Transgenerational epigenetic inheritance in ciliates: proximal causes and long-term evolutionary consequences View Homepage


Ontology type: schema:MonetaryGrant     


Grant Info

YEARS

2013-2016

FUNDING AMOUNT

570000 EUR

ABSTRACT

Epigenetic inheritance in ciliates: mechanisms and evolutionary consequencesMaternal 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 impactSmall 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.Sequencing of micronuclear genomes and rearrangement defects in hybridsA 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. More... »

URL

http://www.agence-nationale-recherche.fr/en/anr-funded-project/?solr=run&tx_lwmsuivibilan_pi2%5BCODE%5D=ANR-12-BSV6-0017

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