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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.

Double-strand breaks (DSBs) may result from endogenous (e.g., reactive oxygen species, variable (diversity) joining, meiotic exchanges, collapsed replication forks, nucleases) or exogenous (e.g., ionizing radiation, chemotherapeutic agents, radiomimetic compounds) events. DSBs disrupt the integrity of DNA and failed or improper DSBs repair may lead to genomic instability and, eventually, mutations, cancer, or cell death. Non-homologous end-joining (NHEJ) is the major pathway used by higher eukaryotic cells to repair these lesions. Given the complexity of NHEJ and the number of proteins and cofactors involved, secondary metabolites from medicinal or food plants might interfere with the process, activating or inhibiting repair. Twelve natural products, arbutin, curcumin, indole-3-carbinol, and nine flavonoids (apigenin, baicalein, chalcone, epicatechin, genistein, myricetin, naringenin, quercetin, sakuranetin) were chosen for their postulated roles in cancer chemoprevention and/or treatment. The effects of these compounds on NHEJ were investigated with an in vitro protocol based on plasmid substrates. Plasmids were linearized by a restriction enzyme, generating cohesive ends, or by a combination of enzymes, generating incompatible ends; plasmids were then incubated with a nuclear extract prepared from normal human small-intestinal cells (FHS 74 Int), either treated with these natural products or untreated (controls). The NHEJ repair complex from nuclear extracts ligates linearized plasmids, resulting in plasmid oligomers that can be separated and quantified by on-chip microelectrophoresis. Some compounds (chalcone, epicatechin, myricetin, sakuranetin and arbutin) clearly activated NHEJ, whereas others (apigenin, baicalein and curcumin) significantly reduced the repair rate of both types of plasmid substrates. Although this in vitro protocol is only partly representative of the in vivo situation, the natural products appear to interfere with NHEJ repair and warrant further investigation.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most frequent hereditary muscle disorders. It is linked to contractions of the D4Z4 repeat array in 4q35. We have characterized the double homeobox 4 (DUX4) gene in D4Z4 and its mRNA transcribed from the distal D4Z4 unit to a polyadenylation signal in the flanking pLAM region. It encodes a transcription factor expressed in FSHD but not healthy muscle cells which initiates a gene deregulation cascade causing differentiation defects, muscle atrophy and oxidative stress. PITX1 was the first identified DUX4 target and encodes a transcription factor involved in muscle atrophy. DUX4 was found expressed in only 1/1000 FSHD myoblasts. We have now shown it was induced upon differentiation and detected in about 1/200 myotube nuclei. The DUX4 and PITX1 proteins presented staining gradients in consecutive myonuclei which suggested a diffusion as known for other muscle nuclear proteins. Both protein half-lifes were regulated by the ubiquitin-proteasome pathway. In addition, we could immunodetect the DUX4 protein in FSHD muscle extracts. As a model, we propose the DUX4 gene is stochastically activated in a small number of FSHD myonuclei. The resulting mRNAs are translated in the cytoplasm around an activated nucleus and the DUX4 proteins diffuse to adjacent nuclei where they activate target genes such as PITX1. The PITX1 protein can further diffuse to additional myonuclei and expand the transcriptional deregulation cascade initiated by DUX4. Together the diffusion and the deregulation cascade would explain how a rare protein could cause the muscle defects observed in FSHD.

