Blanc SVSE 6 - Blanc - SVSE 6 - Génomique, génétique, bioinformatique et biologie systémique

Transgenerational epigenetic inheritance in ciliates: proximal causes and long-term evolutionary consequences – INFERNO

Epigenetic inheritance in ciliates: mechanisms and evolutionary consequences

Maternal small RNAs are required for the elimination of transposons and derived single-copy sequences during development of the somatic nucleus. Their mechanism of action results in non-Mendelian inheritance of alternative genome rearrangements, and of differentiated phenotypes, but should also cause hybrid dysgenesis in crosses between strains showing insertion polymorphisms.

Micronuclear genomes of the 15 P. aurelia species: polymorphic insertions and evolutionary impact

Small RNAs are very often used in eukaryotes as genomic ‘immune systems’ to control transposable elements. They have also been implicated in non-Mendelian inheritance phenomena in a variety of organisms. We propose to use the ciliate P. aurelia to shed some new phylogenetic light on the evolutionary impact of these mechanisms, which remains under-appreciated. In this unicellular eukaryote, germline functions are ensured by the diploid micronuclei and somatic functions by the polyploid macronuclei, the genome of which is rearranged to eliminate transposons and derived single-copy sequences (IESs). These sequences are targeted during development by scnRNAs, germline-derived small RNAs of maternal origin that first probe the maternal somatic genome to identify missing sequences, and then reproduce the same deletions in the zygotic macronucleus. This mechanism results in maternal inheritance of alternative genome rearrangements during conjugation, and was co-opted to ensure epigentic transmission of differentiated characters. But it should theoretically raise a hybrid dysgenesis problem in crosses between strains presenting IES insertion polymorphisms: a strain devoid of a given IES cannot produce homologous scnRNAs, and will not be able to excise this IES when it is introduced through the paternal gamete; if the IES is inserted in an essential gene, further sexual progeny inheriting this allele will not be viable. A significant fraction of the ~45,000 IESs in P. tetraurelia were likely acquired since the divergence of the 15 sibling species of the P. aurelia group. We propose to further study the mechanism of action of scnRNAs and to sequence the micronuclear genome of different species to reconstruct the evolutionary history of transposons, IESs, and micronuclear centromeres, and assess their possible contribution to the phenomenon of speciation.

A major technical difficulty is the purification of micrnuclei in sufficient amounts to allow the sequencing and assembly of genomes, including their repeated fraction. We developed a method based on fluorescence-activated sorting of micronuclei marked by a florescent DNA dye and by a fluorescent fusion protein (CenH3-GFP) that is absent from macronuclei. The method allows te recovery of sufficient amounts of DNA to obtain a good coverage of genomes by high-throughput sequencing techniques, but not to generate the mate pairs required for a full assembly of repeated sequences. To get these mate pairs, we will use DNA from macronuclei developing after depletion of the Pgm endonuclease (the genome of which is amplified to high ploidy but not rearranged), which can be obatined in far greater amounts. After assembly by standard procedures, IESs will be indentified by comparison with the macronuclear genomes of the same strains (sequenced in collaboration with Michael Lynch, Indiana University). The annotation of transposable elements will be done in collaboration with H. Quesneville using a dedicated pipeline and manual curation. Identification of centromeres relies on chromatin immunoprecipitation with an antibody raised against the centromeric histone CenH3, and on the sequencing and analysis of retrieved sequences. The depletion of Pgm (and of other factors potentially involved in DNA rearrangemens or in the scnRNA pathway) uses a simple and efficent RNAi technique in which cells are fed bacteria producing double-stranded RNA homologous to the gene to be inactivated. We will then test our central hypothesis by sequencing the macronuclear genomes of inter-strain and inter-species F1 hybrids, to evaluate the frequency of excision defects for polymorphic IESs in potentially essential genes, and how it correlates with lethality in the F2 generation.

