UMR3244 – Dynamics of Genetic Information

Team Publications

Year of publication 2020

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 Learn more
Summary

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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Year of 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 Learn more
Summary

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 Learn more
Summary

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 Learn more
Summary

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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Ugo Szachnowski, Sara Andus, Dominika Foretek, Antonin Morillon, Maxime Wery (2019 Aug 30)

Endogenous RNAi pathway evolutionarily shapes the destiny of the antisense lncRNAs transcriptome.

Life science alliance : DOI : e201900407 Learn more
Summary

Antisense long noncoding (aslnc)RNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast , lacking RNAi. Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast , Dicer only affecting Xrn1-sensitive aslncRNAs levels in Xrn1-deficient cells. The and mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for Xrn1-sensitive aslncRNAs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in compared with , suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.

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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 Learn more
Summary

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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Brison O., EL-Hilali S., Azar D., Koundrioukoff S., Schmidt M., Naehse-Kumpf V., Jaszczyszyn Y., Lachages A.M., Dutrillaux B., Thermes C., Debatisse M., Chen C.L. (2019 Jul 1)

TRANSCRIPTION-MEDIATED ORGANIZATION OF THE REPLICATION INITIATION PROGRAM ACROSS LARGE GENES SETS UP COMMON FRAGILE SITES GENOME-WIDE

bioRxiv : 714717 : DOI : 10.1101/714717 Learn more
Summary

Common Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts 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, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

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Alexandra Pyatnitskaya, Valérie Borde, Arnaud De Muyt (2019 Jun 26)

Crossing and zipping: molecular duties of the ZMM proteins in meiosis.

Chromosoma : DOI : 10.1007/s00412-019-00714-8 Learn more
Summary

Accurate segregation of homologous chromosomes during meiosis depends on the ability of meiotic cells to promote reciprocal exchanges between parental DNA strands, known as crossovers (COs). For most organisms, including budding yeast and other fungi, mammals, nematodes, and plants, the major CO pathway depends on ZMM proteins, a set of molecular actors specifically devoted to recognize and stabilize CO-specific DNA intermediates that are formed during homologous recombination. The progressive implementation of ZMM-dependent COs takes place within the context of the synaptonemal complex (SC), a proteinaceous structure that polymerizes between homologs and participates in close homolog juxtaposition during prophase I of meiosis. While SC polymerization starts from ZMM-bound sites and ZMM proteins are required for SC polymerization in budding yeast and the fungus Sordaria, other organisms differ in their requirement for ZMM in SC elongation. This review provides an overview of ZMM functions and discusses their collaborative tasks for CO formation and SC assembly, based on recent findings and on a comparison of different model organisms.

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Karen Voelkel-Meiman, Shun-Yun Cheng, Melanie Parziale, Savannah J Morehouse, Arden Feil, Owen R Davies, Arnaud de Muyt, Valérie Borde, Amy J MacQueen (2019 Jun 21)

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein.

PLoS genetics : e1008201 : DOI : 10.1371/journal.pgen.1008201 Learn more
Summary

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.

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Antonin Morillon, Daniel Gautheret (2019 Jun 5)

Bridging the gap between reference and real transcriptomes.

Genome biology : 112 : DOI : 10.1186/s13059-019-1710-7 Learn more
Summary

Genetic, transcriptional, and post-transcriptional variations shape the transcriptome of individual cells, rendering establishing an exhaustive set of reference RNAs a complicated matter. Current reference transcriptomes, which are based on carefully curated transcripts, are lagging behind the extensive RNA variation revealed by massively parallel sequencing. Much may be missed by ignoring this unreferenced RNA diversity. There is plentiful evidence for non-reference transcripts with important phenotypic effects. Although reference transcriptomes are inestimable for gene expression analysis, they may turn limiting in important medical applications. We discuss computational strategies for retrieving hidden transcript diversity.

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Chakraborty A., Jenjaroenpun P., McCulley A., Li J., Hilali S.E., Haarer B., Hoffman E.A., Belak A., Thorland A., Hehnly H., Chen C.l., Kuznetsov V., Feng W. (2019 Jun 1)

Fragile X Mental Retardation Protein regulates R-loop formation and prevents global chromosome fragility

bioRxiv : 601906 : DOI : 10.1101/601906 Learn more
Summary

Fragile X syndrome (FXS) is the most prevalent inherited intellectual disability caused by mutations in the Fragile X Mental Retardation gene (FMR1) and deficiency of its product, FMRP. FMRP is a predominantly cytoplasmic protein thought to bind specific mRNA targets and regulate protein translation. Its potential role in the nucleus is not well understood. We are interested in the global impact on chromosome stability due to FMRP loss. Here we report that compared to an FMRP-proficient normal cell line, cells derived from FXS patients exhibit increased chromosome breaks upon DNA replication stress induced by a DNA polymerase inhibitor, aphidicolin. Moreover, cells from FXS individuals fail to protect genomic regions containing R-loops (co-transcriptional DNA:RNA hybrids) from aphidicolin-induced chromosome breaks. We demonstrate that FMRP is important for abating R-loop accumulation during transcription, particularly in the context of head-on collision with a replication fork, and thereby preventing chromosome breakage. By identifying those FMRP-bound chromosomal loci with overlapping R-loops and fragile sites, we report a list of novel FMRP target loci, many of which have been implicated in neurological disorders. We show that cells from FXS patients have reduced expression of xenobiotics metabolic enzymes, suggesting defective xenobiotics metabolism/excretion might contribute to disease development. Our study provides new insights into the etiological basis of, and enables the discovery of new therapeutic targets for, the FXS.