Oligomerization of linearized plasmids by nuclear proteins extracts, a recognized measure of nonhomologous end-joining (NHEJ) repair capacity, is typically assessed through agarose gel electrophoresis, a labor-intensive procedure. In the current study, a more convenient NHEJ assay was developed using microchips that allow scaled-down separation and quantification. This microchip method allows a considerable reduction in sample amount and analysis time with similar costs and comparable or slightly better precision. Data obtained with quercetin and wortmannin show that the method can be applied to the screening of food components and natural products for positive and negative modulators of NHEJ, potential chemopreventive and indirect genotoxic compounds, respectively.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy after the dystrophinopathies and myotonic dystrophy and is associated with a typical pattern of muscle weakness. Most patients with FSHD carry a large deletion in the polymorphic D4Z4 macrosatellite repeat array at 4q35 and present with 1-10 repeats whereas non-affected individuals possess 11-150 repeats. An almost identical repeat array is present at 10q26 and the high sequence identity between these two arrays can cause difficulties in molecular diagnosis. Each 3.3-kb D4Z4 unit contains a DUX4 (double homeobox 4) gene that, among others, is activated upon contraction of the 4q35 repeat array due to the induction of chromatin remodelling of the 4qter region. A number of 4q subtelomeric sequence variants are now recognised, although FSHD only occurs in association with three 'permissive' haplotypes, each of which is associated with a polyadenylation signal located immediately distal of the last D4Z4 unit. The resulting poly-A tail appears to stabilise DUX4 mRNAs transcribed from this most distal D4Z4 unit in FSHD muscle cells. Synthesis of both the DUX4 transcripts and protein in FSHD muscle cells induces significant cell toxicity. DUX4 is a transcription factor that may target several genes which results in a deregulation cascade which inhibits myogenesis, sensitises cells to oxidative stress and induces muscle atrophy, thus recapitulating many of the key molecular features of FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with D4Z4 repeat contraction on human chromosome 4q35. This genetic lesion does not result in complete loss or mutation of any gene. Consequently, the pathogenic mechanisms underlying FSHD have been difficult to discern. In leading FSHD pathogenesis models, D4Z4 contractions are proposed to cause epigenetic changes, which ultimately increase expression of genes with myopathic potential. Although no gene has been conclusively linked to FSHD development, recent evidence supports a role for the D4Z4-encoded DUX4 gene in FSHD. In this study, our objective was to test the in vivo myopathic potential of DUX4.We delivered DUX4 to zebrafish and mouse muscle by transposon-mediated transgenesis and adeno-associated viral vectors, respectively.Overexpression of DUX4, which encodes a transcription factor, caused abnormalities associated with muscular dystrophy in zebrafish and mice. This toxicity required DNA binding, because a DUX4 DNA binding domain mutant produced no abnormalities. Importantly, we found the myopathic effects of DUX4 were p53 dependent, as p53 inhibition mitigated DUX4 toxicity in vitro, and muscles from p53 null mice were resistant to DUX4-induced damage.Our work demonstrates the myopathic potential of DUX4 in animal muscle. Considering previous studies showed DUX4 was elevated in FSHD patient muscles, our data support the hypothesis that DUX4 overexpression contributes to FSHD development. Moreover, we provide a p53-dependent mechanism for DUX4 toxicity that is consistent with previous studies showing p53 pathway activation in FSHD muscles. Our work justifies further investigation of DUX4 and the p53 pathway in FSHD pathogenesis.

Aims: To examine the immunohistochemical expression of helicase-like transcription factor (HLTF) in relation to the prognosis of hypopharyngeal (HSCCs) and laryngeal (LSCCs) squamous cell carcinomas, and to characterize the HLTF protein variants expressed in biopsy specimens of head and neck squamous cell carcinoma (HNSCC) as well as the HeLa cell line. Methods and results: HLTF expression was determined by immunohistochemistry on a series of 100 hypopharyngeal (stage IV) and 56 laryngeal SCCs (stages I, II and IV). The HLTF variants were defined using reverse transcriptase-polymerase chain reaction and Western blots in 13 fresh HNSCC biopsies and in HeLa cells. High levels of HLTF expression were associated with rapid recurrence rates in a subgroup of 81 stage IV hypopharyngeal SCCs (with complete follow-up). A 95-kDa HLTF variant truncated at the carboxyl-terminal domain was detected in addition to the 115-kDa full-size protein in HNSCC biopsies, while six variants were observed in HeLa cells. Conclusions: Our results demonstrate, for the first time, that hypopharyngeal SCCs presenting high levels of HLTF have a worse prognosis. The quantitative determination of HLTF in hypopharyngeal SCCs was an independent prognostic marker alongside tumour node metastasis staging. HNSCCs expressed the truncated HLTF variant lacking the domains involved in DNA repair.

