Publications by authors named "Laurent Brino"

29 Publications

  • Page 1 of 1

Deubiquitylase UCHL3 regulates bi-orientation and segregation of chromosomes during mitosis.

FASEB J 2020 09 1;34(9):12751-12767. Epub 2020 Aug 1.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of development and stem cells, Illkirch, France.

Equal segregation of chromosomes during mitosis ensures euploidy of daughter cells. Defects in this process may result in an imbalance in the chromosomal composition and cellular transformation. Proteolytic and non-proteolytic ubiquitylation pathways ensure directionality and fidelity of mitotic progression but specific mitotic functions of deubiquitylating enzymes (DUBs) remain less studied. Here we describe the role of the DUB ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) in the regulation of chromosome bi-orientation and segregation during mitosis. Downregulation or inhibition of UCHL3 leads to chromosome alignment defects during metaphase. Frequent segregation errors during anaphase are also observed upon inactivation of UCHL3. Mechanistically, UCHL3 interacts with and deubiquitylates Aurora B, the catalytic subunit of chromosome passenger complex (CPC), known to be critically involved in the regulation of chromosome alignment and segregation. UCHL3 does not regulate protein levels of Aurora B or the binding of Aurora B to other CPC subunits. Instead, UCHL3 promotes localization of Aurora B to kinetochores, suggesting its role in the error correction mechanism monitoring bi-orientation of chromosomes during metaphase. Thus, UCHL3 contributes to the regulation of faithful genome segregation and maintenance of euploidy in human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202000769RDOI Listing
September 2020

High-Throughput Fluorescence-Based Screen Identifies the Neuronal MicroRNA miR-124 as a Positive Regulator of Alphavirus Infection.

J Virol 2020 04 16;94(9). Epub 2020 Apr 16.

Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France

MicroRNAs (miRNAs) are small regulatory RNAs which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs, and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified 16 miRNAs showing a positive effect on Sindbis virus (SINV) expressing green fluorescent protein (GFP), among which were a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 and silent mutations to disrupt this binding site in the viral RNA abolished positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved in other alphaviruses, as its inhibition reduces chikungunya virus (CHIKV) production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection. Arthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arbovirus infection, neurological diseases such as meningitis and encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphavirus infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02145-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163141PMC
April 2020

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

EMBO Mol Med 2019 07 24;11(7):e9982. Epub 2019 May 24.

Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201809982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609913PMC
July 2019

Preferential Response of Basal-Like Head and Neck Squamous Cell Carcinoma Cell Lines to EGFR-Targeted Therapy Depending on -Driven Oncogenic Addiction.

Cancers (Basel) 2019 Jun 8;11(6). Epub 2019 Jun 8.

Université de Strasbourg, Inserm, UMR_S1113, 67200 Strasbourg, France.

The management of locally advanced head and neck squamous cell carcinoma (HNSCC) with Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), achieves only moderate response rates, and clinical trials that evaluated EGFR-blockade with tyrosine kinase inhibitors (TKI) yielded disappointing results. Inter-tumor heterogeneity may hinder the therapeutic efficiency of anti-EGFR treatments. HNSCC heterogeneity was addressed in several studies, which all converged towards the definition of molecular subgroups. They include the basal subgroup, defined by the deregulated expression of factors involved in the EGFR signaling pathway, including the epiregulin EGFR ligand encoded by the gene. These observations indicate that basal tumors could be more sensitive to anti-EGFR treatments. To test this hypothesis, we performed a screen of a representative collection of basal versus non-basal HNSCC cell lines for their sensitivity to several anti-EGFR drugs (Cetuximab, Afatinib, and Gefitinib), tested as monotherapy or in combination with drugs that target closely-linked pathways [Mitogen-activated protein kinase kinase/ extracellular signal-regulated kinases (MEK), mammalian Target of Rapamycine (mTOR) or Human Epidermal growth factor Receptor 2 (HER2)]. Basal-like cell lines were found to be more sensitive to EGFR blockade alone or in combination with treatments that target MEK, mTOR, or HER2. Strikingly, the basal-like status was found to be a better predictor of cell response to EGFR blockade than clinically relevant mutations [e.g., cyclin-dependent kinase Inhibitor 2A ()]. Interestingly, we show that EGFR blockade inhibits expression, and that knock-down decreases basal cell clonogenic survival, suggesting that expression could be a predictive functional marker of sensitivity to EGFR blockade in basal-like HNSCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11060795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627901PMC
June 2019

Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity.

