Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast

Abstract

Ageing-related processes are largely conserved, with simple organisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular ageing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computational methods to study chronological lifespan in Schizosaccharomyces pombe. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of ~700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previously unknown ageing-associated genes, including 46 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent enrichments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an automated colony-forming unit assay to facilitate medium- to high-throughput chronological-lifespan studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analysing cellular ageing in yeast and beyond.

Fission stories: using PomBase to understand Schizosaccharomyces pombe biology

“…Figure 1 shows QuiLT visualization of genes that are conserved in vertebrates and were found to be associated with chronological lifespan in a recent study ( Romila et al 2021 ). The authors compared their list of long-lived mutants with all genes previously associated with the phenotype ‘increased viability in stationary phase’ (FYPO:0001309) to uncover novel aging-associated genes; among the latter genes, they then readily identified conserved genes using the catalog of conserved unstudied genes described above, as well as genes associated with diseases in human.…”

“… QuiLT visualization of 220 S. pombe genes showing viability of the deletion mutant, presence or absence of orthologs in human and budding yeast, and GO annotation. Romila et al (2021) identified 340 genes for which deletion increased chronological lifespan; the visualization uses 220 of those genes annotated in PomBase as conserved in vertebrates. (A) Interactive display controls show or hide annotation types and sort on any visible column.…”

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements

“…We used a recently developed assay to accurately measure lifespans of multiple yeast strains, simultaneously at a medium throughput [ 126 ]. Briefly, after waking strains up on YES plates, 20mL pre-cultures were grown in YES using 100mL flasks.…”

An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence

“…We have previously shown that mutations in nine tested subunits of Ino80C: Ies6, Nht1, Iec1, Iec3, Tfg3, Arp8, Ies2, Ies4, and the putative Ino80C subunit Arp42, lead to quiescence mortality phenotypes (Zahedi et al 2020 ). Mutations in Hap2, Iec1, Arp8, Iec3, Nht1, Arp5, Ies4, and Ies2 were recently shown to be short-lived in stationary phase in fission yeast, implicating Ino80C in chronological ageing (Romila et al 2021 ). Thus, at least six Ino80C subunits: Iec1, Arp8, Iec3, Nht1, Ies4, and Ies2 are implicated both in survival in quiescence and chronological ageing.…”