UMR3244 – Dynamics of Genetic Information

Team Publications

Year of publication 2019

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
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.

Fold up
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.

Fold up
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.

Fold up
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.

Fold up
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.

Fold up
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.

Fold up
Bingning Xie, Emmanuelle Becker, Igor Stuparevic, Maxime Wery, Ugo Szachnowski, Antonin Morillon, Michael Primig (2019 Feb 15)

The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast.

RNA biology : 1-15 : DOI : 10.1080/15476286.2019.1581596 Learn more
Summary

5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3′-5′ exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU’s cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets.

Fold up
Pinskaya M., Saci Z., Gallopin M., Nguyen N.H., Gabriel M., Firlej V., Descrimes M., de la Taille A., Londo~no-Vallejo A., Allory Y., Gautheret D., Morillon A. (2019 Jan 1)

Blind exploration of the unreferenced transcriptome reveals novel RNAs for prostate cancer diagnosis

bioRxiv : DOI : 10.1101/644104 Learn more
Summary

The broad 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 is restricted by the limited power of conventional reference-based protocols relying on uniquely mapped reads and transcript annotations. Here, we implemented a blind reference-free computational protocol, DE-kupl, to directly infer RNA variations of any origin, including yet unreferenced RNAs, from high coverage total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into unannotated putative long noncoding (lnc)RNAs expressed in prostate cancer tissues. Through filtering and visual inspection of 1,179 candidates, we defined 21 lncRNA probes that were further validated for robust tumor-specific expression by NanoString single molecule-based RNA measurements in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling over 90% of true positive detection of cancer in an independent dataset from The Cancer Genome Atlas. Remarkably, this clinical signature made of only 9 unannotated lncRNAs largely outperformed PCA3, the only RNA biomarker approved by the Food and Drug Administration agency, specifically, in detection of high-risk prostate tumors. The proposed reference-free computational workflow is modular, highly sensitive and robust and can be applied to any pathology and any clinical application.

Fold up

Year of 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 Learn more
Summary

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.

Fold up
(2018 May 6)

The Guardian of the Genome Revisited: p53 Downregulates Genes Required for Telomere Maintenance, DNA Repair, and Centromere Structure.

Cancers (Basel) : 10(5) : pii: E135 Learn more
Summary

The p53 protein has been extensively studied for its capacity to prevent proliferation of cells with a damaged genome. Surprisingly, however, our recent analysis of mice expressing a hyperactive mutant p53 that lacks the C-terminal domain revealed that increased p53 activity may alter genome maintenance. We showed that p53 downregulates genes essential for telomere metabolism, DNA repair, and centromere structure and that a sustained p53 activity leads to phenotypic traits associated with dyskeratosis congenita and Fanconi anemia. This downregulation is largely conserved in human cells, which suggests that our findings could be relevant to better understand processes involved in bone marrow failure as well as aging and tumor suppression.

Fold up
Watts BR, Wittmann S, Wery M, Gautier C, Kus K, Birot A, Heo DH, Kilchert C, Morillon A, Vasiljeva L (2018 Mar 26)

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin

Nucleic Acid ResearchHistone deacetylation promotes transcriptional silencing at facultative heterochromatin : DOI : 10.1093/nar/gky232 Learn more
Summary

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

Fold up