LaMarque

Silencing Transposable Elements (TEs) is vital for eukaryotes, but how genome invaders (non-self) are distinguished from cellular genes (self) is ill-understood. The ciliate Paramecium tetraurelia is an excellent model to address this question: the programmed genome rearrangements that occur during its development physically eliminate all non-self sequences genome-wide – a most radical form of TE silencing. This developmental process is linked to the differentiation of distinct types of nuclei for germline and somatic functions. The diploid germline micronucleus (MIC) is transcriptionally silent and only serves to transmit genetic information across sexual generations through meiosis. Genes are expressed from the highly polyploid macronucleus (MAC, ~800n), a somatic nucleus which is not transmitted to sexual progeny; after fertilization, a new MAC differentiates from a copy of the zygotic nucleus. The P. tetraurelia MIC genome contains numerous intragenic TE insertions of various evolutionary ages, called Internal Eliminated Sequences (IESs), that must be precisely excised during development of the somatic nucleus to reconstitute functional genes. Recent insertions are recognized by germline small RNAs that target histone modifications and determine their elimination, but ancient insertions, which have decayed into short single-copy sequences, have switched to a distinct, unknown recognition mechanism. Based on available preliminary data, two non-exclusive hypotheses will be tested, one based on adaptation of aging IES sequences to a set of alternative DNA-binding proteins, the other on acquisition of permanent adenine methylation in the germline. Our project has 3 objectives: i) test hypothesis 1 by characterizing novel protein factors that promote IES excision, ii) test hypothesis 2 by characterizing DNA modifications (N6mA) in the different nuclei and during MAC differentiation and iii) analyse the evolutionary dynamics of IES sequences and transitions in their recognition mechanisms across the Paramecium genus. Our experimental approach, organized in 6 Work Packages, unites functional genomics, molecular biology, quantitative imaging, biochemistry and cutting-edge sequencing technologies and software development. To achieve this program, we have a strong consortium of three leading Paramecium molecular and cellular biology teams and one outstanding evolutionary biology team. Expertise in bioinformatics and high throughput sequence analysis within the partner teams adds to the strength of the consortium. The dissemination of all the datasets generated by the project will be assured through the ParameciumDB public database developed by one of the partners, which for over a decade has integrated the data produced by the international Paramecium research community. Beyond explaining the evolutionary origins of the remarkable DNA splicing system that allows some Paramecium species to tolerate intragenic TE insertions, the LaMarque project is expected to provide an integrated view of the different layers of genetic and epigenetic mechanisms that together allow eukaryotes to keep a precise transgenerational memory of transposable element insertions in the face of constantly evolving sequences.