UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2009

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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier

Année de publication : 2008

Peter Bieling, Stefanie Kandels-Lewis, Ivo A Telley, Juliette van Dijk, Carsten Janke, Thomas Surrey (2008 Dec 29)

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites.

The Journal of cell biology : 1223-33 : DOI : 10.1083/jcb.200809190 En savoir plus
Résumé

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end-binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated alpha-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

Replier
Anne Cammas, Stephen M Lewis, Stephan Vagner, Martin Holcik (2008 Dec 1)

Post-transcriptional control of gene expression through subcellular relocalization of mRNA binding proteins.

Biochemical pharmacology : 1395-403 : DOI : 10.1016/j.bcp.2008.05.022 En savoir plus
Résumé

Eukaryotic cells have developed multiple mechanisms to respond to different physiological cues, such as cellular stress, which allow the cells to adapt themselves to their new environment. The regulation of post-transcriptional gene expression is an important component of the cellular stress response and is mediated by mRNA binding proteins (mRBPs). Recently, several studies have shown that regulated subcellular localization of mRBPs upon diverse stimuli (such as cellular stress) provides an additional level of regulation for gene expression.

Replier
X Mergui, F Leteurtre, M Lipinski, J Bénard, M Amor-Guéret (2008 Nov 1)

Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma.

Biochimie : 1656-66 : DOI : 10.1016/j.biochi.2008.06.008 En savoir plus
Résumé

Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.

Replier
Nuno R dos Santos, Maryvonne Williame, Stéphanie Gachet, Françoise Cormier, Anne Janin, Debra Weih, Falk Weih, Jacques Ghysdael (2008 Jul 2)

RelB-dependent stromal cells promote T-cell leukemogenesis.

PloS one : e2555 : DOI : 10.1371/journal.pone.0002555 En savoir plus
Résumé

The Rel/NF-kappaB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-kappaB activation is found in malignant cells and results from activation of the canonical NF-kappaB pathway, leading to RelA and/or c-Rel activation. Recently, NF-kappaB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-kappaB activation, which leads to RelB activation, has also been reported in breast carcinoma, prostate cancer, and lymphoid leukemia.

Replier
Carsten Janke, Krzysztof Rogowski, Juliette van Dijk (2008 Jul 1)

Polyglutamylation: a fine-regulator of protein function? ‘Protein Modifications: beyond the usual suspects’ review series.

EMBO reports : 636-41 : DOI : 10.1038/embor.2008.114 En savoir plus
Résumé

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.

Replier
Mounira Amor-Guéret, Catherine Dubois-d'Enghien, Anthony Laugé, Rosine Onclercq-Delic, Abdelhamid Barakat, Elbekkay Chadli, Ahmed Aziz Bousfiha, Meriem Benjelloun, Elisabeth Flori, Bérénice Doray, Vincent Laugel, Maria Teresa Lourenço, Rui Gonçalves, Silvia Sousa, Jérôme Couturier, Dominique Stoppa-Lyonnet (2008 Jun 1)

Three new BLM gene mutations associated with Bloom syndrome.

Genetic testing : 257-61 : DOI : 10.1089/gte.2007.0119 En savoir plus
Résumé

Bloom’s syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

Replier
Nassima Temime-Smaali, Lionel Guittat, Thomas Wenner, Emilie Bayart, Céline Douarre, Dennis Gomez, Marie-Josèphe Giraud-Panis, Arturo Londono-Vallejo, Eric Gilson, Mounira Amor-Guéret, Jean-François Riou (2008 May 21)

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres.

The EMBO journal : 1513-24 : DOI : 10.1038/emboj.2008.74 En savoir plus
Résumé

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.

Replier
Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, Bernard Edde, Carsten Janke (2008 Feb 15)

Polyglutamylation is a post-translational modification with a broad range of substrates.

The Journal of biological chemistry : 3915-22 : DOI : 10.1074/jbc.M705813200 En savoir plus
Résumé

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.

Replier
Benoît Froget, Joël Blaisonneau, Sarah Lambert, Giuseppe Baldacci (2008 Feb 1)

Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint.

Molecular biology of the cell : 445-56 : DOI : 10.1091/mbc.E07-07-0728 En savoir plus
Résumé

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

Replier
Hind Medyouf, Jacques Ghysdael (2008 Feb 1)

The calcineurin/NFAT signaling pathway: a novel therapeutic target in leukemia and solid tumors.

Cell cycle (Georgetown, Tex.) : 297-303 : DOI : 10.4161/cc.7.3.5357 En savoir plus
Résumé

The calcineurin/NFAT signaling pathway is unique to vertebrates and clear genetic evidences show that it plays critical roles in orchestrating the intricate cellular interactions that characterize vertebrate development and morphogenesis. In this setting, the transcriptional regulators of the NFAT family function as molecular integrators of specific calcium signals with other signaling pathways, including MAPkinase, WNT or NOTCH. Deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components have recently been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Our studies in mouse models of human T-ALL/lymphoma shows that persistent activation of calcineurin/NFAT signaling is pro-oncogenic in vivo and can be efficiently targeted by well-characterized calcineurin inhibitors. We further discuss facts and hypotheses concerning the molecular events that may act upstream and downstream of calcineurin and/or NFAT activation in different type of cancer cells.

Replier