Summary Objective Prototheca zopfii are colourless algae that can cause mastitis in dairy cattle. Based on sequence analysis of the 18S rRNA gene, three genotypes are described. Material and method To differentiate them, a recent method of diagnostic was proposed based on rDNA amplification by Polymerase Chain Reaction (PCR) and Restriction Fragments Length Polymorphism analysis (RFLP). A total of 30 strains isolated from mastitis cases becoming of various farms were investigated. Results The molecular study allowed the identification of 27 strains belonging to genotype II, three to genotype III and no one to genotype I. Conclusion The simple PCR/RFLP system enabled the rapid, accurate and easily performed identification of genotypes of the species “ zopfii ”.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

The helicase-like transcription factor (HLTF) belongs to the SWI/SNF family of chromatin-remodeling factors. Several SWI/SNF genes are disrupted in cancer, suggesting their possible role as tumor suppressors. Similarly, the HLTF gene was found to be inactivated by hypermethylation in a significant number of colon, gastric and uterine tumors, indicating that HLTF silencing may confer a growth advantage and that HLTF could be considered as a tumor suppressor gene. However, 20-fold HLTF overexpression was detected in various transformed cell lines, suggesting that HLTF could be associated with neoplastic transformation and act more like an oncogene. Moreover, HLTF activation was recently linked to the initial steps of carcinogenesis in an experimental model of estrogen-induced kidney tumors. Those apparently contradictory observations suggest that HLTF might play various roles in cancer. In this review, we will try to reconcile all these data in order to specify the role of HLTF in cancer progression.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder linked to contractions of the D4Z4 repeat array in the subtelomeric region of chromosome 4q. By comparing genome-wide gene expression data from muscle biopsies of patients with FSHD to those of 11 other neuromuscular disorders, paired-like homeodomain transcription factor 1 ( PITX1 ) was found specifically up-regulated in patients with FSHD. In addition, we showed that the double homeobox 4 gene ( DUX4 ) that maps within the D4Z4 repeat unit was up-regulated in patient myoblasts at both mRNA and protein level. We further showed that the DUX4 protein could activate transient expression of a luciferase reporter gene fused to the Pitx1 promoter as well as the endogenous Pitx1 gene in transfected C2C12 cells. In EMSAs, DUX4 specifically interacted with a 30-bp sequence 5′-CGGATGCTGTCTTCTAATTAGTTTGGACCC-3′ in the Pitx1 promoter. Mutations of the TAAT core affected Pitx1-LUC activation in C2C12 cells and DUX4 binding in vitro . Our results suggest that up-regulation of both DUX4 and PITX1 in FSHD muscles may play critical roles in the molecular mechanisms of the disease.

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

Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app) for PS ranging from 6.08x10(10) M to 1.62x10(11)M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app=(1.6+/-0.2)x10(11)M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction.

Double homeodomain (DUX) proteins are encoded by a family of 3.3-kilobase repeated elements dispersed in the human genome. One of these elements named D4Z4 is found in a tandem repeat array on chromosome 4 that is partially deleted in facioscapulohumeral muscular dystrophy. We have evaluated the trafficking and mobility of two DUX proteins, DUX1 and DUX4. We transfected C2C12 myoblasts with cDNA encoding these proteins fused to the green fluorescent protein and studied their intracellular localization and diffusional mobilities using fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We also studied truncated forms of the proteins, containing one or both homeodomains or a region outside the homeodomains. We show that both full-length proteins are actively transported into the nucleus, and that the homeodomains contain the signals required for this localization. DUX1 is more mobile than DUX4 within the nucleus (t(1/2) = 4.8 s for DUX1 and 13.4 s for DUX4), suggesting differences in the way the two proteins interact with nuclear components.

