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Accueil > Départements > Biologie des Génomes > Mireille BETERMIER : Réarrangements programmés du génome

Publications de l’équipe


  • A. Frapporti, C. Miró Pina, O. Arnaiz, D. Holoch, T. Kawaguchi, A. Humbert, E. Eleftheriou, B. Lombard, D. Loew, L. Sperling, K. Guitot, R. Margueron, et S. Duharcourt, « The Polycomb protein Ezl1 mediates H3K9 and H3K27 methylation to repress transposable elements in Paramecium », Nature Communications, vol. 10, nᵒ 1, p. 2710, 2019.
    Résumé : In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.
    Mots-clés : chromatin, complex, DBG, DNA Methylation, DNA Transposable Elements, Gene Silencing, genes, heterochromatin formation, histone methyltransferase activity, Histones, mechanisms, MICMAC, Paramecium tetraurelia, pluripotent, Polycomb Repressive Complex 2, Protein Processing, Post-Translational, specificity, states, structural basis, Transcriptional Activation.
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  • J. Godau, L. P. Ferretti, A. Trenner, E. Dubois, C. von Aesch, A. Marmignon, L. Simon, A. Kapusta, R. Guérois, M. Bétermier, et A. A. Sartori, « Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair », DNA repair, vol. 77, p. 96-108, mars 2019.
    Résumé : DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.
    Mots-clés : AMIG, B3S, CtIP, ctp1, damage response, DBG, DNA double-strand breaks, DNA end resection, end-resection, endonuclease, gene, Homologous recombination, human ctip, Meiosis, MICMAC, mre11 complex, Paramecium tetraurelia, protein, rad32(mre11) nuclease, sae2.

  • I. Nekrasova, V. Nikitashina, S. Bhullar, O. Arnaiz, D. P. Singh, E. Meyer, et A. Potekhin, « Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia », Genes, vol. 10, nᵒ 7, juill. 2019.
    Résumé : A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.
    Mots-clés : chromosome fragile sites, cortical inheritance, DBG, epimutation, maternal inheritance, MICMAC, micronuclear deletion, Paramecium, trichocysts.
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  • S. Bhullar, C. D. Wilkes, O. Arnaiz, M. Nowacki, L. Sperling, et E. Meyer, « A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium », Nucleic Acids Research, vol. 46, nᵒ 18, p. 9550-9562, oct. 2018.
    Résumé : In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of similar to 45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to similar to 60% of IESs are still correctly excised. To get further insight, we designed a mutagenesis screen based on the hypersensitivity of a specific excision event in the mtA gene, which determines mating types. Unlike most IES-containing genes, the active form of mtA is the unexcised one, allowing the recovery of hypomorphic alleles of essential IES recognition/excision factors. Such is the case of one mutation recovered in the Piwi gene PTIWI09, a key player in small RNA-mediated IES recognition. Another mutation identified a novel protein with a C2H2 zinc finger, mtGa, which is required for excision of a small subset of IESs characterized by enrichment in a 5-bp motif. The unexpected implication of a sequence-specific factor establishes a new paradigm for IES recognition and/or excision.
    Mots-clés : ANGE, DBG, developmental excision, differentiation, discovery, genetic-analysis, GTR, internal eliminated sequences, macronuclear development, MICMAC, piwi proteins, rearrangements, rna pathways, tetraurelia.

  • J. Bischerour, S. Bhullar, C. Denby Wilkes, V. Régnier, N. Mathy, E. Dubois, A. Singh, E. Swart, O. Arnaiz, L. Sperling, M. Nowacki, et M. Bétermier, « Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium », eLife, vol. 7, p. e37927, sept. 2018.
    Résumé : The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.
    Mots-clés : ANGE, chromosomes, ciliates, DBG, DNA elimination, gene expression, GTR, IES, MICMAC, piggyBacv, transposées domestication.

  • N. Morellet, X. Li, S. A. Wieninger, J. L. Taylor, J. Bischerour, S. Moriau, E. Lescop, B. Bardiaux, N. Mathy, N. Assrir, M. Bétermier, M. Nilges, A. B. Hickman, F. Dyda, N. L. Craig, et E. Guittet, « Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase », Nucleic Acids Research, vol. 46, nᵒ 5, p. 2660-2677, mars 2018.
    Résumé : The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
    Mots-clés : DBG, MICMAC.
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  • L. Shi, F. Koll, O. Arnaiz, et J. Cohen, « The Ciliary Protein IFT57 in the Macronucleus of Paramecium », The Journal of Eukaryotic Microbiology, vol. 65, nᵒ 1, p. 12-27, janv. 2018.
    Résumé : The intraflagellar transport IFT57 protein is essential for ciliary growth and maintenance. Also known as HIPPI, human IFT57 can be translocated to the nucleus via a molecular partner of the Huntingtin, Hip1, inducing gene expression changes. In Paramecium tetraurelia, we identified four IFT57 genes forming two subfamilies IFT57A/B and IFT57C/D arising from whole genome duplications. The depletion of proteins of the two subfamilies induced ciliary defects and IFT57A and IFT57C localized in basal bodies and cilia. We observed that IFT57A, but not IFT57C, is also present in the macronucleus and able to traffic toward the developing anlage during autogamy. Analysis of chimeric IFT57A-IFT57C-GFP-tagged proteins allowed us to identify a region of IFT57A necessary for nuclear localization. We studied the localization of the unique IFT57 protein of Paramecium caudatum, a species, which diverged from P. tetraurelia before the whole genome duplications. The P. caudatumIFT57C protein was excluded from the nucleus. We also analyzed whether the overexpression of IFT57A in Paramecium could affect gene transcription as the human protein does in HeLa cells. The expression of some genes was indeed affected by overexpression of IFT57A, but the set of affected genes poorly overlaps the set of genes affected in human cells.
    Mots-clés : ANGE, BIOCELL, BIOCIL, cilia, DBG, HIPPI, IFT57 /HIPPI, intraflagellar transport, intraflagellar transport (IFT), Macronucleus, MICMAC, Paramecium.


