Pax3 synergizes with Gli2 and Zic1 in transactivating the Myf5 epaxial somite enhancer

AbstractBoth Glis, the downstream effectors of hedgehog signaling, and Zic transcription factors are required for Myf5 expression in the epaxial somite. Here we demonstrate a novel synergistic interaction between members of both families and Pax3, a paired-domain transcription factor that is essential for both myogenesis and neural crest development. We show that Pax3 synergizes with both Gli2 and Zic1 in transactivating the Myf5 epaxial somite (ES) enhancer in concert with the Myf5 promoter. This synergy is dependent on conserved functional domains of the proteins, as well as on a novel homeodomain motif in the Myf5 promoter and the essential Gli motif in the ES enhancer. Importantly, overexpression of Zic1 and Pax3 in the 10T1/2 mesodermal cell model results in enrichment of these factors at the endogenous Myf5 locus and induction of Myf5 expression. In our previous work, we showed that by enhancing nuclear translocation of Gli factors, Zics provide spatiotemporal patterning for Gli family members in the epaxial induction of Myf5 expression. Our current study indicates a complementary mechanism in which association with DNA-bound Pax3 strengthens the ability of both Zic1 and Gli2 to transactivate Myf5 in the epaxial somite

The Homeobox Transcription Factor Barx2 Regulates Plasticity of Young Primary Myofibers

Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation

DUX4c Is Up-Regulated in FSHD. It Induces the MYF5 Protein and Human Myoblast Proliferation

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology

Approche cellulaire de la dystrophie facioscapulohumérale (développement et caractérisation de cultures primaires musculaires dérivées de patients FSHD)

La dystrophie Facioscapulohumérale (FSHD), troisième maladie neuromusculaire, se caractérise par une dégénérescence progressive de groupes de muscles squelettiques spécifiques à l'âge adulte. Elle est due à une anomalie génétique située en 4q35 mais demeure énigmatique puisqu'aucun gène directement responsable de cette pathologie n'a pu être mis en évidence. Plusieurs hypothèses ont été avancées quant aux voies de signalisation susceptibles d'être impliquées via l'utilisation de cultures primaires musculaires issues de patients atteints de FSHD. Mais les différentes études ont générées de nombreux résultats contradictoires, qui pourraient être du au fait que les cellules FSHD provenaient de muscles présentant des niveaux d'atteinte différents. Aussi, un des objectifs de ces travaux de thèse était de caractériser quatorze cultures primaires musculaires de muscles cliniquement et non cliniquement atteints de patients FSHD par comparaison avec quatorze cultures primaires musculaires de muscles de personnes saines. Ce travail a montré que, quelque soit l'origine musculaire, tous les myoblastes FSHD présentent une sensibilité accrue au stress oxydatif et des anomalies morphologiques en différenciation. Parallèlement, une approche protéomique comparative entre les cultures primaires FSHD et contrôles a été réalisée. L'identification des protéines dont les niveaux d'expression sont spécifiquement altérés dans les cellules FSHD conduira à la détermination des voies spécifiquement mises en jeu dans ces cellules. De plus, les travaux antérieurs du laboratoire réunissant des analyses protéomiques et biochimiques réalisées sur des biopsies musculaires FSHD et contrôles ont permis de proposer des hypothèses quant aux voies de signalisation susceptibles d'être impliquées dans cette pathologie. Grâce à la mise en place et à la caractérisation des cultures primaires FSHD, ces hypothèses sont en cours de testFacioscapulohumeral dystrophy (FSHD), the third neuromuscular disorder, is characterized by a progressive wasting of specific skeletal muscle groups at adult age. It is due to a genetic defect located in 4q35, but the molecular mechanisms involved in the disease are still unknown. Numerous hypotheses have been proposed so far to determine signalling pathways involved in FSHD by using primary muscular cells isolated from FSHD patients, but these studies have led to contradictory results probably because FSHD cells were isolated from muscles with different levels of affection. Thus, one major aim of this thesis work was to characterize fourteen primary muscular cell cultures from affected and non-clinically affected muscles from FSHD patients by comparing them to fourteen primary muscular cell cultures from healthy individuals. This work showed that all FSHD myoblasts were highly sensitive to oxidative stress and were morphologically altered in differentiation. A proteomic approach between FSHD and control primary cultures was also conducted. The identification of proteins specifically altered in FSHD cell cultures will allow the determination of altered pathways within these cells. Moreover, previous work from the lab combining proteomic and biochemical analysis on FSHD and control muscular biopsies led to the identification of putative pathways involved in this pathology. With the characterization of FSHD primary cultures, these hypotheses are currently being testedMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects.: Morphological differentiation defects in FSHD myoblasts

