Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) ