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International audience; The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model. However, a therapeutic application of such nanodiamonds requires their elimination by the organism, particularly in urine, which is impossible for 35 nm particles. Here, we report that hydrogenated cationic nanodiamonds of primary size 7 nm (ND-H) have also a high affinity for siRNA and are capable of delivering them in cells. With siRNA/ND-H complexes, we measured a high inhibition efficacy of EWS/FLI-1 gene expression in Ewing sarcoma cell line. Electron microscopy investigations showed ND-H in endocytosis compartments, and especially in macropinosomes from which they can escape before siRNA degradation occurred. In addition, the association of EWS/FLI-1 silencing by the siRNA/ND-H complex with a vincristine treatment yielded a potentiation of the toxic effect of this chemotherapeutic drug. Therefore ND-H appears as a promising delivery agent in anti-tumoral gene therapy.

Background Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes. Patients and methods Plasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation. Results The miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes. Conclusions Detection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes.

The navigation cue netrin‐1 is well‐documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin‐1. Interestingly, the epitope recognized by NP137 in netrin‐1 shares 90% homology with its counterpart in netrin‐3, the closest member to netrin‐1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin‐3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin‐3 and netrin‐1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL‐1 or NeuroD1 transcription factors in SCLC. Netrin‐3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin‐3 for netrin‐1 receptors and we demonstrated that netrin‐3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin‐3 in NB and SCLC., Expression and function of netrin‐3 have so far never been investigated in human cancers. Based on cohort analyses, this study shows that netrin‐3 is a putative therapeutic target in both small cell lung cancer (SCLC) and neuroblastoma (NB).

International audience; Among the various clay minerals, sepiolite, which is a natural nanofibrous silicate that exhibit a poor cell toxicity, is a potential promising nanocarrier for the non-viral and stable transfer of plasmid DNA into bacteria, mammalian and human cells. We first show here that sepiolite binds to bacteria, which can be useful in decontamination protocols. In a previous research we have shown that is possible to modulate the efficiency of the absorption of different types of DNA molecules onto sepiolite, and that the DNA previously adsorbed could be recovered preserving the DNA structure and biological activity. Taking advantage of both, the sepiolite/bacteria and sepiolite/DNA interactions, we show that pre-assembly of DNA with sepiolite and incubation of bacteria with this obtained biohybrid strongly improve the transformation efficiency, in a rapid, convenient and inexpensive method that doesn't require competent cell preparation. In addition, we also show that the controlled sepiolite and DNA binding capacities can be used to purify plasmids from bacteria, representing an advantageous alternative to onerous commercial kits. All of these results open the way to the use of sepiolite-based bionanohybrids for the development of novel biological models of interest for academic and applied sciences.

International audience; Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.

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// 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.

Oxazaphosphorines including cyclophosphamide, trofosfamide and ifosfamide (IFO) belong to the alkylating agent class and are indicated in the treatment of numerous cancers. However, IFO is subject to limiting side-effects in high-dose protocols. To circumvent IFO drawbacks in clinical practices, preactivated IFO analogs were designed to by-pass the toxic metabolic pathway. Among these IFO analogs, some of them showed the ability to self-assemble due to the use of a poly-isoprenyloxy chain as preactivating moiety. We present here, the in vitro activity of the nanoassembly formulations of preactivated IFO derivatives with a C-4 geranyloxy, farnesyloxy and squalenoxy substituent on a large panel of tumor cell lines. The chemical and colloidal stabilities of the geranyloxy-IFO (G-IFO), farnesyloxy-IFO (F-IFO) and squalenoxy-IFO (SQ-IFO) NAs were further evaluated in comparison to their free formulation. Finally, pharmacokinetic parameters and maximal tolerated dose of the most potent preactivated IFO analog (G-IFO) were determined and compared to IFO, paving the way to in vivo studies.

International audience; The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07\₀0380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

BACKGROUND The enzymes encoded by the GALNT [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNAC-T)] gene family catalyze the first step of O-glycosylation. Little is known about the link between expression of the genes encoding GALNAC-T enzymes and tumor progression in neuroblastoma, a pediatric cancer that can be classified as either low or high risk. We assessed the expression of genes in the GALNT family in a large cohort of neuroblastoma patients and characterized members of this family that might be used as new prognostic markers. METHODS Reverse-transcription PCR analysis of 14 GALNT genes with a panel of neuroblastoma cell lines identified the GALNT9 gene as playing a potential role in disease progression. We used the log-rank test and the multivariable Cox proportional hazards model with a cohort of 122 neuroblastoma patients to analyze the relationship between GALNT9 expression and overall survival or disease-free survival. RESULTS In the high-risk neuroblastoma experimental model IGR-N-91, GALNT9 expression was present in neuroblasts derived from primary tumors but not in neuroblasts from metastatic bone marrow. Moreover, GALNT9 in neuroblastoma cell lines was expressed in substrate adherent (S)-type cell lines but not in neuronal (N)-type lines. In the tumor cohort, GALNT9 expression was associated with high overall survival, independent of the standard risk-stratification covariates. GALNT9 expression was significantly associated with disease-free survival for patients currently classified as at low risk (P < 0.0007). CONCLUSIONS GALNT9 expression correlates with both improved overall survival in low- and high-risk groups and an improved clinical outcome (overall and disease-free survival) in low-risk patients. Thus, the GALNT9 expression may be a prognostic marker for personalized therapy.

