UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2014

Teresa Mendes Maia, Delphine Gogendeau, Carole Pennetier, Carsten Janke, Renata Basto (2014 Feb 15)

Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules.

Biology open : 138-51 : DOI : 10.1242/bio.20146577 En savoir plus
Résumé

Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.

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Lucia Guidugli, Aura Carreira, Sandrine M Caputo, Asa Ehlen, Alvaro Galli, Alvaro N A Monteiro, Susan L Neuhausen, Thomas V O Hansen, Fergus J Couch, Maaike P G Vreeswijk, (2014 Feb 1)

Functional assays for analysis of variants of uncertain significance in BRCA2.

Human mutation : 151-64 : DOI : 10.1002/humu.22478 En savoir plus
Résumé

Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore, alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication, and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review, we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including variants of uncertain significance analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk.

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D Bellanger, V Jacquemin, M Chopin, G Pierron, O A Bernard, J Ghysdael, M-H Stern (2014 Feb 1)

Recurrent JAK1 and JAK3 somatic mutations in T-cell prolymphocytic leukemia.

Leukemia : 417-9 : DOI : 10.1038/leu.2013.271 En savoir plus
Résumé

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Edwin D Hawkins, Jane Oliaro, Kelly M Ramsbottom, Stephen B Ting, Faruk Sacirbegovic, Michael Harvey, Tanja Kinwell, Jacques Ghysdael, Ricky W Johnstone, Patrick O Humbert, Sarah M Russell (2014 Jan 27)

Lethal giant larvae 1 tumour suppressor activity is not conserved in models of mammalian T and B cell leukaemia.

PloS one : e87376 : DOI : 10.1371/journal.pone.0087376 En savoir plus
Résumé

In epithelial and stem cells, lethal giant larvae (Lgl) is a potent tumour suppressor, a regulator of Notch signalling, and a mediator of cell fate via asymmetric cell division. Recent evidence suggests that the function of Lgl is conserved in mammalian haematopoietic stem cells and implies a contribution to haematological malignancies. To date, direct measurement of the effect of Lgl expression on malignancies of the haematopoietic lineage has not been tested. In Lgl1⁻/⁻ mice, we analysed the development of haematopoietic malignancies either alone, or in the presence of common oncogenic lesions. We show that in the absence of Lgl1, production of mature white blood cell lineages and long-term survival of mice are not affected. Additionally, loss of Lgl1 does not alter leukaemia driven by constitutive Notch, c-Myc or Jak2 signalling. These results suggest that the role of Lgl1 in the haematopoietic lineage might be restricted to specific co-operating mutations and a limited number of cellular contexts.

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Année de publication : 2013

S Gachet, E Genescà, D Passaro, M Irigoyen, H Alcalde, C Clémenson, S Poglio, F Pflumio, A Janin, C Lasgi, S Dodier, M Soyer, G Duménil, J Ghysdael (2013 Dec 1)

Leukemia-initiating cell activity requires calcineurin in T-cell acute lymphoblastic leukemia.

Leukemia : 2289-300 : DOI : 10.1038/leu.2013.156 En savoir plus
Résumé

Despite their initial efficient response to induction chemotherapy, relapse remains frequent in patients with T-cell acute lymphoblastic leukemia (T-ALL), an aggressive malignancy of immature T-cell progenitors. We previously reported sustained calcineurin (Cn) activation in human lymphoid malignancies, and showed that Cn inhibitors have antileukemic effects in mouse models of T-ALL. It was unclear, however, from these studies whether these effects resulted from Cn inhibition in leukemic cells themselves or were an indirect consequence of impaired Cn function in the supportive tumor microenvironment. We thus generated a Notch (intracellular Notch 1, ICN1)-induced T-ALL mouse model, in which conditional Cn genetic deletion is restricted to leukemic cells. Ex vivo, Cn deletion altered the adhesive interactions between leukemic cells and their supportive stroma, leukemic cell survival, proliferation, migration and clonogenic potential. In vivo, Cn activation was found to be critical for leukemia initiating/propagating cell activity as demonstrated by the failure of Cn-deficient leukemic cells to transplant the disease to syngeneic recipient mice. Importantly, combination of vincristine treatment with Cre-mediated Cn ablation cooperated to induce long-term remission of ICN1-induced T-ALL. These findings indicate that Cn is a promising target in T-ALL relapse prevention, and call for clinical trials incorporating Cn inhibitors during consolidation therapy.