Gut 2020 02 10;69(2):380-392. Epub 2019 May 10.

Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.

Objective: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle.

Design: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches.

Results: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer.

Conclusion: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2018-317423DOI Listing
February 2020

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

Gut 2020 01 4;69(1):158-167. Epub 2019 Mar 4.

Université de Strasbourg, Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMR_S1110, F-67000 Strasbourg, France.

Objective: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent.

Design: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection.

Results: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets.

Conclusion: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2018-317065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943243PMC
January 2020

Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair.

J Cell Biol 2018 10 27;217(10):3382-3397. Epub 2018 Jul 27.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

DNA repair is critical to maintaining genome integrity, and its dysfunction can cause accumulation of unresolved damage that leads to genomic instability. The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex and the nuclear pore-associated transcription and export complex 2 (TREX-2) couple transcription with mRNA export. In this study, we identify a novel interplay between human TREX-2 and the deubiquitination module (DUBm) of SAGA required for genome stability. We find that the scaffold subunit of TREX-2, GANP, positively regulates DNA repair through homologous recombination (HR). In contrast, DUBm adaptor subunits ENY2 and ATXNL3 are required to limit unscheduled HR. These opposite roles are achieved through monoubiquitinated histone H2B (H2Bub1). Interestingly, the activity of the DUBm of SAGA on H2Bub1 is dependent on the integrity of the TREX-2 complex. Thus, we describe the existence of a functional interaction between human TREX-2 and SAGA DUBm that is key to maintaining the H2B/HB2ub1 balance needed for efficient repair and HR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201803074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168256PMC
October 2018

Genes and Pathways Regulated by Androgens in Human Neural Cells, Potential Candidates for the Male Excess in Autism Spectrum Disorder.

Biol Psychiatry 2018 08 9;84(4):239-252. Epub 2018 Jan 9.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France; Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France; Université de Strasbourg, Illkirch, France; Laboratory of Genetic Diagnostics, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. Electronic address:

Background: Prenatal exposure to androgens during brain development in male individuals may participate to increase their susceptibility to develop neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability. However, little is known about the action of androgens in human neural cells.

Methods: We used human neural stem cells differentiated from embryonic stem cells to investigate targets of androgens.

Results: RNA sequencing revealed that treatment with dihydrotestosterone (DHT) leads to subtle but significant changes in the expression of about 200 genes, encoding proteins of extracellular matrix or involved in signal transduction of growth factors (e.g., insulin/insulin growth factor 1). We showed that the most differentially expressed genes (DEGs), RGCC, RNF144B, NRCAM, TRIM22, FAM107A, IGFBP5, and LAMA2, are reproducibly regulated by different androgens in different genetic backgrounds. We showed, by overexpressing the androgen receptor in neuroblastoma cells SH-SY5Y or knocking it down in human neural stem cells, that this regulation involves the androgen receptor. A chromatin immunoprecipitation combined with direct sequencing analysis identified androgen receptor-bound sequences in nearly half of the DHT-DEGs and in numerous other genes. DHT-DEGs appear enriched in genes involved in ASD (ASXL3, NLGN4X, etc.), associated with ASD (NRCAM), or differentially expressed in patients with ASD (FAM107A, IGFBP5). Androgens increase human neural stem cell proliferation and survival in nutrient-deprived culture conditions, with no detectable effect on regulation of neurite outgrowth.

Conclusions: We characterized androgen action in neural progenitor cells, identifying DHT-DEGs that appear to be enriched in genes related to ASD. We also showed that androgens increase proliferation of neuronal precursors and protect them from death during their differentiation in nutrient-deprived conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biopsych.2018.01.002DOI Listing
August 2018

A molecular roadmap for the emergence of early-embryonic-like cells in culture.

Nat Genet 2018 01 18;50(1):106-119. Epub 2017 Dec 18.

Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, Munich, Germany.

Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extra-embryonic tissues. A rare population of cells resembling 2-cell-stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here we identified intermediate cellular states and molecular determinants during the emergence of 2-cell-like cells. By deploying a quantitative single-cell expression approach, we identified an intermediate population characterized by expression of the transcription factor ZSCAN4 as a precursor of 2-cell-like cells. By using a small interfering RNA (siRNA) screen, we identified epigenetic regulators of 2-cell-like cell emergence, including the non-canonical PRC1 complex PRC1.6 and the EP400-TIP60 complex. Our data shed light on the mechanisms that underlie exit from the ES cell state toward the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-017-0016-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755687PMC
January 2018

Ubiquitin Receptor Protein UBASH3B Drives Aurora B Recruitment to Mitotic Microtubules.