The immunoglobulin heavy chain (IGH) locus is submitted to intra-chromosomal DNA breakages and rearrangements during normal B cell differentiation that create a risk for illegitimate inter-chromosomal translocations leading to a variety of B-cell malignancies. In most Burkitt's and Mantle Cell lymphomas, specific chromosomal translocations juxtapose the IGH locus with a CMYC or Cyclin D1 (CCND1) gene, respectively. 3D-fluorescence in situ hybridization was performed on normal peripheral B lymphocytes induced to mature in vitro from a naive state to the stage where they undergo somatic hypermutation (SHM) and class switch recombination (CSR). The CCND1 genes were found very close to the IGH locus in naive B cells and further away after maturation. In contrast, the CMYC alleles became localized closer to an IGH locus at the stage of SHM/CSR. The colocalization observed between the two oncogenes and the IGH locus at successive stages of B-cell differentiation occurred in the immediate vicinity of the nucleolus, consistent with the known localization of the RAGs and AID enzymes whose function has been demonstrated in IGH physiological rearrangements. We propose that the chromosomal events leading to Mantle Cell lymphoma and Burkitt's lymphoma are favored by the colocalization of CCND1 and CMYC with IGH at the time the concerned B cells undergo VDJ recombination or SHM/CSR, respectively. J. Cell. Biochem. 117: 1506-1510, 2016. © 2016 Wiley Periodicals, Inc.

International audience; Cellular immune reactions against non-self-epitopes require activation of cytotoxic CD8+ T-cells via cross-presentation of MHC class I-restricted peptides by professional antigen presenting cells (pAPCs), with the consequent detection and elimination of cells expressing the same antigens via the endogenous (direct) pathway. The source of peptides for the endogenous pathway is constituted of alternative mRNA translation products; however, it is still unclear which source of peptides is used for cross-presentation. Furthermore, the presentation of non-canonical translation products, produced during a non-conventional translation event, on class I molecules of tumor cells has been reported but how these peptides are generated, presented to pAPCs, and their capacity to stimulate CD8+ T cells is still not known. Here, we report that pioneer translation peptides (PTPs) derived from intron or exon pre-mRNAs can serve as tumor-associated antigens (TA-PTPs) and are delivered from the producing tumor cells to pAPCs via exosomes where they are processed by the cytosolic pathway. Injection of TA-PTPs and tumor-derived exosomes efficiently induce CD8+ T-cell proliferation and prevent tumor growth in mice. Our results show that TA-PTPs represent an efficient source of antigenic peptides for CD8+ T cell activation and that full-length proteins are not required for cross-presentation. These findings can have interesting implications for generating tolerance and for designing vectors to generate vaccines.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants.

International audience; Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.

International audience; Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context.

International audience; Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor with tyrosine kinase activity that was expected to be oncogenic. However, it has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis; and it was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand. Here we show that the TrkC ligand neurotrophin-3 (NT-3) is upregulated in a large fraction of aggressive human neuroblastomas (NBs) and that it blocks TrkC-induced apoptosis of human NB cell lines, consistent with the idea that TrkC is a dependence receptor. Functionally, both siRNA knockdown of NT-3 expression and incubation with a TrkC-specific blocking antibody triggered apoptosis in human NB cell lines. Importantly, disruption of the NT-3 autocrine loop in malignant human neuroblasts triggered in vitro NB cell death and inhibited tumor growth and metastasis in both a chick and a mouse xenograft model. Thus, we believe that our data suggest that NT-3/TrkC disruption is a putative alternative targeted therapeutic strategy for the treatment of NB.

Facio-scapulo-humeral dystrophy (FSHD), a muscular hereditary disease with a prevalence of 1 in 20,000, is caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. Earlier, we demonstrated the existence in the vicinity of the D4Z4 repeat of a nuclear matrix attachment site, FR-MAR, efficient in normal human myoblasts and nonmuscular human cells but much weaker in muscle cells from FSHD patients. We now report that the D4Z4 repeat contains an exceptionally strong transcriptional enhancer at its 5′-end. This enhancer up-regulates transcription from the promoter of the neighboring FRG1 gene. However, an enhancer blocking activity was found present in FR-MAR that in vitro could protect transcription from the enhancer activity of the D4Z4 array. In vivo, transcription from the FRG1 and FRG2 genes could be down- or up-regulated depending on whether or not FR-MAR is associated with the nuclear matrix. We propose a model for an etiological role of the delocalization of FR-MAR in the genesis of FSHD.

International audience; Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) β-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.

International audience; Fascioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder linked to partial deletion of integral numbers of a 3.3 kb polymorphic repeat, D4Z4, within the subtelomeric region of chromosome 4q. Although the relationship between deletions of D4Z4 and FSHD is well established, how this triggers the disease remains unclear. We have mapped the DNA loop domain containing the D4Z4 repeat cluster in human primary myoblasts and in murine–human hybrids. A nuclear matrix attachment site was found located in the vicinity of the repeat. Prominent in normal human myoblasts and nonmuscular human cells, this site is much weaker in muscle cells derived from FSHD patients, suggesting that the D4Z4 repeat array and upstream genes reside in two loops in nonmuscular cells and normal human myoblasts but in only one loop in FSHD myoblasts. We propose a model whereby the nuclear scaffold/matrix attached region regulates chromatin accessibility and expression of genes implicated in the genesis of FSHD.