Translesion synthesis (TLS) with specialized DNA polymerases allows dealing with a base lesion on the template strand during DNA replication; a base is inserted opposite the lesion, correctly or incorrectly, depending on the lesion, the involved DNA polymerase(s) and the sequence context. The major oxidized DNA base 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) is highly mutagenic due to its ability to pair with either cytosine or adenine during DNA synthesis, depending on its conformation and involved DNA polymerases. To measure the correct or mutagenic outcome of lesion bypass, an original quantitative pyrosequencing method was developed and analytically validated. The method was applied to the study of DNA synthesis fidelity through an 8-oxodG or an undamaged guanine. After an in vitro primer-extension through 8-oxodG in the presence of the four deoxynucleotides triphosphates and a total nuclear protein extract, obtained from normal human intestinal epithelial cells (FHs 74 Int cell line), the reaction products were amplified by polymerase chain reaction and analyzed by pyrosequencing to measure nucleotides inserted opposite the lesion. The 8-oxodG bypass fidelity of FHs 74 Int cells nuclear extract is about 85.3%. We calculated within-day and total precisions for both 8-oxodG (2.8% and 2.8%, respectively) and undamaged templates (1.0% and 1.1%, respectively). We also demonstrated that only cytosine is incorporated opposite a normal guanine and that both cytosine and adenine can be incorporated opposite an 8-oxodG lesion. The proposed method is straightforward, fast, reproducible and easily adaptable to other sequences and lesions. It thus has a wide range of applications in the biological field, notably to elucidate TLS mechanisms and modulators.

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.

Objective : to determine if triiodothyronine (T3) regulates the human tissue-type plasminogen activator (t-PA) gene through its upstream DNA sequences. Design : transient transfection analysis of t-PA reporter constructs and expression vectors for thyroid hormone receptor (TR) and retinoid X receptor (RXR) in HT 1080 fibrosarcoma and SK-N-SH neuroblastoma cells. Results : 10 −6 to 10 −9 M T3 was found to inhibit the expression of human t-PA promoter reporter constructs fivefold in transfected HT1080 fibroblast and SK-N-SH neuronal cells. Unliganded TR and RXR were shown to induce the basal level of the t-PA promoter twofold. Repression by T3 required the TR DNA binding domain. The region of the t-PA promoter mediating this repression was located within bp - 110-- 7 and comprised a sequence specifically interacting with TR homodimers. However, deletion of this sequence did not relieve T3 repression. Conclusion : T3 represses the human t-PA promoter activity in HT1080 and SK-N-SH cells. However, binding of TR homodimers to the minimal T3 responsive region was shown not to be involved in the T3 repression, suggesting the existence of an alternative T3 inhibitory mechanism.

Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein–DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione- S -transferase and the DNA-binding domain of the helicase-like transcription factor with its cis -element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant ( K d ) of 1 n m was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.

Summaryα2-Antiplasmin (α2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of α2-AP deficient mice, the murine α2-AP 1 gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the α2-AP 1gene, but failed to identify the 5’ boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5’ boundary of exon 10 is identical in both genes while the 3’ non-coding region is 64 bp longer in the human gene. Introns 2,3,6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5’ flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse α2-AP gene encodes a 491-amino acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-.A targeting vector, ppPNT.α2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.α2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.α2-AP yielded three targeted clones with correct homologous recombination at the 5’- and 3’-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate α2-AP deficient mice.

Translesion synthesis (TLS) relies on a series of specialized DNA polymerases able to insert a base either correctly or incorrectly opposite a lesion on a DNA template strand during replication or post-repair synthesis. To measure the correct or mutagenic outcome of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) bypass by TLS DNA polymerases, a capillary electrophoresis (CE) method with fluorescent label has been developed. Two oligonucleotides were designed and hybridized: (i) a 72-mer oligonucleotide framing one 8-oxodG at position 40 and (ii) the 39-mer oligonucleotide complementary to the first strand from the 3' end to the lesion and labeled at the 5' end with a fluorochrome. After incubation with FHs 74 Int human intestinal epithelial cell nuclear proteins, in the presence of either deoxyadenosine triphosphate (dATP) or deoxycytidine triphosphate (dCTP), and denaturation, the resulting elongated oligomers were analyzed by fluorescent capillary electrophoresis. This primer extension assay was then validated in terms of linearity (linear range=0.5-2.5 nM), detectability (limits of detection and quantification=0.023 and 0.091 nM, respectively), and precision (total precisions=8.1% and 3.7% for dATP and dCTP, respectively, n=9). The addition of some natural phytochemicals to the reaction mix significantly influences the outcome of TLS either in an error-free way or in a mutagenic way.