  • O. Arnaiz, E. Van Dijk, M. Bétermier, M. Lhuillier-Akakpo, A. de Vanssay, S. Duharcourt, E. Sallet, J. Gouzy, et L. Sperling, « Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression », BMC genomics, vol. 18, nᵒ 1, p. 483, juin 2017.
    Résumé : BACKGROUND: The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. RESULTS: We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. CONCLUSIONS: We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( ). TrUC software is freely distributed under a GNU GPL v3 licence ( ).
    Mots-clés : ANGE, Autogamy, Cap-Seq, Ciliate, DBG, Differential gene expression, Gene annotation, MICMAC, RNA-Seq.

  • E. Dubois, N. Mathy, V. Régnier, J. Bischerour, C. Baudry, R. Trouslard, et M. Bétermier, « Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements », Nucleic Acids Research, janv. 2017.
    Résumé : During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.
    Mots-clés : DBG, MICMAC.


  • K. Maliszewska-Olejniczak, J. Gruchota, R. Gromadka, C. Denby Wilkes, O. Arnaiz, N. Mathy, S. Duharcourt, M. Bétermier, et J. K. Nowak, « TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements », PLoS genetics, vol. 11, nᵒ 7, p. e1005383, juill. 2015.
    Résumé : Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.
    Mots-clés : ANGE, Cell Lineage, DBG, Genome, germ cells, High-Throughput Nucleotide Sequencing, MICMAC, Paramecium tetraurelia, RNA Polymerase II, RNA, Long Noncoding, Transcription, Genetic, Transcriptional Elongation Factors.
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Principales publications avant 2015

- Marmignon, A., Bischerour, J., Silve, A., Fojcik, C., Dubois, E., Arnaiz, O., Kapusta, A., Malinsky, S., Bétermier, M. (2014) Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia. PLoS Genet. 10(8):e1004552

- Bétermier, M., Bertrand, P. Lopez, B.S. (2014). Is non-homologous end-joining really an inherently error-prone process ? PLoS Genet. 10(1) : e1004086.

- Arnaiz, O., Mathy, N., Baudry, C., Malinsky, S., Aury, J.M., Denby-Wilkes, C., Garnier, O., Labadie, K., Lauderdale, B., Le Mouël, A., Marmignon, A., Nowacki, M., Poulain, J., Prajer, M., Wincker, P., Meyer, E., Duharcourt, S., Duret, L., Bétermier, M.*, Sperling, L.* (2012) The Paramecium germline genome provides a niche for intragenic parasitic DNA : Evolutionary Dynamics of Internal Eliminated Sequences. PLoS Genet. 8(10):e1002984. * co-corresponding authors.

- Dubois, E., Bischerour, J., Marmignon, A., Mathy, N., Régnier, V., Bétermier, M. (2012) Transposon invasion of the Paramecium germline genome countered by a domesticated PiggyBac transposase and the NHEJ pathway. International Journal of Evolutionary Biology. 2012:436196. Epub 2012 Jul 22.

- Kapusta, A.*, Matsuda, A.*, Marmignon, A., Ku, M., Silve, A., Meyer, E., Forney, J.D., Malinsky, S., Bétermier, M. (2011) Highly precise and developmentally programmed genome assembly in Paramecium requires Ligase IV-dependent end joining. PLoS Genet. 7(4) :e1002049. * equal contribution.

- Baudry, C.*, Malinsky, S.*, Restituito, M., Kapusta, A., Rosa, S., Meyer, E., Bétermier, M. (2009) PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia. Genes & Dev. 23 : 2478-2483.

- Gratias, A., Lepère, G., Garnier, O., Rosa, S., Duharcourt, S., Malinsky, S., Meyer, E., Bétermier, M. (2008) Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites. Nucl. Acids Res. 36 :3244-3251.

- Aury, J.M., Jaillon, O., Duret, L., Noel, B., Jubin, C., Porcel, B.M., Segurens, B., Daubin, V., Anthouard, V., Aiach, N., Arnaiz, O., Billaut, A., Beisson, J., Blanc, I., Bouhouche, K., Camara, F., Duharcourt, S., Guigo, R., Gogendeau, D., Katinka, M., Keller, A.M., Kissmehl, R., Klotz, C., Koll, F., Le Mouel, A., Lepere, G., Malinsky, S., Nowacki, M., Nowak, J.K., Plattner, H., Poulain, J., Ruiz, F., Serrano, V., Zagulski, M., Dessen, P., Bétermier, M., Weissenbach, J., Scarpelli, C., Schachter, V., Sperling, L., Meyer, E., Cohen, J., Wincker, P. (2006) Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444 :171-178.

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