UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2010

Robin Jeannet, Jérôme Mastio, Alejandra Macias-Garcia, Attila Oravecz, Todd Ashworth, Anne-Solen Geimer Le Lay, Bernard Jost, Stéphanie Le Gras, Jacques Ghysdael, Thomas Gridley, Tasuku Honjo, Freddy Radtke, Jon C Aster, Susan Chan, Philippe Kastner (2010 Dec 16)

Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL.

Blood : 5443-54 : DOI : 10.1182/blood-2010-05-286658 En savoir plus
Résumé

The Notch pathway is frequently activated in T-cell acute lymphoblastic leukemias (T-ALLs). Of the Notch receptors, Notch1 is a recurrent target of gain-of-function mutations and Notch3 is expressed in all T-ALLs, but it is currently unclear how these receptors contribute to T-cell transformation in vivo. We investigated the role of Notch1 and Notch3 in T-ALL progression by a genetic approach, in mice bearing a knockdown mutation in the Ikaros gene that spontaneously develop Notch-dependent T-ALL. While deletion of Notch3 has little effect, T cell-specific deletion of floxed Notch1 promoter/exon 1 sequences significantly accelerates leukemogenesis. Notch1-deleted tumors lack surface Notch1 but express γ-secretase-cleaved intracellular Notch1 proteins. In addition, these tumors accumulate high levels of truncated Notch1 transcripts that are caused by aberrant transcription from cryptic initiation sites in the 3′ part of the gene. Deletion of the floxed sequences directly reprograms the Notch1 locus to begin transcription from these 3′ promoters and is accompanied by an epigenetic reorganization of the Notch1 locus that is consistent with transcriptional activation. Further, spontaneous deletion of 5′ Notch1 sequences occurs in approximately 75% of Ikaros-deficient T-ALLs. These results reveal a novel mechanism for the oncogenic activation of the Notch1 gene after deletion of its main promoter.

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Krzysztof Rogowski, Juliette van Dijk, Maria M Magiera, Christophe Bosc, Jean-Christophe Deloulme, Anouk Bosson, Leticia Peris, Nicholas D Gold, Benjamin Lacroix, Montserrat Bosch Grau, Nicole Bec, Christian Larroque, Solange Desagher, Max Holzer, Annie Andrieux, Marie-Jo Moutin, Carsten Janke (2010 Nov 12)

A family of protein-deglutamylating enzymes associated with neurodegeneration.

Cell : 564-78 : DOI : 10.1016/j.cell.2010.10.014 En savoir plus
Résumé

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

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Ryan B Jensen, Aura Carreira, Stephen C Kowalczykowski (2010 Oct 7)

Purified human BRCA2 stimulates RAD51-mediated recombination.

Nature : 678-83 : DOI : 10.1038/nature09399 En savoir plus
Résumé

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.

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Vincent Mackiewicz, Anne Cammas, Delphine Desbois, Eric Marchadier, Sandra Pierredon, Frédérik Beaulieux, Elisabeth Dussaix, Stephan Vagner, Anne-Marie Roque-Afonso (2010 Oct 1)

Nucleotide variability and translation efficiency of the 5′ untranslated region of hepatitis A virus: update from clinical isolates associated with mild and severe hepatitis.

Journal of virology : 10139-47 : DOI : 10.1128/JVI.02598-09 En savoir plus
Résumé

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% +/- 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.

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Kenza Lahkim Bennani-Belhaj, Géraldine Buhagiar-Labarchède, Nada Jmari, Rosine Onclercq-Delic, Mounira Amor-Guéret (2010 Sep 8)

BLM Deficiency Is Not Associated with Sensitivity to Hydroxyurea-Induced Replication Stress.

Journal of nucleic acids : DOI : 10.4061/2010/319754 En savoir plus
Résumé

Bloom’s syndrome (BS) displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU), which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

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Xénia Mergui, Marie-Line Puiffe, Dominique Valteau-Couanet, Marc Lipinski, Jean Bénard, Mounira Amor-Guéret (2010 Sep 2)

p21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma.

BMC cancer : 473 : DOI : 10.1186/1471-2407-10-473 En savoir plus
Résumé

Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21Waf1 cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients’ tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.

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Sarah Lambert, Ken'ichi Mizuno, Joël Blaisonneau, Sylvain Martineau, Roland Chanet, Karine Fréon, Johanne M Murray, Antony M Carr, Giuseppe Baldacci (2010 Aug 13)

Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange.

Molecular cell : 346-59 : DOI : 10.1016/j.molcel.2010.07.015 En savoir plus
Résumé

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

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Carsten Janke, Matthias Kneussel (2010 Aug 1)

Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton.

Trends in neurosciences : 362-72 : DOI : 10.1016/j.tins.2010.05.001 En savoir plus
Résumé

In the past decades, a range of post-translational modifications has been discovered on tubulins, the major constituents of microtubules. Pioneering studies have described the occurrence and dynamics of these modifications and provided first insights into their potential functions in regulating the microtubule cytoskeleton. By contrast, several tubulin-modifying enzymes were only discovered in the last few years, and studies on molecular mechanisms and cellular functions of tubulin modifications are just beginning to emerge. This review highlights the roles of tubulin modifications in neurons. Recent studies are also discussed in relation to how the combinatorial use of tubulin modifications could generate a dynamic microtubule code, and how such a code might regulate basic as well as higher-order neuronal functions.

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Martin Dutertre, Stephan Vagner, Didier Auboeuf (2010 Jul 1)

Alternative splicing and breast cancer.