International audienceFacioscapulohumeral dystrophy (FSHD) is a muscular hereditary disease with a prevalence of 1 in 20,000 caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. However, very little is known about the pathogenesis as well as the molecular and biochemical changes linked to the progressive muscle degeneration observed in these patients. Several studies have investigated possible pathophysiological pathways in FSHD myoblasts and mature muscle cells but some of these reports were apparently in contradiction. The discrepancy between these studies may be explained by differences between the sources of myoblasts. Therefore, we decided to thoroughly analyze affected and unaffected muscles from patients with FSHD in terms of vulnerability to oxidative stress, differentiation capacity and morphological abnormalities. We have established a panel of primary myoblast cell cultures from patients affected with FSHD and matched healthy individuals. Our results show that primary myoblasts are more susceptible to an induced oxidative stress than control myoblasts. Moreover, we demonstrate that both types of FSHD primary myoblasts differentiate into multi-nucleated myotubes, which present morphological abnormalities. Whereas control myoblasts fuse to form branched myotubes with aligned nuclei, FSHD myoblasts fuse to form either thin and branched myotubes with aligned nuclei or large myotubes with random nuclei distribution. In conclusion, we postulate that these abnormalities could be responsible for muscle weakness in patients with FSHD and provide an important marker for FSHD myoblasts

Caprine Arthritis Encephalitis Virus Is Associated with Renal Lesions

Caprine arthritis encephalitis virus (CAEV) is a monocyte/macrophage-tropic lentivirus that primarily infects goats resulting in a well-recognized set of chronic inflammatory syndromes focused on the joint synovium, tissues of the central nervous system, pulmonary interstitium and mammary gland. Clinically affected animals generally manifest with one or more of these classic CAEV-associated tissue lesions; however, CAEV-associated renal inflammation in goats has not been reported in the peer-reviewed literature. Here we describe six goats with chronic, multisystemic CAEV infections in conjunction with CAEV-associated renal lesions. One of the animals had CAEV antigen-associated thrombotic arteritis resulting in infarction of both the kidney and heart. These goats had microscopic evidence of inflammatory renal injury (interstitial nephritis) with detectable renal immunolabeling for CAEV antigen in three of six animals and amplifiable proviral sequences consistent with CAEV in all six animals. Cardiac lesions (vascular, myocardial or endocardial) were also identified in four of six animals. Within the viral promoter (U3) region, known transcription factor binding sites (TFBSs) were generally conserved, although one viral isolate had a duplication of the U3 A region encoding a second gamma-activated site (GAS). Despite the TFBS conservation, the isolates demonstrated a degree of phylogenetic diversity. At present, the clinical consequence of CAEV-associated renal injury is not clear

Caprine Arthritis Encephalitis Virus Is Associated with Renal Lesions.

Caprine arthritis encephalitis virus (CAEV) is a monocyte/macrophage-tropic lentivirus that primarily infects goats resulting in a well-recognized set of chronic inflammatory syndromes focused on the joint synovium, tissues of the central nervous system, pulmonary interstitium and mammary gland. Clinically affected animals generally manifest with one or more of these classic CAEV-associated tissue lesions; however, CAEV-associated renal inflammation in goats has not been reported in the peer-reviewed literature. Here we describe six goats with chronic, multisystemic CAEV infections in conjunction with CAEV-associated renal lesions. One of the animals had CAEV antigen-associated thrombotic arteritis resulting in infarction of both the kidney and heart. These goats had microscopic evidence of inflammatory renal injury (interstitial nephritis) with detectable renal immunolabeling for CAEV antigen in three of six animals and amplifiable proviral sequences consistent with CAEV in all six animals. Cardiac lesions (vascular, myocardial or endocardial) were also identified in four of six animals. Within the viral promoter (U3) region, known transcription factor binding sites (TFBSs) were generally conserved, although one viral isolate had a duplication of the U3 A region encoding a second gamma-activated site (GAS). Despite the TFBS conservation, the isolates demonstrated a degree of phylogenetic diversity. At present, the clinical consequence of CAEV-associated renal injury is not clear

DUX4 expression in FSHD muscle cells: how could such a rare protein cause a myopathy?

International audienc

Differentiating myoblasts and maturating myotubes have different levels of Barx2 expression.

<p><b>A.</b> Cultured primary myoblasts were induced to differentiate by serum withdrawal and changes in cell shape, actin remodeling and myogenin expression were monitored over the first 6 hours. <b>B–D.</b> Myotube maturation was examined between 24 and 72 hours post serum-withdrawal. At 24 hours myotubes were thin and appeared immature (<b>B</b>). Between 48 and 72 hours, thicker myotubes appeared often with local aggregations of nuclei (<b>C</b> and <b>D</b>) and frequent strong contractions. <b>E.</b> Observations of myotube maturation in culture presented schematically. <b>F.</b> Barx2 expression was measured by RT-PCR at different stages of differentiation. Scale bars represent (A–C) - 20 µm, D–50 µm.</p