DNA from P. tetraurelia micronuclei purified by Partner 2 has been sequenced and is currently being assembled at the Genoscope. This constitutes the pilot phase of the France Génomique project, which will allow us to adjust the methodology to be used for other strains and species. In parallel, we have started to sequence micronuclear DNA from other species, and to prepare macronuclear DNA samples from 2 strains for each species, and from F1 hybrids of some of these strains. Concerning centromeres, a first CenH3 ChIPseq trial was carried out and analysed; further trials have been planned. Mutants obtained in our screens designed to hit the siRNA or scnRNA pathways are being identified and studied, as is the role of candidate factors. A more focused study of the cross between strains 51 and 32 of P. tetraurelia showed that IES retention in F1 heterozygotes deriving from the naive parent can be generalized to at least one other IES, among the 7 IESs shown to be present in one strain but absent from the other. As is the case for the mtB gene IES, retention is stronger on the naive side of the cross but can also be detected on the other side, which cannot be explained by the lack of maternal scnRNAs. We are making the hypothesis that the hemizygous state of these sequences in F1s results in another problem impairing IES excision, which may be related to ‘Meiotic Silencing of Unpaired DNA’. Preliminary results suggest that these are indeed two distinct effects: the depletion of Ptiwi03, a paralog of unknown function of the proteins loaded with scnRNAs, appears to increase the rate of retention specifically on the naive side, suggesting it could be involved in exchange of scnRNAs between conjugants, while that of Dcl5, a protein putatively involved in a mechanism of amplification of scnRNA action during macronuclear development, increases this frequency on both sides.

Validating our central hypothsesis would represent a major advance: the mechnism involved would be remarkably similar to the hybrid dysgenesis observed in Drosophila, which results from the requirement of maternal scnRNAs to control transposons introduced through the paternal gamete. Thus the mechanism could be a general one, contributing to speciation in many eukaryotes. Preliminary evidence suggest the existence of a distinct effect, due to the hemizygous state of IES sequences rather than to the lack of maternal scnRNAs, but this would similarly contribute to hybrid dysgenesis. Preliminary evidence that new factors seem to counteract these deleterious effects raise the very interesting hypothesis that P. aurelia species have adapted to these difficulties by evolving new mechanisms, possibly involving new small RNA pathways, that allow the exchange of epigenetic information (and not only of the Mendelian genome) between sexual partners.

1. Singh, D.P., Saudemont, S., Guglielmi, G., Arnaiz, O., Goût, J.F., Prajer, M., Potekhin, A., Przybòs, E., Aubusson-Fleury, A., Bhullar, S., Bouhouche, K., Lhuillier-Akakpo, M., Tanty, V., Blugeon, C., Alberti, A., Labadie, K., Aury, J.M., Sperling, L., Duharcourt, S., Meyer, E. (2014) Genome-defence small RNAs exapted for epigenetic mating-type inheritance. Nature 509:447-452. Epub 2014 May 7. Selected by Faculty of 1000.

Cette étude de l’hérédité maternelle des types sexuels chez 3 espèces P. aurelia montre que si le type E est toujours déterminé par l’expression de la protéine transmembranaire mtA, les mécanismes développementaux produisant des clones de type O diffèrent : P. tetraurelia et P. octaurelia excisent le promoteur de mtA comme une IES, tandis que P. septaurelia excise des segments de séquence codante du gène mtB, qui code un facteur de transcription spécifiquement requis pour l’expression de mtA. Ce sont les premiers exemples de co-optation de la voie des scnRNAs et de la machinerie d’excision des IES pour la régulation de gènes cellulaires et l’hérédité épigénétique de polymorphismes phénotypiques essentiels.

2. Marker, S., Carradec, Q. , Tanty, V., Arnaiz, O., and Meyer, E. (2014) A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia. Nucleic Acids Res. 42:7268-7280. Epub 2014 May 23.

3. Marmignon A, Bischerour J, Silve A, Fojcik C, Dubois E, et al. (2014) Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia. PLoS Genet 10(8): e1004552. doi:10.1371/journal.pgen.1004552. eCollection 2014 Aug.