BACKGROUND The Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression. METHODS CDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided. RESULTS CDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03). CONCLUSIONS Together, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs.

Muscular dystrophy is a condition potentially predisposing for cancer; however, currently, only Myotonic dystrophy patients are known to have a higher risk of cancer. Here, we have searched for a link between facioscapulohumeral dystrophy (FSHD) and cancer by comparing published transcriptome signatures of FSHD and various malignant tumours and have found a significant enrichment of cancer-related genes among the genes differentially expressed in FSHD. The analysis has shown that gene expression profiles of FSHD myoblasts and myotubes resemble that of Ewing's sarcoma more than that of other cancer types tested. This is the first study demonstrating a similarity between FSHD and cancer cell expression profiles, a finding that might indicate the existence of a common step in the pathogenesis of these two diseases.

International audience; HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.

International audience; Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast-and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared. Citation: Pirozhkova I, Barat A, Dmitriev P, Kim E, Robert T, et al. (2013) Differences in Transcription Patterns between Induced Pluripotent Stem Cells Produced from the Same Germ Layer Are Erased upon Differentiation.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder linked to the deletion of an integral number of 3.3-kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of chromosome 4q. Most genes identified in this region are overexpressed in FSHD myoblasts, including the double homeobox genes DUX4 and DUX4c. We have carried out a simultaneous miRNome/transcriptome analysis of FSHD and control primary myoblasts. Of 365 microRNAs (miRNAs) analyzed in this study, 29 were found to be differentially expressed between FSHD and normal myoblasts. Twenty-one microRNAs (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331, miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, and miR-660) were up-regulated, and eight were down-regulated (miR-15b, miR-20b, miR-21, miR-25, miR-100, miR-155, miR-345, and miR-594). Twelve of the miRNAs up-regulated in FHSD were also up-regulated in the cells ectopically expressing DUX4c, suggesting that this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206 were highly expressed in FSHD myoblasts, which nonetheless did not prematurely enter myogenic differentiation. This could be accounted for by the fact that in FSHD myoblasts, functionally important target genes, including cell cycle, DNA damage, and ubiquitination-related genes, escape myogenic microRNA-induced repression.

Résumé en anglais : Our team is working on the mechanisms of apoptosis induced by nutlin-3, a small molecule which binds to MDM2 and activates p53, in different lymphomas associated with Epstein-Barr virus such as Burkitt lymphoma (BL) or Post-transplant lymphoproliferative disorder (PTLD). Our results show that nutlin-3 strongly induce apoptosis in EBV (-) cells whereas EBV (+) latency III cells are much more resistant. The aim of my PhD project was to study the mechanisms involved in the resistance of EBV (+) latency III cells to apoptosis and to develop new therapeutic strategies to bypass these mechanisms. A transcriptomic analysis was realized after treatment with nutlin-3 of two cell lines which only differs by their EBV status. Based on the results obtained, a study was performed which allow us to show that: 1) autophagy is induced after nutlin-3 treatment in EBV (+) latency III cells; 2) these cells strongly expressed beclin-1 and present a constitutively high level of autophagy; 3) autophagy is involved in the resistance of apoptosis observed in these cells. Furthermore, our results demonstrate that Bcl-2 also contributes to the resistance of EBV (+) latency III cells and that treatment with ABT-737, a Bcl-2 inhibitor, restores their susceptibility to nutlin-3 treatment. We thus assessed the efficiency of this compound in vivo, in monotherapy or associated with conventional treatments (Cyclophosphamide for BL and Rituximab for PTLD). Results obtained during these pre-clinical studies show that: 1) ABT-737 reduces tumor growth and increase the overall survival of mice xenografted with a lymphoblastoïd cell line (LCL, used as a model for PTLD studies) but has no effects on mice xenografed with BL cell lines; 2) the association ABT-737/Cyclophosphamide reduces tumor growth during treatment but doesn’t improve the overall survival of mice xenografed with BL cell lines; 3) the association ABT-737/Rituximab is very efficient and induces 70% of complete remission in mice xenografted with LCL.; Résumé en français : Notre équipe étudie les mécanismes de l’apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d’activer la p53, dans différents types de lymphomes associés au virus d’Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l’apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d’une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l’autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie contribue à la résistance de ces cellules à l’apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l’utilisation d’ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L’efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d’une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu’il n’a pas d’effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n’améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l’association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL

