UMR3244 – Dynamique de l’information génétique

Publications de l’équipe

Année de publication : 2020

Arijita Chakraborty, Piroon Jenjaroenpun, Jing Li, Sami El Hilali, Andrew McCulley, Brian Haarer, Elizabeth A Hoffman, Aimee Belak, Audrey Thorland, Heidi Hehnly, Carl Schildkraut, Chun-Long Chen, Vladimir A Kuznetsov, Wenyi Feng (2020 Sep 23)

Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome.

Cell reports : 108179 : DOI : S2211-1247(20)31168-2 En savoir plus
Résumé

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

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Zhiming Li, Xu Hua, Albert Serra-Cardona, Xiaowei Xu, Songlin Gan, Hui Zhou, Wen-Si Yang, Chun-Long Chen, Rui-Ming Xu, Zhiguo Zhang (2020 Sep 14)

DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands.

Science advances : eabb5820 : DOI : 10.1126/sciadv.abb5820 En savoir plus
Résumé

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication-linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

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Sanchez Aurore, Borde Valérie (2020 Sep 1)

Methods to Map Meiotic Recombination Proteins in Saccharomyces cerevisiae

Methods in Molecular BiologyHomologous Recombination : 2153 : 295-306 : DOI : 10.1007/978-1-0716-0644-5_21 En savoir plus
Résumé

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), catalyzed by the type II topoisomerase-like Spo11 protein. Meiotic DSBs are repaired by homologous recombination, which produces either crossovers or noncrossovers, this decision being linked to the binding of proteins specific of each pathway. Mapping the binding of these proteins along chromosomes in wild type or mutant yeast background is extremely useful to understand how and at which step the decision to repair a DSB with a crossover is taken. It is now possible to obtain highly synchronous yeast meiotic populations, which, combined with appropriate negative controls, enable to detect by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) the transient binding of diverse recombination proteins with high sensitivity and resolution.

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Gnan Stefano, Liu Yaqun, Spagnuolo Manuela, Chen Chun-Long (2020 Aug 31)

The impact of transcription-mediated replication stress on genome instability and human disease

Genome Instability & Disease : 1 : 207-234 : DOI : 10.1007/s42764-020-00021-y En savoir plus
Résumé

DNA replication is a vital process in all living organisms. At each cell division, > 30,000 replication origins are activated in a coordinated manner to ensure the duplication of > 6 billion base pairs of the human genome. During differentiation and development, this program must adapt to changes in chromatin organization and gene transcription: its deregulation can challenge genome stability, which is a leading cause of many diseases including cancers and neurological disorders. Over the past decade, great progress has been made to better understand the mechanisms of DNA replication regulation and how its deregulation challenges genome integrity and leads to human disease. Growing evidence shows that gene transcription has an essential role in shaping the landscape of genome replication, while it is also a major source of endogenous replication stress inducing genome instability. In this review, we discuss the current knowledge on the various mechanisms by which gene transcription can impact on DNA replication, leading to genome instability and human disease.

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Elda Cannavo, Aurore Sanchez, Roopesh Anand, Lepakshi Ranjha, Jannik Hugener, Céline Adam, Ananya Acharya, Nicolas Weyland, Xavier Aran-Guiu, Jean-Baptiste Charbonnier, Eva R Hoffmann, Valérie Borde, Joao Matos, Petr Cejka (2020 Aug 21)

Regulation of the MLH1-MLH3 endonuclease in meiosis.

Nature : DOI : 10.1038/s41586-020-2592-2 En savoir plus
Résumé

During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes.

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Alexy Promonet, Ismaël Padioleau, Yaqun Liu, Lionel Sanz, Anna Biernacka, Anne-Lyne Schmitz, Magdalena Skrzypczak, Amélie Sarrazin, Clément Mettling, Maga Rowicka, Krzysztof Ginalski, Frédéric Chedin, Chun-Long Chen, Yea-Lih Lin, Philippe Pasero (2020 Aug 10)

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

Nature communications : 3940 : DOI : 10.1038/s41467-020-17858-2 En savoir plus
Résumé

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

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Eléonore Toufektchan, Vincent Lejour, Romane Durand, Neelam Giri, Irena Draskovic, Boris Bardot, Pierre Laplante, Sara Jaber, Blanche P Alter, José-Arturo Londono-Vallejo, Sharon A Savage, Franck Toledo (2020 Apr 18)

Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance.

Science advances : eaay3511 : DOI : 10.1126/sciadv.aay3511 En savoir plus
Résumé

Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of , a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.

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Franck Toledo and Michelle Debatisse (2020 Apr 17)

Mechanisms generating cancer genome complexity : a look back at the interphase breakage model

Science : Vol. 368, Issue 6488 : eaba0712 : DOI : 10.1126/science.aba0712 En savoir plus
Résumé

Understanding the mechanims responsible for cancer genome complexity has been an important goal for many decades. Umbreit et al. recently combined live cell imaging and single cell genome sequencing to analyze the cascade of genome rearrangements following the formation of a chromosome bridge in human cells (1). Their results suggest that this bridge leads to an initial breakage-fusion-bridge (BFB) cycle, followed by additional BFB cycles interwoven with episodes of micronucleation and chromothripsis, to generate complex genome rearrangements (1).
This conclusion is strikingly consistent with the previously proposed “interphase breakage model” (2). In the 1990s, fluorescent in situ hybridization (FISH) provided unprecedented insights into the mechanisms underlying gene amplification in mammalian cells. FISH made it possible to analyze the distribution of amplified genes at the level of a single cell, just a few cell divisions after initiation of the amplification process, in model systems of cultured cells selected for resist

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Année de publication : 2019

Olivier Brison, Sami El-Hilali, Dana Azar, Stéphane Koundrioukoff, Mélanie Schmidt, Viola Nähse, Yan Jaszczyszyn, Anne-Marie Lachages, Bernard Dutrillaux, Claude Thermes, Michelle Debatisse, Chun-Long Chen (2019 Dec 15)

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Nature communications : 5693 : DOI : 10.1038/s41467-019-13674-5 En savoir plus
Résumé

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

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Mireille Bétermier, Valérie Borde, Jean-Pierre de Villartay (2019 Dec 11)

Coupling DNA Damage and Repair: an Essential Safeguard during Programmed DNA Double-Strand Breaks?

