UMR3244 – Dynamics of Genetic Information

Team Publications

Year of publication 2021

Yaqun Liu, Yea-Lih Lin, Philippe Pasero, Chun-Long Chen (2021 Feb 8)

Topoisomerase I prevents transcription-replication conflicts at transcription termination sites.

Molecular & cellular oncology : 1843951 : DOI : 10.1080/23723556.2020.1843951 Learn more
Summary

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops, we mapped RNA:DNA hybrids, markers of replication fork stalling and DNA double-strand breaks along the human genome. This analysis indicates that transient replication fork pausing occurs at the transcription termination sites of highly expressed genes enriched in R-loops and prevents head-on conflicts with transcription, in a topoisomerase I-dependent manner.

Fold up

Year of publication 2020

Aurore Sanchez, Céline Adam, Felix Rauh, Yann Duroc, Lepakshi Ranjha, Bérangère Lombard, Xiaojing Mu, Mélody Wintrebert, Damarys Loew, Alba Guarné, Stefano Gnan, Chun-Long Chen, Scott Keeney, Petr Cejka, Raphaël Guérois, Franz Klein, Jean-Baptiste Charbonnier, Valérie Borde (2020 Nov 17)

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Proceedings of the National Academy of Sciences of the United States of America : DOI : 202013012 Learn more
Summary

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

Fold up
Pacôme Prompsy, Pia Kirchmeier, Justine Marsolier, Marc Deloger, Nicolas Servant, Céline Vallot (2020 Nov 12)

Interactive analysis of single-cell epigenomic landscapes with ChromSCape.

Nature communications : 5702 : DOI : 10.1038/s41467-020-19542-x Learn more
Summary

Chromatin modifications orchestrate the dynamic regulation of gene expression during development and in disease. Bulk approaches have characterized the wide repertoire of histone modifications across cell types, detailing their role in shaping cell identity. However, these population-based methods do not capture cell-to-cell heterogeneity of chromatin landscapes, limiting our appreciation of the role of chromatin in dynamic biological processes. Recent technological developments enable the mapping of histone marks at single-cell resolution, opening up perspectives to characterize the heterogeneity of chromatin marks in complex biological systems over time. Yet, existing tools used to analyze bulk histone modifications profiles are not fit for the low coverage and sparsity of single-cell epigenomic datasets. Here, we present ChromSCape, a user-friendly interactive Shiny/R application distributed as a Bioconductor package, that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations. ChromSCape analyses the distribution of repressive and active histone modifications as well as chromatin accessibility landscapes from single-cell datasets. Using ChromSCape, we deconvolve chromatin landscapes within the tumor micro-environment, identifying distinct H3K27me3 landscapes associated with cell identity and breast tumor subtype.

Fold up
Brison O., Gnan S., Azar D., Schmidt M., Koundrioukoff S., El-Hilali S., Jaszczyszyn Y., Lachages A.M., Thermes C., Chen C.L., Debatisse M. (2020 Nov 1)

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

bioRxiv : 2020.11.19.390054 : DOI : 10.1101/2020.11.19.390054 Learn more
Summary

Genome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs) however escapes surveillance, which triggers chromosome breakage. Using human cells, we asked here whether such leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition suppresses CFS instability. Repli-Seq and molecular combing analyses consistently showed a burst of replication initiations in mid S phase across large origin-poor domains shaped by transcription, including CFSs. Strikingly, CDC6 or CDT1 depletion or CDC7-DBF4 inhibition during the S phase prevented both extra-initiations and CFS rescue, showing that CDK1 inhibition promotes targeted and mistimed building of functional extra-origins. In addition to delay mitotic onset, checkpoint activation therefore advances replication completion of chromosome domains at risk of under-replication, two complementary roles preserving genome stability. ### Competing Interest Statement The authors have declared no competing interest.

Fold up
Md Maminur Rahman, Mohiuddin Mohiuddin, Islam Shamima Keka, Kousei Yamada, Masataka Tsuda, Hiroyuki Sasanuma, Jessica Andreani, Raphael Guerois, Valérie Borde, Jean-Baptiste Charbonnier, Shunichi Takeda (2020 Oct 3)

Genetic Evidence for the Involvement of Mismatch Repair Proteins, PMS2 and MLH3, in a Late Step of Homologous Recombination.

The Journal of biological chemistry : DOI : jbc.RA120.013521 Learn more
Summary

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in non-meiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5 times decrease in the frequency of heteroallelic HR-dependent repair of a restriction-enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of g-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and three times decrease in the number of cisplatin-induced sister chromatid exchange (SCE). The ectopic expression of the Gen1 HJ resolvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

Fold up
Ming-Jun Shi, Xiang-Yu Meng, Jacqueline Fontugne, Chun-Long Chen, François Radvanyi, Isabelle Bernard-Pierrot (2020 Sep 29)

Identification of new driver and passenger mutations within APOBEC-induced hotspot mutations in bladder cancer.

Genome medicine : 85 : DOI : 10.1186/s13073-020-00781-y Learn more
Summary

APOBEC-driven mutagenesis and functional positive selection of mutated genes may synergistically drive the higher frequency of some hotspot driver mutations compared to other mutations within the same gene, as we reported for FGFR3 S249C. Only a few APOBEC-associated driver hotspot mutations have been identified in bladder cancer (BCa). Here, we systematically looked for and characterised APOBEC-associated hotspots in BCa.

Fold up
Felix Raimundo, Celine Vallot, Jean-Philippe Vert (2020 Aug 25)

Tuning parameters of dimensionality reduction methods for single-cell RNA-seq analysis.

Genome biology : 212 : DOI : 10.1186/s13059-020-02128-7 Learn more
Summary

Many computational methods have been developed recently to analyze single-cell RNA-seq (scRNA-seq) data. Several benchmark studies have compared these methods on their ability for dimensionality reduction, clustering, or differential analysis, often relying on default parameters. Yet, given the biological diversity of scRNA-seq datasets, parameter tuning might be essential for the optimal usage of methods, and determining how to tune parameters remains an unmet need.

Fold up
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.

Fold up

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.

Fold up
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.

Fold up
(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.

Fold up
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.

Fold up
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.

Fold up
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.

Fold up
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.

Fold up