Dev Cell 2016 Jan;36(1):63-78

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique UMR 7104, Institut National de la Santé et de la Recherche Médicale U964, Université de Strasbourg, 67404 Illkirch, France. Electronic address:

Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2015.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400057PMC
January 2016

PI4K-beta and MKNK1 are regulators of hepatitis C virus IRES-dependent translation.

Sci Rep 2015 Sep 1;5:13344. Epub 2015 Sep 1.

Department of Medicine II, University of Freiburg, Freiburg, Germany.

Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep13344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555030PMC
September 2015

A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses.

Hepatology 2016 Jan 6;63(1):35-48. Epub 2015 Oct 6.

Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.

Unlabelled: Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses.

Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus-glypican 5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.28013DOI Listing
January 2016

The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin.

Cell Rep 2015 Apr 26;11(1):149-63. Epub 2015 Mar 26.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104 Centre National de la Recherche Scientifique (CNRS), UdS, INSERM U964, BP 10142, 67404 Illkirch Cedex, Strasbourg, France. Electronic address:

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results in the sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.03.005DOI Listing
April 2015

A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair.

Nucleic Acids Res 2014 May 25;42(9):5689-701. Epub 2014 Mar 25.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104 CNRS, UdS, INSERM U964, BP 10142, F-67404 Illkirch Cedex, CU de Strasbourg, France

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027216PMC
May 2014

A small molecule screen identifies an inhibitor of DNA repair inducing the degradation of TFIIH and the chemosensitization of tumor cells to platinum.

Chem Biol 2014 Mar 6;21(3):398-407. Epub 2014 Feb 6.

IGBMC, Department of Functional Genomics and Cancer, Equipe Labellisée Ligue 2014, CNRS/INSERM/Université de Strasbourg, BP 163, 67404 Illkirch Cedex, CU Strasbourg, France. Electronic address:

Nucleotide excision repair (NER) removes DNA lesions resulting from exposure to UV irradiation or chemical agents such as platinum-based drugs used as anticancer molecules. Pharmacological inhibition of NER is expected to enhance chemosensitivity but nontoxic NER inhibitors are rare. Using a drug repositioning approach, we identify spironolactone (SP), an antagonist of aldosterone, as a potent NER inhibitor. We found that SP promotes a rapid and reversible degradation of XPB, a subunit of transcription/repair factor TFIIH. Such degradation depends both on ubiquitin-activating enzyme and on the 26S proteasome. Supplementation of extracts from SP-treated cells with purified TFIIH restored TFIIH-dependent repair and transcription activities in vitro, demonstrating the specific impact of SP on two fundamental functions of TFIIH. Finally, SP potentiated the cytotoxicity of platinum derivatives toward tumor cells, making it a potential therapeutic and research tool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2013.12.014DOI Listing
March 2014

The human TREX-2 complex is stably associated with the nuclear pore basket.

J Cell Sci 2013 Jun 16;126(Pt 12):2656-67. Epub 2013 Apr 16.

Cellular Signaling and Nuclear Dynamics Program, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U964, Université de Strasbourg, BP 10142 - 67404 ILLKIRCH Cedex, CU de Strasbourg, France.

In eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC. Here, we characterize the dynamics of human TREX-2 and show that it stably associates with the NPC basket. Moreover, the association of TREX-2 with the NPC requires the basket nucleoporins NUP153 and TPR, but is independent of transcription. Differential profiles of mRNA nuclear accumulation reveal that TREX-2 functions similarly to basket nucleoporins, but differently from NXF1. Thus, our results show that TREX-2 is an NPC-associated complex in mammalian cells and suggest that it is involved in putative NPC basket-related functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.118000DOI Listing
June 2013

HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex.

Cell Host Microbe 2013 Mar;13(3):302-13

Inserm, U1110, Institut de Virologie, 67000 Strasbourg, France.

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin β1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2013.02.006DOI Listing
March 2013

Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions.

PLoS Pathog 2012 28;8(6):e1002761. Epub 2012 Jun 28.

Unité de Génétique, Papillomavirus et Cancer Humain, Institut Pasteur, Paris, France.

Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1002761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386243PMC
November 2012

Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response.

Proc Natl Acad Sci U S A 2011 Dec 6;108(51):20603-8. Epub 2011 Dec 6.

