conducted a bibliographical study. This work i) identifies novel regulators of myogenesis, and ii) helps understanding the resistance of muscle stem cells to genotoxic stress. It has potential implications for improving cellular therapies for muscle dysfunction by acting on the newly discovered regulators.; Les cassures double-brin (DSB) sont des dommages dangereux de l’ADN et représentent un facteur de risque pour la stabilité du génome. Le maintien de l'intégrité du génome est essentiel pour les cellules souches adultes, qui sont responsables de la régénération des tissus endommagés et de l'homéostasie tissulaire tout au long de la vie. La régénération musculaire chez l'adulte repose sur les cellules souches musculaires (cellules satellites, SCs) qui possèdent une remarquable capacité de réparation des DSB, mais dont le mécanisme sous-jacent reste inconnu. Ce projet de thèse consistait à étudier comment la différenciation musculaire est affectée lorsque la réparation des DSB est altérée, et quels sont le(s) mécanisme(s) et les conséquences de ce défaut de réparation sur la régénération musculaire. Au cours de cette étude, il est apparu de façon originale que les facteurs de réparation des DSB peuvent affecter la myogenèse, indépendamment de leur fonction dans la réparation de l'ADN. La présente étude a porté sur le rôle de la protéine kinase dépendante de l'ADN (DNA-PK), un facteur crucial pour la réparation non-homologue des DSBs (NHEJ), au cours de la différenciation musculaire chez la souris. L’étude a ciblé l'activation des SCs et la régénération musculaire in vitro et in vivo et a également abordé la régulation de cette kinase. Le rôle "canonique" de la DNA-PK, et donc du NHEJ, dans les SCs a également été étudié en présence de lésions de l'ADN radio-induites. Le rôle d’ATM, une kinase qui orchestre les réponses cellulaires aux DSB, a également été abordé dans le contexte de la régénération musculaire. Ces résultats confirment la notion émergente du rôle multifonctionnel des protéines de réparation de l’ADN dans d’autres processus physiologiques que la réparation elle-même, ce qui m’a également permis de réaliser une étude bibliographique. Ce travail i) identifie de nouveaux régulateurs de la myogenèse et ii) contribue à la compréhension de la résistance des cellules souches musculaires au stress génotoxique. Ces résultats pourraient avoir des implications dans l'amélioration des thérapies cellulaires de la dysfonction musculaire en agissant sur les régulateurs nouvellement découverts.