With its nuclear dualism, the ciliate Paramecium constitutes an original model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MIC) and a polyploid somatic macronucleus (MAC) that develops from the MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of ~45000 TE-derived Internal Eliminated Sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by non-coding RNAs and repressive chromatin marks. A subset of IESs are excised independently of this epigenetic control, raising the question of how they are targeted for elimination. To gain insight into the determinants of IES excision, we determined the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with next-generation sequencing. Essentially all IESs are excised within one endoduplication round only (32C to 64C), while TEs are eliminated at a later stage. We show that time, rather than replication, controls the progression of DNA elimination. Further analyses defined four IES classes according to excision timing and revealed that the earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision, by acquiring stronger sequence determinants and escaping epigenetic control.

This study was also supported by grants from the Agence Nationale de la Recherche (ANR-21-CE12-0019-01) and the Fondation pour la Recherche Médicale (FRM EQU202103012766).