UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2011

Aura Carreira, Stephen C Kowalczykowski (2011 Jun 28)

Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms.

Proceedings of the National Academy of Sciences of the United States of America : 10448-53 : DOI : 10.1073/pnas.1106971108 En savoir plus
Résumé

The human tumor suppressor protein BRCA2 plays a key role in recombinational DNA repair. BRCA2 recruits RAD51 to sites of DNA damage through interaction with eight conserved motifs of approximately 35 amino acids, the BRC repeats; however, the specific function of each repeat remains unclear. Here, we investigated the function of the individual BRC repeats by systematically analyzing their effects on RAD51 activities. Our results reveal the existence of two categories of BRC repeats that display unique functional characteristics. One group, comprising BRC1, -2, -3, and -4, binds to free RAD51 with high affinity. The second group, comprising BRC5, -6, -7, and -8, binds to free RAD51 with low affinity but binds to the RAD51-ssDNA filament with high affinity. Each member of the first group reduces the ATPase activity of RAD51, whereas none of the BRC repeats of the second group affects this activity. Thus, through different mechanisms, both types of BRC repeats bind to and stabilize the RAD51 nucleoprotein filament on ssDNA. In addition, members of the first group limit binding of RAD51 to duplex DNA, where members of the second group do not. Only the first group enhances DNA strand exchange by RAD51. Our results suggest that the two groups of BRC repeats have differentially evolved to ensure efficient formation of a nascent RAD51 filament on ssDNA by promoting its nucleation and growth, respectively. We propose that the BRC repeats cooperate in a partially redundant but reinforcing manner to ensure a high probability of RAD51 filament formation.

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Pauline Chabosseau, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Sarah Lambert, Michelle Debatisse, Olivier Brison, Mounira Amor-Guéret (2011 Jun 28)

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome.

Nature communications : 368 : DOI : 10.1038/ncomms1363 En savoir plus
Résumé

Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.

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Benjamin Lacroix, Carsten Janke (2011 Jun 25)

Generation of differentially polyglutamylated microtubules.

Methods in molecular biology (Clifton, N.J.) : 57-69 : DOI : 10.1007/978-1-61779-252-6_4 En savoir plus
Résumé

Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of microtubules is the posttranslational modification of tubulin molecules. Two tubulin modifications, polyglutamylation and polyglycylation, generate amino acid side chains of different length on tubulin. These modifications are thought to regulate interactions between microtubules and their associated proteins; however, detailed studies of this potential mechanism have not been performed. The investigation of the potential regulatory role of polyglutamylation requires in vitro tools to visualize the molecular events that could be affected by this modification. Classically, in vitro work with microtubules is performed with tubulin from brain tissue; however, this tubulin is highly posttranslationally modified. Here, we describe a method for the purification of tubulin carrying controlled levels of polyglutamylation, which can be used in basic in vitro assays.

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Pierre Zindy, Yann Bergé, Ben Allal, Thomas Filleron, Sandra Pierredon, Anne Cammas, Samantha Beck, Loubna Mhamdi, Li Fan, Gilles Favre, Jean-Pierre Delord, Henri Roché, Florence Dalenc, Magali Lacroix-Triki, Stéphan Vagner (2011 Jun 15)

Formation of the eIF4F translation-initiation complex determines sensitivity to anticancer drugs targeting the EGFR and HER2 receptors.

Cancer research : 4068-73 : DOI : 10.1158/0008-5472.CAN-11-0420 En savoir plus
Résumé

Elucidating how cancer cells respond to antagonists of HER receptor family members is critical to understanding mechanisms of therapeutic resistance that arise in patients. In large part, resistance to such agents appears to arise from deregulation of the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway. mTOR-dependent phosphorylation of the translation repressor 4E-BP1 leads to its dissociation from eIF4E, thereby causing an increase in the formation of the eIF4F complex, which also comprises eIF4G and eIF4A. In this study, we show that trastuzumab, cetuximab, and erlotinib all decrease the formation of the eIF4F complex in breast, colon, and head and neck cancer cells, respectively. Ectopic expression of eIF4E restores the trastuzumab-dependent defect in eIF4F formation, renders cells resistant to the trastuzumab-mediated decrease in cell proliferation, and rescues breast cancer xenografts from inhibition by trastuzumab. In breast tumor specimens, the level of eIF4E expression is associated with the therapeutic response to a trastuzumab-based regimen. Together, our findings suggest that formation of the eIF4F complex may be a critical determinant of the response to anticancer drugs that target HER2 and epidermal growth factor receptor.

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Marie-Jo Moutin, Annie Andrieux, Carsten Janke (2011 May 25)

[Microtubule polyglutamylation and neurodegeneration].

Médecine sciences : M/S : 464-7 : DOI : 10.1051/medsci/2011275006 En savoir plus
Résumé

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Adrien Decorsière, Anne Cayrel, Stéphan Vagner, Stefania Millevoi (2011 Feb 1)

Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3′-end processing and function during DNA damage.

Genes & development : 220-5 : DOI : 10.1101/gad.607011 En savoir plus
Résumé

Following DNA damage, mRNA 3′-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3′-end formation when normal mechanisms of pre-mRNA 3′-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3′-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.

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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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