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Emilia Puig Lombardi, Arturo Londoño-Vallejo, Alain Nicolas (2019 May 30)

Relationship Between G-Quadruplex Sequence Composition in Viruses and Their Hosts.

Molecules (Basel, Switzerland) : DOI : E1942 Learn more
Summary

A subset of guanine-rich nucleic acid sequences has the potential to fold into G-quadruplex (G4) secondary structures, which are functionally important for several biological processes, including genome stability and regulation of gene expression. Putative quadruplex sequences (PQSs) GNGNGNG are widely found in eukaryotic and prokaryotic genomes, but the base composition of the N loops is biased across species. Since the viruses partially hijack their hosts’ cellular machinery for proliferation, we examined the PQS motif size, loop length, and nucleotide compositions of 7370 viral genome assemblies and compared viral and host PQS motifs. We studied seven viral taxa infecting five distant eukaryotic hosts and created a resource providing a comprehensive view of the viral quadruplex motifs. Overall, short-looped PQSs are predominant and with a similar composition across viral taxonomic groups, albeit subtle trends emerge upon classification by hosts. Specifically, there is a higher frequency of pyrimidine loops in viruses infecting animals irrespective of the viruses’ genome type. This observation is confirmed by an in-depth analysis of the Herpesviridae family of viruses, which showed a distinctive accumulation of thermally stable C-looped quadruplexes in viruses infecting high-order vertebrates. The occurrence of viral C-looped G4s, which carry binding sites for host transcription factors, as well as the high prevalence of viral TTA-looped G4s, which are identical to vertebrate telomeric motifs, provide concrete examples of how PQSs may help viruses impinge upon, and benefit from, host functions. More generally, these observations suggest a co-evolution of virus and host PQSs, thus underscoring the potential functional significance of G4s.

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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 Learn more
Summary

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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Ming-Jun Shi, Xiang-Yu Meng, Philippe Lamy, A Rouf Banday, Jie Yang, Aura Moreno-Vega, Chun-Long Chen, Lars Dyrskjøt, Isabelle Bernard-Pierrot, Ludmila Prokunina-Olsson, François Radvanyi (2019 Apr 13)

APOBEC-mediated Mutagenesis as a Likely Cause of FGFR3 S249C Mutation Over-representation in Bladder Cancer.

European urology : 9-13 : DOI : S0302-2838(19)30261-1 Learn more
Summary

FGFR3 is one of the most frequently mutated genes in bladder cancer and a driver of an oncogenic dependency. Here we report that only the most common recurrent FGFR3 mutation, S249C (TCC→TGC), represents an APOBEC-type motif and is probably caused by the APOBEC-mediated mutagenic process, accounting for its over-representation. We observed significant enrichment of the APOBEC mutational signature and overexpression of AID/APOBEC gene family members in bladder tumors with S249C compared to tumors with other recurrent FGFR3 mutations. Analysis of replication fork directionality suggests that the coding strand of FGFR3 is predominantly replicated as a lagging strand template that could favor the formation of hairpin structures, facilitating mutagenic activity of APOBEC enzymes. In vitro APOBEC deamination assays confirmed S249 as an APOBEC target. We also found that the FGFR3 S249C mutation was common in three other cancer types with an APOBEC mutational signature, but rare in urothelial tumors without APOBEC mutagenesis and in two diseases probably related to aging. PATIENT SUMMARY: We propose that APOBEC-mediated mutagenesis can generate clinically relevant driver mutations even within suboptimal motifs, such as in the case of FGFR3 S249C, one of the most common mutations in bladder cancer. Knowledge about the etiology of this mutation will improve our understanding of the molecular mechanisms of bladder cancer.

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Aria Ronsmans, Maxime Wery, Ugo Szachnowski, Camille Gautier, Marc Descrimes, Evelyne Dubois, Antonin Morillon, Isabelle Georis (2019 Mar 1)

Transcription-dependent spreading of the Dal80 yeast GATA factor across the body of highly expressed genes.

PLoS genetics : e1007999 : DOI : 10.1371/journal.pgen.1007999 Learn more
Summary

GATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well defined in vitro, the in vivo selectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites, correlating with nitrogen- and/or Dal80-sensitive gene expression. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies requires active transcription. Consistently, Dal80 co-immunoprecipitated with the initiating and post-initiation forms of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.

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