RNA biology : 403-11 : DOI : 10.4161/rna.7.4.12152 En savoir plus
Résumé

Alternative splicing is a key molecular event in the gene expression process. It allows for the synthesis of different products from the same gene, and consequently increases the complexity of the proteome encoded by a limited number of genes. Although alterations of alternative splicing are among the myriad of alterations present in tumor cells, increasing evidence indicates that cancer-associated splicing variants play an important role in tumor initiation and progression. Therefore, alternative splicing studies are opening new avenues of research in basic and translational molecular oncology.

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Tony Durand, Gaëtana Di Liberto, Hélène Colman, Anne Cammas, Sebastien Boni, Patrick Marcellin, Annie Cahour, Stephan Vagner, Cyrille Féray (2010 Jul 1)

Occult infection of peripheral B cells by hepatitis C variants which have low translational efficiency in cultured hepatocytes.

Gut : 934-42 : DOI : 10.1136/gut.2009.192088 En savoir plus
Résumé

Plasma hepatitis C virus (HCV) originates from hepatocytes. However, in certain subjects, B cells may harbour both plasma strains and occult HCV strains tha t are not detected in the plasma. The internal ribosome entry site (IRES) of these latter strains is mutated, suggesting that the efficiency of viral translation could drive the cellular tropism of HCV.

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Benjamin Lacroix, Juliette van Dijk, Nicholas D Gold, Julien Guizetti, Gudrun Aldrian-Herrada, Krzysztof Rogowski, Daniel W Gerlich, Carsten Janke (2010 Jun 14)

Tubulin polyglutamylation stimulates spastin-mediated microtubule severing.

The Journal of cell biology : 945-54 : DOI : 10.1083/jcb.201001024 En savoir plus
Résumé

Posttranslational glutamylation of tubulin is present on selected subsets of microtubules in cells. Although the modification is expected to contribute to the spatial and temporal organization of the cytoskeleton, hardly anything is known about its functional relevance. Here we demonstrate that glutamylation, and in particular the generation of long glutamate side chains, promotes the severing of microtubules. In human cells, the generation of long side chains induces spastin-dependent microtubule disassembly and, consistently, only microtubules modified by long glutamate side chains are efficiently severed by spastin in vitro. Our study reveals a novel control mechanism for microtubule mass and stability, which is of fundamental importance to cellular physiology and might have implications for diseases related to microtubule severing.

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C Virely, S Moulin, C Cobaleda, C Lasgi, A Alberdi, J Soulier, F Sigaux, S Chan, P Kastner, J Ghysdael (2010 Jun 1)

Haploinsufficiency of the IKZF1 (IKAROS) tumor suppressor gene cooperates with BCR-ABL in a transgenic model of acute lymphoblastic leukemia.

Leukemia : 1200-4 : DOI : 10.1038/leu.2010.63 En savoir plus
Résumé

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Kotoe Kashiwaya, Hidewaki Nakagawa, Masayo Hosokawa, Yasuo Mochizuki, Koji Ueda, Lianhua Piao, Suyoun Chung, Ryuji Hamamoto, Hidetoshi Eguchi, Hiroaki Ohigashi, Osamu Ishikawa, Carsten Janke, Yasuhisa Shinomura, Yusuke Nakamura (2010 May 15)

Involvement of the tubulin tyrosine ligase-like family member 4 polyglutamylase in PELP1 polyglutamylation and chromatin remodeling in pancreatic cancer cells.

Cancer research : 4024-33 : DOI : 10.1158/0008-5472.CAN-09-4444 En savoir plus
Résumé

Polyglutamylation is a new class of posttranslational modification in which glutamate side chains are formed in proteins, although its biological significance is not well known. Through our genome-wide gene expression profile analyses of pancreatic ductal adenocarcinoma (PDAC) cells, we identified the overexpression of tubulin tyrosine ligase-like family member 4 (TTLL4) in PDAC cells. Subsequent reverse transcription-PCR and Northern blot analyses confirmed its upregulation in several PDACs. TTLL4 belongs to the TTLL family which was reported to have polyglutamylase activity. Knockdown of TTLL4 by short hairpin RNA in PDAC cells attenuated the growth of PDAC cells and exogenous introduction of TTLL4 enhanced cell growth. We also found that TTLL4 expression was correlated with polyglutamylation levels of a glutamate stretch region of the proline, glutamate, and leucine-rich protein 1 (PELP1) that was shown to interact with various proteins such as histone H3, and was involved in several signaling pathways through its function as a scaffold protein. PELP1 polyglutamylation could influence its interaction with histone H3 and affect histone H3 acetylation. We also identified the interaction of PELP1 with LAS1L and SENP3, components of the MLL1-WDR5 supercomplex involving chromatin remodeling. Our findings imply that TTLL4 could play important roles in pancreatic carcinogenesis through its polyglutamylase activity and subsequent coordination of chromatin remodeling, and might be a good molecular candidate for the development of new therapeutic strategies for pancreatic cancer.

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Stefania Millevoi, Stéphan Vagner (2010 May 1)

Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation.

Nucleic acids research : 2757-74 : DOI : 10.1093/nar/gkp1176 En savoir plus
Résumé

Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3′ end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3′ end processing regulation by proposing several overlapping models of regulation.

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Kenza Lahkim Bennani-Belhaj, Sébastien Rouzeau, Géraldine Buhagiar-Labarchède, Pauline Chabosseau, Rosine Onclercq-Delic, Emilie Bayart, Fabrice Cordelières, Jérôme Couturier, Mounira Amor-Guéret (2010 Mar 9)

The Bloom syndrome protein limits the lethality associated with RAD51 deficiency.

Molecular cancer research : MCR : 385-94 : DOI : 10.1158/1541-7786.MCR-09-0534 En savoir plus
Résumé

Little is known about the functional interaction between the Bloom’s syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family.

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