UMR3244 – Dynamique de l’information génétique

Publications de l’équipe

Année de publication : 2011

Mathieu Tisseur, Marta Kwapisz, Antonin Morillon (2011 May 3)

Pervasive transcription – Lessons from yeast.

Biochimie : 1889-96 : DOI : 10.1016/j.biochi.2011.07.001 En savoir plus
Résumé

Pervasive transcription is now accepted to be a general feature of eukaryotic genomes, generating short and long non-coding RNAs (ncRNAs). Growing number of examples have shown that regulatory ncRNAs can control gene expression and chromatin domain formation. In this review, we discuss recent reports that show that Saccharomyces cerevisiae’s genome also supports pervasive transcription, which is strongly controlled by RNA decay pathways and nucleosome positioning. We therefore propose that S. cerevisiae is an excellent model for studying large ncRNAs, which has already provided important examples of antisense-mediated transcriptional silencing.

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Emmanuelle Martini, Valérie Borde, Matthieu Legendre, Stéphane Audic, Béatrice Regnault, Guillaume Soubigou, Bernard Dujon, Bertrand Llorente (2011 Apr 28)

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

PLoS genetics : e1002305 : DOI : 10.1371/journal.pgen.1002305 En savoir plus
Résumé

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

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Isabelle Ourliac-Garnier, Arturo Londoño-Vallejo (2011 Apr 5)

Telomere strand-specific length analysis by fluorescent in situ hybridization (Q-CO-FISH).

Methods in molecular biology (Clifton, N.J.) : 33-46 : DOI : 10.1007/978-1-61779-092-8_4 En savoir plus
Résumé

The implementation of quantitative approaches in telomere chromosome-oriented FISH (telomeric CO-FISH) allows the assessment of the relative efficiency of lagging versus leading strand telomere replication and thus provides information on the implicated mechanisms. Here, we describe a simple method for telomere strand-specific analyses and discuss its potential applications.

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Isabelle Ourliac-Garnier, Arturo Londoño-Vallejo (2011 Apr 5)

Telomere length analysis by quantitative fluorescent in situ hybridization (Q-FISH).

Methods in molecular biology (Clifton, N.J.) : 21-31 : DOI : 10.1007/978-1-61779-092-8_3 En savoir plus
Résumé

Length is a functional parameter of telomeres, the nucleoprotein structures that protect chromosome ends. The availability of highly specific, high-affinity probes for telomeric repeated sequences allowed the development of quantitative approaches aimed at measuring telomere length directly on chromosomes or in interphase nuclei. Here, we describe a general method for telomere quantitative FISH on metaphase chromosomes and discuss its most common applications in research.

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Année de publication : 2010

E L van Dijk, C L Chen, Y d'Aubenton-Carafa, S Gourvennec, M Kwapisz, V Roche, C Bertrand, M Silvain, P Legoix-Né, S Loeillet, A Nicolas, C Thermes, A Morillon (2010 Dec 31)

XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast.

Nature : 114-7 : DOI : 10.1038/nature10118 En savoir plus
Résumé

Non-coding (nc)RNAs are key players in numerous biological processes such as gene regulation, chromatin domain formation and genome stability. Large ncRNAs interact with histone modifiers and are involved in cancer development, X-chromosome inactivation and autosomal gene imprinting. However, despite recent evidence showing that pervasive transcription is more widespread than previously thought, only a few examples mediating gene regulation in eukaryotes have been described. In Saccharomyces cerevisiae, the bona-fide regulatory ncRNAs are destabilized by the Xrn1 5′-3′ RNA exonuclease (also known as Kem1), but the genome-wide characterization of the entire regulatory ncRNA family remains elusive. Here, using strand-specific RNA sequencing (RNA-seq), we identify a novel class of 1,658 Xrn1-sensitive unstable transcripts (XUTs) in which 66% are antisense to open reading frames. These transcripts are polyadenylated and RNA polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in lithium-containing media, indicating that they might have a role in adaptive responses to changes in growth conditions. Notably, RNAPII chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis of Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over 273 genes with antisense XUTs. These genes show an unusual bias for H3K4me3 marks and require the Set1 histone H3 lysine 4 methyl-transferase for silencing. Furthermore, abolishing H3K4me3 triggers the silencing of other genes with antisense XUTs, supporting a model in which H3K4me3 antagonizes antisense ncRNA repressive activity. Our results demonstrate that antisense ncRNA-mediated regulation is a general regulatory pathway for gene expression in S. cerevisiae.