Trends in cell biology : DOI : S0962-8924(19)30201-6 En savoir plus
Résumé

DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the ‘price to pay’ to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.

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(2019 Nov 15)

Reference-free transcriptome exploration reveals novel RNAs for prostate cancer diagnosis.

Life Sci Alliance : DOI : 10.26508/lsa.201900449 En savoir plus
Résumé

The use of RNA-sequencing technologies held a promise of improved diagnostic tools based on comprehensive transcript sets. However, mining human transcriptome data for disease biomarkers in clinical specimens are restricted by the limited power of conventional reference-based protocols relying on unique and annotated transcripts. Here, we implemented a blind reference-free computational protocol, DE-kupl, to infer yet unreferenced RNA variations from total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into putative long noncoding (lnc)RNAs expressed in prostate cancer. Through filtering of 1,179 candidates, we defined 21 lncRNAs that were further validated by NanoString for robust tumor-specific expression in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling more than 90% of true-positive detections of cancer in an independent The Cancer Genome Atlas cohort. Remarkably, this clinical signature made of only nine unannotated lncRNAs largely outperformed PCA3, the only used prostate cancer lncRNA biomarker, in detection of high-risk tumors. This modular workflow is highly sensitive and can be applied to any pathology or clinical application.

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Emilia Puig Lombardi, Allyson Holmes, Daniela Verga, Marie-Paule Teulade-Fichou, Alain Nicolas, Arturo Londoño-Vallejo (2019 Jul 9)

Thermodynamically stable and genetically unstable G-quadruplexes are depleted in genomes across species.

Nucleic acids research : 47 : 6098-6113 : DOI : 10.1093/nar/gkz463 En savoir plus
Résumé

G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.

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Kevin Grosselin, Adeline Durand, Justine Marsolier, Adeline Poitou, Elisabetta Marangoni, Fariba Nemati, Ahmed Dahmani, Sonia Lameiras, Fabien Reyal, Olivia Frenoy, Yannick Pousse, Marcel Reichen, Adam Woolfe, Colin Brenan, Andrew D. Griffiths*, Céline Vallot* & Annabelle Gérard* (2019 May 31)

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer

Nature Genetics : 1060–1066 : DOI : 10.1038/s41588-019-0424-9 En savoir plus
Résumé

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(2019 May 1)

Long non-coding RNAs: towards urinary diagnosis for prostate cancer

Bulletin de l'Académie Nationale de Médecine : DOI : https://doi.org/10.1016/j.banm.2018.03.001 En savoir plus
Résumé

With nearly 54,000 new cases per year in France, prostate cancer is the most common cancer in men and is the third leading cause of cancer deaths. Nowadays, the early diagnosis of prostate cancer is done by a blood test of the Prostate Specific Antigen marker (PSA) and a digital rectal examination. However, the diagnosis is based on prostate biopsies that can be sources of infection (less than 5% risk) and can be negative in 55% of cases. The search for new, more robust markers is therefore necessary. High-throughput sequencing of the human genome and transcriptome combined with bioinformatics has completely changed understanding of the genome’s organization. Only 2% of the genome is transcribed into proteins-encoding mRNA, 66% into non-coding RNA, including long non-coding RNAs (lncRNAs). These RNA, of more than 200 nucleotides, are specific for a given cell or tissue and may have oncogenic or tumor suppressive functions. They can also be diagnostic and prognostic biomarkers and therapeutic targets in oncology. Among the different classes of lncRNA, the antisense transcripts (aslncRNA), encoded by the DNA strand complementary to that of a mRNA, are the least described. We have identified, by high-throughput sequencing, many non-annotated aslncRNAs, three of which were very significantly increased in prostate tumors compared to normal prostate tissues. This result has been validated by a hybridization technique (NanoString) on a cohort of 166 tumors and a preliminary study on urine from patients with prostate cancer seems very promising. Our purpose is to develop a specific, non-invasive, early, rapid and robust urinary test to diagnose prostate cancer or to direct patients to biopsies with much greater relevance.

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Année de publication : 2018

Antoine Hocher, Myriam Ruault, Petra Kaferle, Marc Descrimes, Mickaël Garnier, Antonin Morillon, Angela Taddei (2018 Oct 26)

Expanding heterochromatin reveals discrete subtelomeric domains delimited by chromatin landscape transitions.

Genome research : DOI : gr.236554.118 En savoir plus
Résumé

The eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast fly and human. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at varying levels in yeast and found that Sir3 spreads into Extended Silent Domains (ESDs), eventually reaching saturation at subtelomeres. We observed the spread of Sir3 into subtelomeric domains associated with specific histone marks in wild-type cells and stopping at zones of histone mark transitions including H3K79 tri-methylation levels. Our study shows that the conserved H3K79 methyltransferase Dot1 is essential in restricting Sir3 spread beyond ESDs, thus ensuring viability upon overexpression of Sir3. Lastly, our analyses of published data demonstrate how ESDs unveil uncharacterized discrete domains isolating structural and functional subtelomeric features from the rest of the genome. Our work offers a new approach on how to separate subtelomeres from the core chromosome.

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