Facioscapulohumeral muscular dystrophy (FSHD), the most frequent muscular dystrophy, is an autosomal dominant disease. In most individuals with FSHD, symptoms are restricted to muscles of the face, arms, legs, and trunk. FSHD is genetically linked to contractions of the D4Z4 repeat array causing activation of several genes. One of these maps in the repeat itself and expresses the DUX4 (the double homeobox 4) transcription factor causing a gene deregulation cascade. In addition, analyses of the RNA or protein expression profiles in muscle have indicated deregulations in the oxidative stress response. Since oxidative stress affects peripheral muscle function, we investigated mitochondrial function and oxidative stress in skeletal muscle biopsies and blood samples from patients with FSHD and age-matched healthy controls, and evaluated their association with physical performances. We show that specifically, oxidative stress (lipid peroxidation and protein carbonylation), oxidative damage (lipofuscin accumulation), and antioxidant enzymes (catalase, copper-zinc-dependent superoxide dismutase, and glutathione reductase) were higher in FSHD than in control muscles. FSHD muscles also presented abnormal mitochondrial function (decreased cytochrome c oxidase activity and reduced ATP synthesis). In addition, the ratio between reduced (GSH) and oxidized glutathione (GSSG) was strongly decreased in all FSHD blood samples as a consequence of GSSG accumulation. Patients with FSHD also had reduced systemic antioxidative response molecules, such as low levels of zinc (a SOD cofactor), selenium (a GPx cofactor involved in the elimination of lipid peroxides), and vitamin C. Half of them had a low ratio of gamma/alpha tocopherol and higher ferritin concentrations. Both systemic oxidative stress and mitochondrial dysfunction were correlated with functional muscle impairment. Mitochondrial ATP production was significantly correlated with both quadriceps endurance (T(LimQ)) and maximal voluntary contraction (MVC(Q)) values (rho=0.79, P=0.003; rho=0.62, P=0.05, respectively). The plasma concentration of oxidized glutathione was negatively correlated with the T(LimQ), MVC(Q) values, and the 2-min walk distance (MWT) values (rho=-0.60, P=0.03; rho=-0.56, P=0.04; rho=-0.93, P0.0001, respectively). Our data characterized oxidative stress in patients with FSHD and demonstrated a correlation with their peripheral skeletal muscle dysfunction. They suggest that antioxidants that might modulate or delay oxidative insult may be useful in maintaining FSHD muscle functions.

International audience; pAp (3'-5' phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.

International audience; Translational repression is achieved by protein complexes that typically bind 3′ UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5′ extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.

Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We here report that the enhancer within the D4Z4 repeat binds the Krüppel-like factor KLF15. KLF15 was found to be upregulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15 binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.; La dystrophie musculaire facioscapulohumérale (FSHD) est la troisième myopathie la plus fréquente en Europe. La maladie atteint progressivement les muscles du visage, des bras et des jambes. Les symptômes apparaissent dans la majorité des cas avant 20 ans et la maladie, souvent douloureuse, provoque fréquemment un handicap majeur qui nécessite de se déplacer en fauteuil roulant. J'ai démontré que la protéine KLF15 est surexprimée dans les tissus des patients FSHD et dans les myoblastes cultivés in vitro et prélevés sur des patients FSHD. Des résultats préliminaires indiquent que la surexpression du facteur KLF15 pourrait être expliquée par le stress oxydant induit par DUX4 et la surexpression de certains facteurs de myogenèse induits par DUX4c. J'ai démontré que KLF15 interagit directement avec les répétitions D4Z4 et contrôle leur fonction d'activation de transcription. Ainsi KLF15 sert de médiateur entre les répétitions D4Z4 et deux gènes dans la région 4q35: FRG2 et DUX4c ce que explique la surexpression de ces deux gènes dans la FSHD. La surexpression du gène DUX4c, homologue du DUX4, perturbe le programme de différentiation musculaire en activant certains facteurs de myogenèse (myomiRs ou microRNAs myogéniques). En somme, la découverte du rôle du facteur KLF15 dans la FSHD met en évidence une boucle de rétrocontrôle positif qui relie la surexpression du facteur KLF15 avec la surexpression des gènes codés dans la région 4q35. Le rôle central du facteur KLF15 dans ce nouveau modèle de la maladie permet d'envisager une nouvelle piste thérapeutique pour la dystrophie FSHD basé sur l'inhibition du facteur KLF15.