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Céline Vallot, Nicolas Stransky, Isabelle Bernard-Pierrot, Aurélie Hérault, Jessica Zucman-Rossi, Elodie Chapeaublanc, Dimitri Vordos, Agnès Laplanche, Simone Benhamou, Thierry Lebret, Jennifer Southgate, Yves Allory, François Radvanyi (2010 Dec 20)

A novel epigenetic phenotype associated with the most aggressive pathway of bladder tumor progression.

Journal of the National Cancer Institute : 47-60 : DOI : 10.1093/jnci/djq470 En savoir plus
Résumé

Epigenetic silencing can extend to whole chromosomal regions in cancer. There have been few genome-wide studies exploring its involvement in tumorigenesis.

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N S Zhdanova, Iu M Minina, T V Karamysheva, N B Rubtsov, J-A Londono-Vallejo (2010 Nov 11)

[The structure of long telomeres in chromosomes of the Iberian shrew].

Genetika : 1222-5 En savoir plus
Résumé

It is shown that the size, localization, and structure of telomeres in the Iberian shrew (Sorex granarius) are not characteristic of mammals. In this species, long telomeres of an average size of 213 kb are localized on the short arms of all 32 acrocentrics; ribosomal blocks and active nucleolus-organizing regions (NORs) were also discovered there. At the remaining chromosome ends the average size of telomeres is 3.8 kb. However, in a closely related species, Sorex araneus, all telomeres have size similar to that of human telomeres, i.e., 6.8-15.2 kb. Despite the fact that some long telomeres contain ribosomal repeats in addition to telomeric ones, the long telomeres have preserved asymmetry of G- and C-rich strands as in functional telomeres. It is probable that long telomeres were formed in meiosis at the stage of chromosome bouquet as a result of global reorganization of the chromosome ends. The provoking factors for such reorganization might be the fission of several metacentrics and the necessity of telomerization of the resulting acrocentrics.

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Jing Ye, Christelle Lenain, Serge Bauwens, Angela Rizzo, Adelaïde Saint-Léger, Anaïs Poulet, Delphine Benarroch, Frédérique Magdinier, Julia Morere, Simon Amiard, Els Verhoeyen, Sébastien Britton, Patrick Calsou, Bernard Salles, Anna Bizard, Marc Nadal, Erica Salvati, Laure Sabatier, Yunlin Wu, Annamaria Biroccio, Arturo Londoño-Vallejo, Marie-Josèphe Giraud-Panis, Eric Gilson (2010 Jul 27)

TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage.

Cell : 230-42 : DOI : 10.1016/j.cell.2010.05.032 En savoir plus
Résumé

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5′ exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres.

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Sophie Georgin-Lavialle, Achille Aouba, Luc Mouthon, J Arturo Londono-Vallejo, Yves Lepelletier, Anne-Sophie Gabet, Olivier Hermine (2010 May 4)

The telomere/telomerase system in autoimmune and systemic immune-mediated diseases.

Autoimmunity reviews : 646-51 : DOI : 10.1016/j.autrev.2010.04.004 En savoir plus
Résumé

Telomeres are specialized nucleoproteic structures that cap and protect the ends of chromosomes. They can be elongated by the telomerase enzyme, but in telomerase negative cells, telomeres shorten after each cellular division because of the end replicating problem. This phenomenon leads ultimately to cellular senescence, conferring to the telomeres a role of biological clock. Oxidative stress, inflammation and increased cell renewal are supplementary environmental factors that accelerate age-related telomere shortening. Similar to other types of DNA damage, very short/dysfunctional telomeres activate a DNA response pathway leading to different outcomes: DNA repair, cell senescence or apoptosis. During the last 10 years, studies on the telomere/telomerase system in autoimmune and/or systemic immune-mediated diseases have revealed its involvement in relevant physiopathological processes. Here, we present a literature review of telomere and telomerase homeostasis in systemic inflammatory diseases including systemic lupus erythematosus, rheumatoid arthritis and granulomatous diseases. The available data indicate that both telomerase activity and telomere length are modified in various systemic immune-mediated diseases and appear to be connected with premature immunosenescence. Studies on the telomere/telomerase system open new research avenues for the basic understanding and for therapeutic approaches of these pathologies.

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Johans Fakhoury, Delphine Tamara Marie-Egyptienne, José-Arturo Londoño-Vallejo, Chantal Autexier (2010 Apr 30)

Telomeric function of mammalian telomerases at short telomeres.

