UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2010

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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Michael G Kemp, Aaron C Mason, Aura Carreira, Joyce T Reardon, Stuart J Haring, Gloria E O Borgstahl, Stephen C Kowalczykowski, Aziz Sancar, Marc S Wold (2010 Feb 12)

An alternative form of replication protein a expressed in normal human tissues supports DNA repair.

The Journal of biological chemistry : 4788-97 : DOI : 10.1074/jbc.M109.079418 En savoir plus
Résumé

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.

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Martin Dutertre, Magali Lacroix-Triki, Keltouma Driouch, Pierre de la Grange, Lise Gratadou, Samantha Beck, Stefania Millevoi, Jamal Tazi, Rosette Lidereau, Stephan Vagner, Didier Auboeuf (2010 Feb 1)

Exon-based clustering of murine breast tumor transcriptomes reveals alternative exons whose expression is associated with metastasis.

Cancer research : 896-905 : DOI : 10.1158/0008-5472.CAN-09-2703 En savoir plus
Résumé

In the field of bioinformatics, exon profiling is a developing area of disease-associated transcriptome analysis. In this study, we performed a microarray-based transcriptome analysis at the single exon level in mouse 4T1 primary mammary tumors with different metastatic capabilities. A novel bioinformatics platform was developed that identified 679 genes with differentially expressed exons in 4T1 tumors, many of which were involved in cell morphology and movement. Of 152 alternative exons tested by reverse transcription-PCR, 97 were validated as differentially expressed in primary tumors with different metastatic capability. This analysis revealed candidate progression genes, hinting at variations in protein functions by alternate exon usage. In a parallel effort, we developed a novel exon-based clustering analysis and identified alternative exons in tumor transcriptomes that were associated with dissemination of primary tumor cells to sites of pulmonary metastasis. This analysis also revealed that the splicing events identified by comparing primary tumors were not aberrant events. Lastly, we found that a subset of differentially spliced variant transcripts identified in the murine model was associated with poor prognosis in a large clinical cohort of patients with breast cancer. Our findings illustrate the utility of exon profiling to define novel theranostic markers for study in cancer progression and metastasis.

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Année de publication : 2009

Ken'Ichi Mizuno, Sarah Lambert, Giuseppe Baldacci, Johanne M Murray, Antony M Carr (2009 Dec 15)

Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism.

Genes & development : 2876-86 : DOI : 10.1101/gad.1863009 En savoir plus
Résumé

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.

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Aura Carreira, Stephen C Kowalczykowski (2009 Nov 1)

BRCA2: Shining light on the regulation of DNA-binding selectivity by RAD51.

Cell cycle (Georgetown, Tex.) : 3445-7 : DOI : 10.4161/cc.8.21.9748 En savoir plus
Résumé

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Gérard Maillot, Magali Lacroix-Triki, Sandra Pierredon, Lise Gratadou, Sabine Schmidt, Vladimir Bénès, Henri Roché, Florence Dalenc, Didier Auboeuf, Stefania Millevoi, Stéphan Vagner (2009 Nov 1)

Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth.

Cancer research : 8332-40 : DOI : 10.1158/0008-5472.CAN-09-2206 En savoir plus
Résumé

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17beta-estradiol (E(2)). We show that the expression of a broad set of miRNAs decreases following E(2) treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E(2)-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E(2)-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer.

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Stefania Millevoi, Adrien Decorsière, Clarisse Loulergue, Jason Iacovoni, Sandra Bernat, Michael Antoniou, Stéphan Vagner (2009 Aug 1)

A physical and functional link between splicing factors promotes pre-mRNA 3′ end processing.

Nucleic acids research : 4672-83 : DOI : 10.1093/nar/gkp470 En savoir plus
Résumé

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3′ end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3′ end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3′ UTR is abrogated by a 3′ end processing-inactivating mutation. We show that PTB promotes both in vitro 3′ end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

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Dorota Wloga, Danielle M Webster, Krzysztof Rogowski, Marie-Hélène Bré, Nicolette Levilliers, Maria Jerka-Dziadosz, Carsten Janke, Scott T Dougan, Jacek Gaertig (2009 Jun 15)

TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

Developmental cell : 867-76 : DOI : 10.1016/j.devcel.2009.04.008 En savoir plus
Résumé

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer’s vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

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Krzysztof Rogowski, François Juge, Juliette van Dijk, Dorota Wloga, Jean-Marc Strub, Nicolette Levilliers, Daniel Thomas, Marie-Hélène Bré, Alain Van Dorsselaer, Jacek Gaertig, Carsten Janke (2009 Jun 12)

Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation.

Cell : 1076-87 : DOI : 10.1016/j.cell.2009.05.020 En savoir plus
Résumé

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

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Stephanie Gachet, Jacques Ghysdael (2009 Jun 12)

Calcineurin/NFAT signaling in lymphoid malignancies.

General physiology and biophysics : F47-54 En savoir plus
Résumé

Deregulated calcium signaling is observed at different stages of tumorigenic processes. An important signaling pathway activated in response to calcium involves the protein phosphatase calcineurin and NFAT transcriptional factors. We review here recent data that indicate an important role of the calcineurin/NFAT pathway in lymphoma/leukemogenesis and discuss the potential therapeutic implications of these findings.

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Susannah L Hewitt, Bu Yin, Yanhong Ji, Julie Chaumeil, Katarzyna Marszalek, Jeannette Tenthorey, Giorgia Salvagiotto, Natalie Steinel, Laura B Ramsey, Jacques Ghysdael, Michael A Farrar, Barry P Sleckman, David G Schatz, Meinrad Busslinger, Craig H Bassing, Jane A Skok (2009 Jun 1)

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.

Nature immunology : 655-64 : DOI : 10.1038/ni.1735 En savoir plus
Résumé

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

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Christoph Maas, Dorthe Belgardt, Han Kyu Lee, Frank F Heisler, Corinna Lappe-Siefke, Maria M Magiera, Juliette van Dijk, Torben J Hausrat, Carsten Janke, Matthias Kneussel (2009 May 26)

Synaptic activation modifies microtubules underlying transport of postsynaptic cargo.

Proceedings of the National Academy of Sciences of the United States of America : 8731-6 : DOI : 10.1073/pnas.0812391106 En savoir plus
Résumé

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

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Aura Carreira, Jovencio Hilario, Ichiro Amitani, Ronald J Baskin, Mahmud K K Shivji, Ashok R Venkitaraman, Stephen C Kowalczykowski (2009 Mar 20)

The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51.

Cell : 1032-43 : DOI : 10.1016/j.cell.2009.02.019 En savoir plus
Résumé

The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifested by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA-binding selectivity.

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