Department of Functional Genomics and Cancer, Institut National de la Santé et de la Recherche Médicale U964, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Université de Strasbourg, BP 10142, 67404 Illkirch Cedex, France.

SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3-based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1102572108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251120PMC
December 2011

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.

Nat Med 2011 May 24;17(5):589-95. Epub 2011 Apr 24.

Institut National de la Santé et de la Recherche Médicale, U748, Strasbourg, France.

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.2341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938446PMC
May 2011

Nondenaturing chemical proteomics for protein complex isolation and identification.

Chembiochem 2010 Nov;11(17):2359-61

Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du Rhin, 67401 Illkirch-Graffenstaden, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201000574DOI Listing
November 2010

A chemical labeling strategy for proteomics under nondenaturing conditions.

Chembiochem 2010 Jan;11(1):79-82

Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du Rhin, Illkirch-Graffenstaden, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.200900641DOI Listing
January 2010

E6 proteins from diverse papillomaviruses self-associate both in vitro and in vivo.

J Mol Biol 2010 Feb 13;396(1):90-104. Epub 2009 Nov 13.

Ecole Supérieure de Biotechnologie de Strasbourg (IREBS, FRE 3211), Boulevard Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France.

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2009.11.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900769PMC
February 2010

The PHD domain of Np95 (mUHRF1) is involved in large-scale reorganization of pericentromeric heterochromatin.

Mol Biol Cell 2008 Aug 28;19(8):3554-63. Epub 2008 May 28.

Department of Structural and Functional Biology, University of Insubria, 21052 Busto Arsizio (VA), Italy.

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.e07-10-1059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2488286PMC
August 2008

RNAi: a powerful tool to unravel hepatitis C virus-host interactions within the infectious life cycle.

J Hepatol 2008 Mar 31;48(3):523-5. Epub 2007 Dec 31.

Inserm, U 748, Strasbourg, France.

Cellular cofactors affecting hepatitis C virus infection and replication. Randall G, Panis M, Cooper JD, Tellinghuisen TL, Sukhodolets KE, Pfeffer S, Landthaler M, Landgraf P, Kan S, Lindenbach BD, Chien M, Weir DB, Russo JJ, Ju J, Brownstein MJ, Sheridan R, Sander C, Zavolan M, Tuschl T, Rice CM. Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target DICER, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target DICER inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh7.5 cells, and Huh7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV. [Abstract reproduced by permission of Proc Natl Acad Sci USA 2007;104:12884-12889]
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2007.12.007DOI Listing
March 2008

RReportGenerator: automatic reports from routine statistical analysis using R.

Bioinformatics 2008 Jan 24;24(2):276-8. Epub 2007 Nov 24.

Laboratoire de Bioinformatique et Génomique Intégratives, IGBMC, UMR 7104, 67404 Illkirch, France.

Unlabelled: With the establishment of high-throughput (HT) screening methods there is an increasing need for automatic analysis methods. Here we present RReportGenerator, a user-friendly portal for automatic routine analysis using the statistical platform R and Bioconductor. RReportGenerator is designed to analyze data using predefined analysis scenarios via a graphical user interface (GUI). A report in pdf format combining text, figures and tables is automatically generated and results may be exported. To demonstrate suitable analysis tasks we provide direct web access to a collection of analysis scenarios for summarizing data from transfected cell arrays (TCA), segmentation of CGH data, and microarray quality control and normalization.

Availability: RReportGenerator, a user manual and a collection of analysis scenarios are available under a GNU public license on http://www-bio3d-igbmc.u-strasbg.fr/~wraff
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btm556DOI Listing
January 2008

A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift.

Yeast 2004 Jan;21(1):25-39

Natural Sciences Section, Södertörns University College, S-141 89 Huddinge, Sweden.

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/yea.1053DOI Listing
January 2004

A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

BMC Genomics 2003 Sep 22;4(1):38. Epub 2003 Sep 22.

Institut Pasteur, Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université PM Curie), 25 rue du Docteur Roux, F-75724 Paris cedex 15, France.

Background: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data.

Results: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences.

Conclusions: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-4-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC239980PMC
September 2003

Localization of the yeast RNA polymerase I-specific subunits.

EMBO J 2002 Aug;21(15):4136-44

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP163, F-67404 Illkirch Cedex, France.

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126139PMC
http://dx.doi.org/10.1093/emboj/cdf392DOI Listing
August 2002