Telomere healing seals the broken ends of DNA double-strand breaks by adding de novo telomeres. In the ciliate Paramecium, telomere healing results in chromosome fragmentation, which ultimately leads to clonal ageing. Despite its importance, where and how chromosomes fragment and undergo telomere healing, and to what extent sequence preferences exist, remain poorly understood. To investigate chromosome fragmentation, we enriched for telomere-containing sequences from the complex, highly polyploid genome of Paramecium by developing Subtle-seq (Subtelomere Enrichment-sequencing). Subtle-seq reads of young Paramecium mapped to chromosome termini and shifted toward proximal sites with increasing divisions, recapitulating chromosome fragmentation during ageing. Transcriptome analysis identified downregulated differentially expressed genes (DEGs) in aged cells, and Subtle-seq reads enriched in or around these genes. Moreover, we identified nucleosome-occupied DNA as hotspots of telomere healing. Nucleosome occupancy has been reported to correlate with gene expression in Paramecium, and indeed, the downregulated DEGs were predominantly highly expressed and showed higher nucleosome densities. Hence, the density of nucleosomes needed for high expression of genes likely results in chromosome fragmentation as "collateral damage" and may contribute to the reduced transcription in aged cells. Our method allows the detection of rare telomere-healing events and sheds light on the hitherto unrecognized mechanism of telomere healing and consequent ageing in Paramecium.
Matsubara et al. (Fri,) studied this question.