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Antony M Carr, Sarah Lambert, Aura Carreira (2013 Nov 29)

Replication stress-induced genome instability: the dark side of replication maintenance by homologous recombination.

Journal of molecular biology : 4733-44 : DOI : 10.1016/j.jmb.2013.04.023 En savoir plus
Résumé

Homologous recombination (HR) is an evolutionary-conserved mechanism involved in a subtle balance between genome stability and diversity. HR is a faithful DNA repair pathway and has been largely characterized in the context of double-strand break (DSB) repair. Recently, multiple functions for the HR machinery have been identified at arrested forks. These are evident across different organisms and include replication fork-stabilization and fork-restart functions. Interestingly, a DSB appears not to be a prerequisite for HR-mediated replication maintenance. HR has the ability to rebuild a replisome at inactivated forks, but perhaps surprisingly, the resulting replisome is liable to intrastrand and interstrand switches leading to replication errors. Here, we review our current understanding of the replication maintenance function of HR. The error proneness of these pathways leads us to suggest that the origin of replication-associated genome instability should be re-evaluated.

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Michaela Waibel, Vanessa S Solomon, Deborah A Knight, Rachael A Ralli, Sang-Kyu Kim, Kellie-Marie Banks, Eva Vidacs, Clemence Virely, Keith C S Sia, Lauryn S Bracken, Racquel Collins-Underwood, Christina Drenberg, Laura B Ramsey, Sara C Meyer, Megumi Takiguchi, Ross A Dickins, Ross Levine, Jacques Ghysdael, Mark A Dawson, Richard B Lock, Charles G Mullighan, Ricky W Johnstone (2013 Nov 27)

Combined targeting of JAK2 and Bcl-2/Bcl-xL to cure mutant JAK2-driven malignancies and overcome acquired resistance to JAK2 inhibitors.

Cell reports : 1047-59 : DOI : 10.1016/j.celrep.2013.10.038 En savoir plus
Résumé

To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2) and the effector stage (Bcl-2/Bcl-xL), is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.

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Maria M Magiera, Carsten Janke (2013 Aug 27)

Investigating tubulin posttranslational modifications with specific antibodies.

Methods in cell biology : 247-67 : DOI : 10.1016/B978-0-12-407757-7.00016-5 En savoir plus
Résumé

Microtubules play highly diverse and essential roles in every eukaryotic cell. While built from conserved dimers of α- and β-tubulin, microtubules can be diversified by posttranslational modifications in order to fulfill specific functions in cells. The tubulin posttranslational modifications: acetylation, detyrosination, polyglutamylation, and polyglycylation play important roles in microtubule functions; however, only little functional and mechanistic insight has been gained so far. The modification state of microtubules can be visualized with specific antibodies. A drawback is that detailed information about the specificities and limitations of these antibodies are not easily accessible in the literature. We provide here a comprehensive description of the currently available set of antibodies specific to tubulin modifications. Focusing on glutamylation antibodies, we discuss specific protocols that allow using these antibodies to gain semi-quantitative information on the levels and distribution of tubulin modifications in immunocytochemistry and immunoblot.

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Montserrat Bosch Grau, Gloria Gonzalez Curto, Cecilia Rocha, Maria M Magiera, Patricia Marques Sousa, Tiziana Giordano, Nathalie Spassky, Carsten Janke (2013 Aug 5)

Tubulin glycylases and glutamylases have distinct functions in stabilization and motility of ependymal cilia.

The Journal of cell biology : 441-51 : DOI : 10.1083/jcb.201305041 En savoir plus
Résumé

Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.

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Hélène Colman, Catherine Le Berre-Scoul, Céline Hernandez, Sandra Pierredon, Audrey Bihouée, Rémi Houlgatte, Stephan Vagner, Arielle R Rosenberg, Cyrille Féray (2013 Jun 1)

Genome-wide analysis of host mRNA translation during hepatitis C virus infection.

