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Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2′-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.

Muscular dystrophies (MDs) are a group of inherited degenerative muscle disorders characterized by a progressive skeletal muscle wasting. Respiratory impairments and subsequent hypoxemia are encountered in a significant subgroup of patients in almost all MD forms. In response to hypoxic stress, compensatory mechanisms are activated especially through Hypoxia-Inducible Factor 1 α (HIF-1α). In healthy muscle, hypoxia and HIF-1α activation are known to affect oxidative stress balance and metabolism. Recent evidence has also highlighted HIF-1α as a regulator of myogenesis and satellite cell function. However, the impact of HIF-1α pathway modifications in MDs remains to be investigated. Multifactorial pathological mechanisms could lead to HIF-1α activation in patient skeletal muscles. In addition to the genetic defect per se, respiratory failure or blood vessel alterations could modify hypoxia response pathways. Here, we will discuss the current knowledge about the hypoxia response pathway alterations in MDs and address whether such changes could influence MD pathophysiology.

Dystrophia myotonica type 1 (DM1) results from nuclear sequestration of splicing factors by a messenger RNA (mRNA) harboring a large (CUG)n repeat array transcribed from the causal (CTG)n DNA amplification. Several compounds were previously shown to bind the (CUG)n RNA and release the splicing factors. We now investigated for the first time the interaction of an aliphatic polycarbonate carrying guanidinium functions to DM1 DNA/RNA model probes by affinity capillary electrophoresis. The apparent association constants (Ka) were in the range described for reference compounds such as pentamidine. Further macromolecular engineering could improve association specificity. The polymer presented no toxicity in cell culture at concentrations of 1.6–100.0 μg/mL as evaluated both by MTT and real-time monitoring xCELLigence method. These promising results may lay the foundation for a new branch of potential therapeutic agents for DM1.

// Petr Dmitriev 1, 2, * , Ekaterina Kiseleva 2, 3, * , Olga Kharchenko 2, 3 , Evgeny Ivashkin 2, 3 , Andrei Pichugin 2, 3, 7 , Philippe Dessen 4 , Thomas Robert 4 , Frederique Coppee 5 , Alexandra Belayew 5 , Gilles Carnac 6 , Dalila Laoudj-Chenivesse 6 , Marc Lipinski 1, 2 , Andrei Vasiliev 3 , Yegor S. Vassetzky 1, 2, 3 1 UMR 8126, Univ. Paris-Sud, CNRS, Institut de Cancerologie Gustave-Roussy, Villejuif, France 2 LIA1066 Laboratoire Franco-Russe de Recherches en Oncologie, Villejuif, France 3 N.K. Koltzov Institute of Developmental Biology, RAS, Moscow, Russia 4 Functional Genomics Unit, Institut de Cancerologie Gustave-Roussy, Villejuif, France 5 Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium 6 PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, Montpellier, France 7 Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia * These authors contributed equally to this work Correspondence to: Yegor S. Vassetzky, email: vassetzky@igr.fr Keywords: DUX4, CXCR4, SDF1, signalling, migration Received: April 01, 2016 Accepted: August 10, 2016 Published: August 18, 2016 ABSTRACT We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c- transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts. In a Transwell cell migration assay, human bone marrow-derived mesenchymal stem cells (BMSCs) were migrating more efficiently towards human immortalized myoblasts overexpressing DUX4 as compared to controls; the migration efficiency of DUX4-transfected BMSCs was also increased. DUX4c overexpression in myoblasts or in BMSCs had no impact on the rate of BMSC migration. Antibodies against SDF1 and CXCR4 blocked the positive effect of DUX4 overexpression on BMSC migration. We propose that DUX4 controls the cellular migration of mesenchymal stem cells through the CXCR4 receptor.

Unfortunately the name of Jean Jacques Vanden Eynde was missing as co-author of this contribution. The correct list of authors is: Ioan O. Neaga, Stephanie Hambye, Ede Bodoki, Claudio Palmieri, Jean Jacques Vanden Eynde, Eugenie Ansseau, Alexandra Belayew, Radu Oprean, Bertrand Blankert.

Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4–5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors’ knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment.