In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.; Le génôme des cellules eucaryotes est condensé au sein d'une structure complexe hiérarchiquement organisée : la chromatine. La chromatine est composée d'ADN, de protéines histone et non-histone. Cette thèse a pour but d'étudier le rôle de la chromatine dans la réponse cellulaire aux dommages de l'ADN (DDR) par les méthodologies de génomique fonctionnelle et de protéomique. Nous avons tout d'abord analysé les modifications post-traductionnelles (PTM) des histones dans le cadre des pontages inter-brins (ou "Interstrand Crosslinks", ICL), type particulier de lésions de l'ADN, en choisissant le modèle de l'Anémie de Fanconi (FA). Ceci a été réalisé grâce aux techniques de protéomique quantitative SILAC (Stable Isotope Labeling of Amino acid during Cell culture) et de spectrométrie de masse (MS). Nous avons ainsi réussi à identifier et à quantifier de nombreuses PTMs dans les histones H3 et H4, et à démontrer que certaines de ces PTM sont dépendantes d'une voie fonctionnelle de la signalisation de FA. Nous avons également approfondi l'étude des DDR dans les cellules de FA par une approche de génomique fonctionnelle. Pour cela, nous avons analysé le profil l'expression d'enzymes associées à l'acétylation et à la méthylation des histones. Nos résultats suggèrent l'existence de corrélations entre le profil d'expression de ces enzymes et les PTMs des histones. Des études complémentaires sont nécessaires en vue de confirmer ces corrélations. Nous avons également comparé le transcriptome de deux lignées cellulaires de FA (mutée en FANCC et corrigée en FANCC) après induction de dommages à l'ADN. Afin de différencier les changements spécifiquement associés à la voie de signalisation de FA en réponse aux ICL de l'ADN des réponses plus générales aux dommages de l'ADN, nous avons inclus des cellules traitées par rayonnement ionisant. En réalisant une analyse d'interactions factorielles, nous avons pu identifier une réponse transcriptionnelle aux dommages de l'ADN nécessitant une voie fonctionnelle de la signalisation de FA. Nous avons également tenté de pallier aux limitations rencontrées dans l'analyse des PTMs des histones. En effet, les PTMs des histones que nous avons identifiées représentent l'ensemble des modifications, c'est-à-dire les PTMs concernant les histones se trouvant immédiatement à proximité du site du dommage et en relation directe avec celui-ci, et les PTMs se trouvant à distance du dommage et pouvant ne pas être en relation directe avec celui-ci. Les approches courantes pour identifier les PTMs se trouvant à des loci particuliers sont basées sur l'immunoprécipitation classique de la chromatine où l'utilisation de formaldéhyde altère les protéines, ce qui en rend impossible l'analyse par MS. Nous avons proposé une nouvelle méthodologie basée sur la biotinylation expérimentale d'histones situées à proximité d'une protéine particulière, suivie de la purification des nucléosomes contenant ces histones biotinylées. Contrairement àl'immunoprécipiatation classique de la chromatine, cette méthode n'induit pas d'altération des protéines, permettant ainsi de purifier les histones à partir d'un locus spécifique et d'analyser à grande échelle leurs PTMs par MS. Cette approche permet aussi de suivre dans le temps les PTMs d'une fraction des histones juste après leur biotinylation. Enfin, elle présente l'avantage de pouvoir étudier le profil des PTMs de différents états fonctionnels de la chromatine grâce à l'utilisation de variants d'histones.

In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.; Le génôme des cellules eucaryotes est condensé au sein d'une structure complexe hiérarchiquement organisée : la chromatine. La chromatine est composée d'ADN, de protéines histone et non-histone. Cette thèse a pour but d'étudier le rôle de la chromatine dans la réponse cellulaire aux dommages de l'ADN (DDR) par les méthodologies de génomique fonctionnelle et de protéomique. Nous avons tout d'abord analysé les modifications post-traductionnelles (PTM) des histones dans le cadre des pontages inter-brins (ou "Interstrand Crosslinks", ICL), type particulier de lésions de l'ADN, en choisissant le modèle de l'Anémie de Fanconi (FA). Ceci a été réalisé grâce aux techniques de protéomique quantitative SILAC (Stable Isotope Labeling of Amino acid during Cell culture) et de spectrométrie de masse (MS). Nous avons ainsi réussi à identifier et à quantifier de nombreuses PTMs dans les histones H3 et H4, et à démontrer que certaines de ces PTM sont dépendantes d'une voie fonctionnelle de la signalisation de FA. Nous avons également approfondi l'étude des DDR dans les cellules de FA par une approche de génomique fonctionnelle. Pour cela, nous avons analysé le profil l'expression d'enzymes associées à l'acétylation et à la méthylation des histones. Nos résultats suggèrent l'existence de corrélations entre le profil d'expression de ces enzymes et les PTMs des histones. Des études complémentaires sont nécessaires en vue de confirmer ces corrélations. Nous avons également comparé le transcriptome de deux lignées cellulaires de FA (mutée en FANCC et corrigée en FANCC) après induction de dommages à l'ADN. Afin de différencier les changements spécifiquement associés à la voie de signalisation de FA en réponse aux ICL de l'ADN des réponses plus générales aux dommages de l'ADN, nous avons inclus des cellules traitées par rayonnement ionisant. En réalisant une analyse d'interactions factorielles, nous avons pu identifier une réponse transcriptionnelle aux dommages de l'ADN nécessitant une voie fonctionnelle de la signalisation de FA. Nous avons également tenté de pallier aux limitations rencontrées dans l'analyse des PTMs des histones. En effet, les PTMs des histones que nous avons identifiées représentent l'ensemble des modifications, c'est-à-dire les PTMs concernant les histones se trouvant immédiatement à proximité du site du dommage et en relation directe avec celui-ci, et les PTMs se trouvant à distance du dommage et pouvant ne pas être en relation directe avec celui-ci. Les approches courantes pour identifier les PTMs se trouvant à des loci particuliers sont basées sur l'immunoprécipitation classique de la chromatine où l'utilisation de formaldéhyde altère les protéines, ce qui en rend impossible l'analyse par MS. Nous avons proposé une nouvelle méthodologie basée sur la biotinylation expérimentale d'histones situées à proximité d'une protéine particulière, suivie de la purification des nucléosomes contenant ces histones biotinylées. Contrairement àl'immunoprécipiatation classique de la chromatine, cette méthode n'induit pas d'altération des protéines, permettant ainsi de purifier les histones à partir d'un locus spécifique et d'analyser à grande échelle leurs PTMs par MS. Cette approche permet aussi de suivre dans le temps les PTMs d'une fraction des histones juste après leur biotinylation. Enfin, elle présente l'avantage de pouvoir étudier le profil des PTMs de différents états fonctionnels de la chromatine grâce à l'utilisation de variants d'histones.