Journal of cell science : 1693-704 : DOI : 10.1242/jcs.063636 En savoir plus
Résumé

Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) known as TERT and an RNA known as TR. We reconstituted heterologous mouse (m) and human (h) TERT-TR complexes and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component. The presence of hTERT-hTR, but not heterologous TERT-TR complexes or chimeric mTERT-hTERT-hTR complexes, significantly reduced the percentage of chromosomes without telomeric signals in ALT cells. Moreover, heterologous and chimeric complexes were defective in recruitment to telomeres. Our results suggest a requirement for several hTERT domains and interaction with multiple proteins for proper recruitment of telomerase to the shortest telomeres in human ALT cells. Late-passage mTERT(-/-) mouse embryonic stem (ES) cells ectopically expressing hTERT or mTERT harboured fewer chromosome ends without telomeric signals and end-to-end fusions than typically observed in late-passage mTERT(-/-) ES cells. The ability of hTERT to function at mouse telomeres and the inability of mTERT to function at human telomeres suggest that mechanisms regulating the recruitment and activity of hTERT at mouse telomeres might be less stringent than the mechanisms regulating mTERT at human telomeres.

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Nausica Arnoult, Caroline Schluth-Bolard, Anne Letessier, Irena Drascovic, Rachida Bouarich-Bourimi, Judith Campisi, Sahn-Ho Kim, Amina Boussouar, Alexandre Ottaviani, Frédérique Magdinier, Eric Gilson, Arturo Londoño-Vallejo (2010 Apr 28)

Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear localization.

PLoS genetics : e1000920 : DOI : 10.1371/journal.pgen.1000920 En savoir plus
Résumé

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

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Année de publication : 2009

Nausica Arnoult, Carole Saintome, Isabelle Ourliac-Garnier, Jean-François Riou, Arturo Londoño-Vallejo (2009 Dec 17)

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN.

Genes & development : 2915-24 : DOI : 10.1101/gad.544009 En savoir plus
Résumé

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.

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J Arturo Londoño-Vallejo (2009 Dec 3)

[Hundred-years-old Nobel celebrates telomers and telomerase].

Médecine sciences : M/S : 973-6 : DOI : 10.1051/medsci/20092511973 En savoir plus
Résumé

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Hajime Murakami, Valérie Borde, Alain Nicolas, Scott Keeney (2009 Oct 6)

Gel electrophoresis assays for analyzing DNA double-strand breaks in Saccharomyces cerevisiae at various spatial resolutions.

Methods in molecular biology (Clifton, N.J.) : 117-42 : DOI : 10.1007/978-1-59745-527-5_9 En savoir plus
Résumé

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-labeling) applied to samples of genomic DNA from sporulating cultures of budding and fission yeast. Such assays are extremely useful for quantifying and characterizing many aspects of the initiation of meiotic recombination, including the timing of DSB formation relative to other events, the distribution of DSBs across the genome, and the influence on DSB formation of mutations in recombination factors and other gene products. By varying the type of gel electrophoresis and other parameters, the spatial resolution of DSB analysis can range from single nucleotides up to whole yeast chromosomes.

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Irena Draskovic, Nausica Arnoult, Villier Steiner, Silvia Bacchetti, Patrick Lomonte, Arturo Londoño-Vallejo (2009 Sep 1)

Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination.

Proceedings of the National Academy of Sciences of the United States of America : 15726-31 : DOI : 10.1073/pnas.0907689106 En savoir plus
Résumé

Promyelocytic leukemia (PML) bodies (also called ND10) are dynamic nuclear structures implicated in a wide variety of cellular processes. ALT-associated PML bodies (APBs) are specialized PML bodies found exclusively in telomerase-negative tumors in which telomeres are maintained by recombination-based alternative (ALT) mechanisms. Although it has been suggested that APBs are directly implicated in telomere metabolism of ALT cells, their precise role and structure have remained elusive. Here we show that PML bodies in ALT cells associate with chromosome ends forming small, spatially well-defined clusters, containing on average 2-5 telomeres. Using an innovative approach that gently enlarges PML bodies in living cells while retaining their overall organization, we show that this physical enlargement of APBs spatially resolves the single telomeres in the cluster, but does not perturb the potential of the APB to recruit chromosome extremities. We show that telomere clustering in PML bodies is cell-cycle regulated and that unique telomeres within a cluster associate with recombination proteins. Enlargement of APBs induced the accumulation of telomere-telomere recombination intermediates visible on metaphase spreads and connecting heterologous chromosomes. The strand composition of these recombination intermediates indicated that this recombination is constrained to a narrow time window in the cell cycle following replication. These data provide strong evidence that PML bodies are not only a marker for ALT cells but play a direct role in telomere recombination, both by bringing together chromosome ends and by promoting telomere-telomere interactions between heterologous chromosomes.

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