Background Facioscapulohumeral muscular dystrophy (FSHD) is associated with DNA hypomethylation at the 4q35 D4Z4 repeat array. Both the causal gene DUX4 and its homolog DUX4c are induced. DUX4c is immunodetected in every myonucleus of proliferative cells, while DUX4 is present in only 1/1000 of myonuclei where it initiates a gene deregulation cascade. FSHD primary myoblasts differentiate into either atrophic or disorganized myotubes. DUX4 expression induces atrophic myotubes and associated FSHD markers. Although DUX4 silencing normalizes the FSHD atrophic myotube phenotype, this is not the case for the disorganized phenotype. DUX4c overexpression increases the proliferation rate of human TE671 rhabdomyosarcoma cells and inhibits their differentiation, suggesting a normal role during muscle differentiation. Methods By gain- and loss-of-function experiments in primary human muscle cells, we studied the DUX4c impact on proliferation, differentiation, myotube morphology, and FSHD markers. Results In primary myoblasts, DUX4c overexpression increased the staining intensity of KI67 (a proliferation marker) in adjacent cells and delayed differentiation. In differentiating cells, DUX4c overexpression led to the expression of some FSHD markers including β-catenin and to the formation of disorganized myotubes presenting large clusters of nuclei and cytoskeletal defects. These were more severe when DUX4c was expressed before the cytoskeleton reorganized and myofibrils assembled. In addition, endogenous DUX4c was detected at a higher level in FSHD myotubes presenting abnormal clusters of nuclei and cytoskeletal disorganization. We found that the disorganized FSHD myotube phenotype could be rescued by silencing of DUX4c, not DUX4. Conclusion Excess DUX4c could disturb cytoskeletal organization and nuclear distribution in FSHD myotubes. We suggest that DUX4c up-regulation could contribute to DUX4 toxicity in the muscle fibers by favoring the clustering of myonuclei and therefore facilitating DUX4 diffusion among them. Defining DUX4c functions in the healthy skeletal muscle should help to design new targeted FSHD therapy by DUX4 or DUX4c inhibition without suppressing DUX4c normal function. Electronic supplementary material The online version of this article (10.1186/s13395-017-0148-4) contains supplementary material, which is available to authorized users.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with an activation of the double homeobox 4 (DUX4) gene, which we previously identified within the D4Z4 repeated elements in the 4q35 subtelomeric region. The pathological DUX4 mRNA is derived from the most distal D4Z4 unit and extends unexpectedly within the flanking pLAM region, which provides an intron and polyadenylation signal. The conditions that are required to develop FSHD are a permissive allele providing the polyadenylation signal and hypomethylation of the D4Z4 repeat array compared with the healthy muscle. The DUX4 protein is a 52-kDa transcription factor that initiates a large gene deregulation cascade leading to muscle atrophy, inflammation, differentiation defects, and oxidative stress, which are the key features of FSHD. DUX4 is a retrogene that is normally expressed in germline cells and is submitted to repeat-induced silencing in adult tissues. Since DUX4 mRNAs have been detected in human embryonic and induced pluripotent stem cells, we investigated whether they could also be expressed in human mesenchymal stromal cells (hMSCs). We found that DUX4 mRNAs were induced during the differentiation of hMSCs into osteoblasts and that this process involved DUX4 and new longer protein forms (58 and 70 kDa). A DUX4 mRNA with a more distant 5' start site was characterized that presented a 60-codon reading frame extension and encoded the 58-kDa protein. Transfections of hMSCs with an antisense oligonucleotide targeting DUX4 mRNAs decreased both the 52- and 58-kDa protein levels and confirmed their identity. Gain- and loss-of-function experiments in hMSCs suggested these DUX4 proteins had opposite roles in osteogenic differentiation as evidenced by the alkaline phosphatase activity and calcium deposition. Differentiation was delayed by the 58-kDa DUX4 expression and it was increased by 52-kDa DUX4. These data indicate a role for DUX4 protein forms in the osteogenic differentiation of hMSCs.

Ethnopharmacological relevance: in the 1990s, a Belgian cohort of more than 100 patients reported cases of Aristolochic Acid Nephropathy (AAN). This progressive renal and interstitial fibrosis, frequently associated with urothelial malignancies, was consecutive to the Chinese-herbs based slimming capsules intake where a plant Stephania tetrandra S. Moore was replaced by a highly genotoxic Aristolochia species. 70% of the Belgian patients evolved into end-stage renal disease, requiring dialysis or renal transplantation. Furthermore the prevalence of upper urinary tract carcinoma was found alarmingly high in these patients. The Aristolochia adulteration was blamed for the intoxication cases and, to the best of our knowledge, the prescription itself has not been further investigated. Aim of the study This work proposes to evaluate the in vitro cytotoxicity and genotoxicity of Aristolochia and Magnolia traditional aqueous decoctions and their association. Materials and methods The cytotoxicity of extracts has been assessed by a MTT cell proliferation assay and the genotoxicity by measuring the presence of γ-H2AX, a phosphorylated histone associated with DNA damages. Results Treating cells for 24 h with a mixture 1:1 of Magnolia officinalis and Aristolochia baetica decoctions led to an increase in the production of γ-H2AX. Conclusions This genotoxic potentiation warrants further studies but may lead to an explanatory factor for the “Chinese herb nephropathy” cases.

The Helicase-like Transcription Factor (HLTF) belongs to the SWI/SNF family of proteins involved in chromatin remodeling. In addition to its role in gene transcription, HLTF has been implicated in DNA repair, which suggests that this protein acts as a tumor suppressor. Accumulating evidence indicates that HLTF expression is altered in various cancers via two mechanisms: gene silencing through promoter hypermethylation or alternative mRNA splicing, which leads to the expression of truncated proteins that lack DNA repair domains. In either case, the alteration of HLTF expression in cancer has a poor prognosis. In this review, we gathered published clinical and molecular data on HLTF. Our purposes are (a) to address whether HLTF alterations could be considered as cancer drivers or passengers and (b) to determine whether its different functions (transcription or DNA repair) could be diverted in clonal selection during cancer progression.

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPIpositive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c., SCOPUS: ar.j, info:eu-repo/semantics/published

The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.