The study of tumoral microenvironment should take into account different modes of intercellular communications: direct contacts between extracellular membranes, secretion and uptake of cytokines and finally emission and uptake of complex biological objects like exosomes and microvesicles.Epstein-Barr virus (EBV) is associated with several human malignancies of epithelial origin (Nasopharyngeal carcinoma or NPC) or of lymphoïd origin (post-transplant lymphoproliferative disorder or PTLD). In these tumors, malignant cells are latently infected by EBV and release exosomes and microvesicles containing viral nucleic acids and proteins. Studying them will enable a better understanding of tumor-host interactions and the discovery of new markers which could be useful for early diagnostic and the follow-up of the disease under treatment.The first aim of this thesis was to study the release by malignant cells of EBV microRNAs belonging to the BART family and their blood diffusion in patients bearing NPC tumors. For the first time, I’ve shown that exosomes released by NPC cells in vitro contain EBV miR-BART microRNAs. Moreover, ebv-miR-BART7 can be detected in the plasma of NPC patients. Unlike what is observed in vitro, circulating BART microRNAs are not carried by exosomes. Recent data from studies in xenografted mice show that they are carried by extra-cellular complexes which can be immunoprecipitated by anti-Ago2 antibodies. We are currently trying to confirm these data in plasma from NPC patients. This work will ease the use of miR-BARTs as potential biomarkers.The second aim was to study the proteome modifications induced by the EBV Latent Membrane Protein 1 protein (LMP1). I’ve shown that LMP1 expression in lymphoid or epithelial cells infected or not by EBV induces the release of PARP1 in the extra-cellular space. This extra-cellular PARP1 is not carried by exosomes or microvesicles but is embedded in non-vesicular nano-objects containing histones and DNA. We have called these objects “DNA-proteins complexes”. We don’t know how they are produced and released by cells. We think that they are not only secreted by apoptotic cells. Recent data show that this release of extra-cellular PARP1 is associated with PARP1 activation by LMP1 oncoprotein expression. We are trying to prove this hypothesis using cell lines expressing wild type or mutated LMP1. The release and the activation of PARP1 induced by LMP1 expression will help to understand the mechanisms of EBV-associated oncogenesis and auto-immunity.; En pathologie tumorale, l’étude du micro-environnement tumoral doit prendre en compte différents modes de communication cellulaire : contacts directs entre membranes plasmiques, émission et réception de cytokines et enfin émission et internalisation d’objets biologiques plus complexes comme les microvésicules et les exosomes qui peuvent être assimilés à de véritables organites extra-cellulaires. Le virus d’Epstein-Barr (EBV) participe à l’oncogenèse de plusieurs affections malignes humaines d’origine épithéliale (carcinomes nasopharyngés ou NPC) ou lymphocytaire (lymphomes post-transplantation). Dans ces tumeurs, les cellules malignes qui sont infectées de façon latente par EBV libèrent des exosomes et des microvésicules qui contiennent des protéines et des acides nucléiques d’origine virale. L’étude de ces éléments doit permettre de mieux comprendre les interactions hôte-tumeur et de mettre en évidence de nouveaux biomarqueurs utiles pour le diagnostic précoce et la surveillance de la maladie sous traitement. Le premier objectif de ma thèse consistait à étudier la sécrétion par les cellules malignes d’une famille de microARN viraux appelés miR-BART et leur diffusion dans le sang périphérique chez les sujets porteurs de tumeurs associées à EBV. Pour la première fois j’ai mis en évidence une sécrétion d’exosomes porteurs de miR-BART par les cellules de NPC en culture in vitro. J’ai également montré que les miR-BART, particulièrement miR-BART7, sont détectables dans le plasma de sujets porteurs de NPC. Contrairement à ce qui se passe in vitro les miR-BART plasmatiques ne sont pas transportés par des exosomes. Des données obtenues chez la souris montrent qu’ils peuvent être transportés par des complexes extra-cellulaires que l’on peut précipiter au moyen d’anticorps anti-ago2. Nous cherchons à confirmer ces données sur des échantillons de plasma provenant de patients porteurs de NPC. Ces données pourront guider à l’avenir l’utilisation des miR-BART circulants comme source de biomarqueurs.Le deuxième volet de ma thèse avait pour but d’étudier les modifications du protéome des exosomes induites par une oncoprotéine du virus d’Epstein-Barr appelée LMP1 (latent membrane protein 1). J’ai montré que la LMP1, lorsqu’elle est exprimée dans les cellules lymphocytaires ou épithéliales, infectées ou non par EBV, induit la libération de la protéine PARP1 dans le milieu extra-cellulaire. Cette PARP1 extra-cellulaire n’est pas associée aux exosomes ni aux microvésicules mais à des nano-objets non-vésiculaires contenant notamment des histones et de l’ADN. Nous avons désignés ces objets sous le terme de complexes ADN-protéines extra-cellulaires. Nous ne savons presque rien de la biogenèse de ces complexes ; nous pensons qu’ils ne proviennent pas uniquement de cellules en apoptose. En revanche, des expériences préliminaires suggèrent que la présence de PARP1 dans ces complexes coïncide avec une activation permanente de la PARP1 induite dans les cellules productrices par l’expression de l’oncoprotéine LMP1. Cette hypothèse est en cours de vérification grâce à des expériences menées sur des lignées cellulaires exprimant différentes formes sauvages ou mutées de la LMP1. Ces données sur l’activation de la PARP1 et sur sa sécrétion induite par la LMP1 auront des retombées intéressantes pour notre compréhension des mécanismes d’oncogenèse et d’auto-immunité liés à l’infection par le virus d’Epstein-Barr.