4. Lhuillier-Akakpo M*, Frapporti A*, Denby Wilkes C, Matelot M, Vervoort M, Sperling L, Duharcourt S. (2014). Local effect of Enhancer of Zeste-like reveals cooperation of epigenetic and cis-acting determinants for zygotic genome rearrangements. PLoS Genet. In press. (

Recent developments in epigenetics suggest the widespread use of small-RNA pathways as a genomic immune system allowing eukaryotes to control molecular parasites such as transposable elements (TEs). Small RNAs have also been shown to mediate non-Mendelian inheritance in divergent species, resurrecting the Lamarckian idea of inheritance of acquired traits. To understand the basic biological logic that may underlie the connection between these themes, and identify fundamental aspects despite the confounding diversity of mechanisms uncovered in different phyla, we propose to address this issue from a phylogenetically unique angle. The ciliate Paramecium tetraurelia – at an equal evolutionary distance from plants and animals – was one of the first organisms in which transgenerational epigenetic inheritance was clearly demonstrated, and recent molecular studies have shown that many cases can be explained by the role of a meiosis-specific class of small RNAs, the scnRNAs, in epigenetic regulation of the genome rearrangements that occur during the development of the somatic macronucleus (MAC) from the germline micronucleus (MIC), in each sexual generation.
Rearrangements include the elimination of all TEs as well as the precise excision of ~45.000 single-copy Internal Eliminated Sequences (IESs), which are believed to be degenerate remnants of ancient TE insertions. MIC centromeres may also be eliminated since they are not active in the MAC, which divides by a non-mitotic mechanism. During meiosis, scnRNAs are produced from the entire MIC genome and mediate a genome-wide comparison of germline and somatic sequences, allowing the zygotic MAC to eliminate any germline sequence not present in the maternal MAC. The system has probably evolved as an efficient mechanism to detect any new TE insertion in the germline, and has further been co-opted to ensure the non-Mendelian inheritance of essential phenotypic polymorphisms, such as mating types. But the mechanism should also in principle make any new insertion potentially lethal if it is introduced by conjugation into a naive cell - an effect that is reminiscent of hybrid dysgenesis in Drosophila, the devastating effects of TEs introduced by the male gamete when the mother cannot produce homologous piRNAs to repress transposition.
By comparing the IES content of paralogous gene pairs of different ages derived from 3 successive whole-genome duplications, we recently obtained evidence that a substantial fraction of IESs have been acquired since the last duplication. This is the time when the Paramecium ancestor underwent numerous speciation events, resulting in a group of 15 sibling species that are morphologically indistinguishable but sexually incompatible. This raises the possibility that the scnRNA-based mechanism for recognition and elimination of TEs, which decreases the burden on host fitness and allows them to persist in the genome and degenerate into single-copy IESs, has been a major force driving speciation.
The main objectives of this project are to reconstruct the evolutionary history of IESs and other MIC specific sequences such as TEs and centromeres, and to experimentally test the hypothesis that polymorphisms in these elements result in sexual incompatibility. We propose to (i) sequence and assemble the entire MIC genome of P. tetraurelia, since very little is currently known about the diversity and copy number of TEs; (ii) explore further the scnRNA pathway mechanism, by testing the role of known protein factors in the recognition of the genome-wide set of ~45,000 IESs, by identifying new ones, and by deep-sequencing scnRNA populations during sexual events to describe their dynamics; (iii) identify IESs and centromeres in different strains and species to study their evolution; and (iv) experimentally determine the effects of IES polymorphisms (presence/absence, divergence of sequences) on genome rearrangements in sexual progeny of interstrain or interspecies crosses.

Project coordinator

Monsieur Eric Meyer (CNRS Institut de Biologie de l'Ecole Normale Superieure) –

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CNRS IBENS CNRS Institut de Biologie de l'Ecole Normale Superieure
CGM Centre de Génétique Moléculaire
LBBE Laboratoire de Biométrie et Biologie Evolutive

Help of the ANR 570,000 euros
Beginning and duration of the scientific project: December 2012 - 48 Months

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