Journal of virology : 6668-77 : DOI : 10.1128/JVI.00538-13 En savoir plus
Résumé

In the model of Huh-7.5.1 hepatocyte cells infected by the JFH1 hepatitis C virus (HCV) strain, transcriptomic and proteomic studies have revealed modulations of pathways governing mainly apoptosis and cell cycling. Differences between transcriptomic and proteomic studies pointed to regulations occurring at the posttranscriptional level, including the control of mRNA translation. In this study, we investigated at the genome-wide level the translational regulation occurring during HCV infection. Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Translationally regulated mRNAs were found to correspond to genes enriched in specific pathways, including vesicular transport and posttranscriptional regulation. Interestingly, the strongest translational regulation was found for mRNAs encoding proteins involved in pre-mRNA splicing, mRNA translation, and protein folding. Strikingly, these pathways were not previously identified, through transcriptomic studies, as being modulated following HCV infection. Importantly, the observed changes in host mRNA translation were directly due to HCV replication rather than to HCV entry, since they were not observed in JFH1-infected Huh-7.5.1 cells treated with a potent HCV NS3 protease inhibitor. Overall, this study highlights the need to consider, beyond transcriptomic or proteomic studies, the modulation of host mRNA translation as an important aspect of HCV infection.

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Sarah Lambert, Antony M Carr (2013 Apr 1)

Replication stress and genome rearrangements: lessons from yeast models.

Current opinion in genetics & development : 132-9 : DOI : 10.1016/j.gde.2012.11.009 En savoir plus
Résumé

Replication failures induced by replication fork barriers (RFBs) or global replication stress generate many of the chromosome rearrangement (CR) observed in human genomic disorders and cancer. RFBs have multiple causes and cells protect themselves from the consequences of RFBs using three general strategies: preventing expression of RFB activity, stabilising the arrested replisome and, in the case of replisome failure, shielding the fork DNA to allow rebuilding of the replisome. Yeast models provide powerful tools to understand the cellular response to RFBs, delineate pathways that suppress genome instability and define mechanisms by which CRs occur when these fail. Recent progress has identified key features underlying RFBs activity and is beginning to uncover the DNA dynamics that bring about genome instability.

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Virginie Quidville, Samar Alsafadi, Aïcha Goubar, Frédéric Commo, Véronique Scott, Catherine Pioche-Durieu, Isabelle Girault, Sonia Baconnais, Eric Le Cam, Vladimir Lazar, Suzette Delaloge, Mahasti Saghatchian, Patricia Pautier, Philippe Morice, Philippe Dessen, Stéphan Vagner, Fabrice André (2013 Apr 1)

Targeting the deregulated spliceosome core machinery in cancer cells triggers mTOR blockade and autophagy.

Cancer research : 2247-58 : DOI : 10.1158/0008-5472.CAN-12-2501 En savoir plus
Résumé

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology.

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Sarah Lambert, Antony M Carr (2013 Mar 1)

Impediments to replication fork movement: stabilisation, reactivation and genome instability.

Chromosoma : 33-45 : DOI : 10.1007/s00412-013-0398-9 En savoir plus
Résumé

Maintaining genome stability is essential for the accurate transmission of genetic material. Genetic instability is associated with human genome disorders and is a near-universal hallmark of cancer cells. Genetic variation is also the driving force of evolution, and a genome must therefore display adequate plasticity to evolve while remaining sufficiently stable to prevent mutations and chromosome rearrangements leading to a fitness disadvantage. A primary source of genome instability are errors that occur during chromosome replication. More specifically, obstacles to the movement of replication forks are known to underlie many of the gross chromosomal rearrangements seen both in human cells and in model organisms. Obstacles to replication fork progression destabilize the replisome (replication protein complex) and impact on the integrity of forked DNA structures. Therefore, to ensure the successful progression of a replication fork along with its associated replisome, several distinct strategies have evolved. First, there are well-orchestrated mechanisms that promote continued movement of forks through potential obstacles. Second, dedicated replisome and fork DNA stabilization pathways prevent the dysfunction of the replisome if its progress is halted. Third, should stabilisation fail, there are mechanisms to ensure damaged forks are accurately fused with a converging fork or, when necessary, re-associated with the replication proteins to continue replication. Here, we review what is known about potential barriers to replication fork progression, how these are tolerated and their impact on genome instability.

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Andrea E Prota, Maria M Magiera, Marijn Kuijpers, Katja Bargsten, Daniel Frey, Mara Wieser, Rolf Jaussi, Casper C Hoogenraad, Richard A Kammerer, Carsten Janke, Michel O Steinmetz (2013 Feb 4)

Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

The Journal of cell biology : 259-70 : DOI : 10.1083/jcb.201211017 En savoir plus
Résumé

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.

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Année de publication : 2012

Audrey Costes, Sarah A E Lambert (2012 Dec 27)

Homologous recombination as a replication fork escort: fork-protection and recovery.

Biomolecules : 39-71 : DOI : 10.3390/biom3010039 En savoir plus
Résumé

Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

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