The study of tumoral microenvironment should take into account different modes of intercellular communications: direct contacts between extracellular membranes, secretion and uptake of cytokines and finally emission and uptake of complex biological objects like exosomes and microvesicles.Epstein-Barr virus (EBV) is associated with several human malignancies of epithelial origin (Nasopharyngeal carcinoma or NPC) or of lymphoïd origin (post-transplant lymphoproliferative disorder or PTLD). In these tumors, malignant cells are latently infected by EBV and release exosomes and microvesicles containing viral nucleic acids and proteins. Studying them will enable a better understanding of tumor-host interactions and the discovery of new markers which could be useful for early diagnostic and the follow-up of the disease under treatment.The first aim of this thesis was to study the release by malignant cells of EBV microRNAs belonging to the BART family and their blood diffusion in patients bearing NPC tumors. For the first time, I’ve shown that exosomes released by NPC cells in vitro contain EBV miR-BART microRNAs. Moreover, ebv-miR-BART7 can be detected in the plasma of NPC patients. Unlike what is observed in vitro, circulating BART microRNAs are not carried by exosomes. Recent data from studies in xenografted mice show that they are carried by extra-cellular complexes which can be immunoprecipitated by anti-Ago2 antibodies. We are currently trying to confirm these data in plasma from NPC patients. This work will ease the use of miR-BARTs as potential biomarkers.The second aim was to study the proteome modifications induced by the EBV Latent Membrane Protein 1 protein (LMP1). I’ve shown that LMP1 expression in lymphoid or epithelial cells infected or not by EBV induces the release of PARP1 in the extra-cellular space. This extra-cellular PARP1 is not carried by exosomes or microvesicles but is embedded in non-vesicular nano-objects containing histones and DNA. We have called these objects “DNA-proteins complexes”. We don’t know how they are produced and released by cells. We think that they are not only secreted by apoptotic cells. Recent data show that this release of extra-cellular PARP1 is associated with PARP1 activation by LMP1 oncoprotein expression. We are trying to prove this hypothesis using cell lines expressing wild type or mutated LMP1. The release and the activation of PARP1 induced by LMP1 expression will help to understand the mechanisms of EBV-associated oncogenesis and auto-immunity.; En pathologie tumorale, l’étude du micro-environnement tumoral doit prendre en compte différents modes de communication cellulaire : contacts directs entre membranes plasmiques, émission et réception de cytokines et enfin émission et internalisation d’objets biologiques plus complexes comme les microvésicules et les exosomes qui peuvent être assimilés à de véritables organites extra-cellulaires. Le virus d’Epstein-Barr (EBV) participe à l’oncogenèse de plusieurs affections malignes humaines d’origine épithéliale (carcinomes nasopharyngés ou NPC) ou lymphocytaire (lymphomes post-transplantation). Dans ces tumeurs, les cellules malignes qui sont infectées de façon latente par EBV libèrent des exosomes et des microvésicules qui contiennent des protéines et des acides nucléiques d’origine virale. L’étude de ces éléments doit permettre de mieux comprendre les interactions hôte-tumeur et de mettre en évidence de nouveaux biomarqueurs utiles pour le diagnostic précoce et la surveillance de la maladie sous traitement. Le premier objectif de ma thèse consistait à étudier la sécrétion par les cellules malignes d’une famille de microARN viraux appelés miR-BART et leur diffusion dans le sang périphérique chez les sujets porteurs de tumeurs associées à EBV. Pour la première fois j’ai mis en évidence une sécrétion d’exosomes porteurs de miR-BART par les cellules de NPC en culture in vitro. J’ai également montré que les miR-BART, particulièrement miR-BART7, sont détectables dans le plasma de sujets porteurs de NPC. Contrairement à ce qui se passe in vitro les miR-BART plasmatiques ne sont pas transportés par des exosomes. Des données obtenues chez la souris montrent qu’ils peuvent être transportés par des complexes extra-cellulaires que l’on peut précipiter au moyen d’anticorps anti-ago2. Nous cherchons à confirmer ces données sur des échantillons de plasma provenant de patients porteurs de NPC. Ces données pourront guider à l’avenir l’utilisation des miR-BART circulants comme source de biomarqueurs.Le deuxième volet de ma thèse avait pour but d’étudier les modifications du protéome des exosomes induites par une oncoprotéine du virus d’Epstein-Barr appelée LMP1 (latent membrane protein 1). J’ai montré que la LMP1, lorsqu’elle est exprimée dans les cellules lymphocytaires ou épithéliales, infectées ou non par EBV, induit la libération de la protéine PARP1 dans le milieu extra-cellulaire. Cette PARP1 extra-cellulaire n’est pas associée aux exosomes ni aux microvésicules mais à des nano-objets non-vésiculaires contenant notamment des histones et de l’ADN. Nous avons désignés ces objets sous le terme de complexes ADN-protéines extra-cellulaires. Nous ne savons presque rien de la biogenèse de ces complexes ; nous pensons qu’ils ne proviennent pas uniquement de cellules en apoptose. En revanche, des expériences préliminaires suggèrent que la présence de PARP1 dans ces complexes coïncide avec une activation permanente de la PARP1 induite dans les cellules productrices par l’expression de l’oncoprotéine LMP1. Cette hypothèse est en cours de vérification grâce à des expériences menées sur des lignées cellulaires exprimant différentes formes sauvages ou mutées de la LMP1. Ces données sur l’activation de la PARP1 et sur sa sécrétion induite par la LMP1 auront des retombées intéressantes pour notre compréhension des mécanismes d’oncogenèse et d’auto-immunité liés à l’infection par le virus d’Epstein-Barr.

Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We report here that the enhancer within the D4Z4 repeat binds the Kruppel-like factor KLF15. KLF15 was found to be up-regulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts, the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15-binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes, and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and it thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.

BACKGROUND: Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. METHODS: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). RESULTS: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. CONCLUSIONS: Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

International audience; Apoptosis-Inducing factor (AIF) plays important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. Here, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria where they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal (IMSS) encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 upon exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been "designed" to be retained in mitochondria and to minimize its potential neurotoxic activity.

International audience; Facioscapulohumeral 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.

International audience; Most of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease.

International audience; Facioscapulohumeral 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.

International audience; Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models.

We report here the first realization of an artificial branched DNA template where a single wall carbon nanotube is positioned with the necessary geometry of an individually gated field effect transistor.

International audience; The similarities between essential molecular mechanisms in Archaea and Eukarya make it possible to discover, using comparative genomics, new fundamental mechanisms conserved between these two domains. We are studying a complex of two proteins conserved in Archaea and Eukarya whose precise biological role and biochemical function remain unknown. One of them is a universal protein known as Kae1 (kinase-asociated endopeptidase 1). The second protein is a serine/threonine kinase corresponding to the proteins Bud32 in Saccharomyces cerevisiae and PRPK (p53-related protein kinase) in humans. The genes encoding the archaeal orthologues of Kae1 and PRPK are either contiguous or even fused in many archaeal genomes. In S. cerevisiae, Kae1 and Bud32 (PRPK) belong to a chromatin-associated complex [KEOPS (kinase, endopeptidase and other proteins of small size)/EKC (endopeptidase-like kinase chromatin-associated)] that is essential for telomere elongation and transcription of essential genes. Although Kae1 is annotated as O-sialoglycoprotein endopeptidase in most genomes, we found that the Kae1 protein from Pyrococcus abyssi has no protease activity, but is an atypical DNA-binding protein with an AP (apurinic) lyase activity. The structure of the fusion protein from Methanocaldococcus jannaschii revealed that Kae1 maintains the ATP-binding site of Kae1 in an inactive configuration. We have in fact found that Kae1 inhibits the kinase activity of Bud32 (PRPK) in vitro. Understanding the precise biochemical function and biological role of these two proteins (which are probably essential for genome maintenance) remains a major challenge.

International audience; Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn(-) strain. A triple mutant (nrnA(-), nrnB(-), yhaM(-)) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5'-to-3' exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.

Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alphavbeta3 and alphavbeta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.Gene Therapy advance online publication, 12 June 2008; doi:10.1038/gt.2008.100.

International audience; Invasive ductal carcinomas (IDCs) and invasive lobular carcinomas (ILCs) are the two major pathological types of breast cancer. Epidemiological and histoclinical data suggest biological differences, but little is known about the molecular alterations involved in ILCs. We undertook a comparative large-scale study by both array-compared genomic hybridization and cDNA microarray of a set of 50 breast tumors (21 classic ILCs and 29 IDCs) selected on homogeneous histoclinical criteria. Results were validated on independent tumor sets, as well as by quantitative RT-PCR. ILCs and IDCs presented differences at both the genomic and expression levels with ILCs being less rearranged and heterogeneous than IDCs. Supervised analysis defined a 75-BACs signature discriminating accurately ILCs from IDCs. Expression profiles identified two subgroups of ILCs: typical ILCs ( approximately 50%), which were homogeneous and displayed a normal-like molecular pattern, and atypical ILCs, more heterogeneous with features intermediate between ILCs and IDCs. Supervised analysis identified a 75-gene expression signature that discriminated ILCs from IDCs, with many genes involved in cell adhesion, motility, apoptosis, protein folding, extracellular matrix and protein phosphorylation. Although ILCs and IDCs share common alterations, our data show that ILCs and IDCs could be distinguished on the basis of their genomic and expression profiles suggesting that they evolve along distinct genetic pathways.

Background A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification. Results We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/μ L) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/μ L), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. Conclusion We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis.

International audience; Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.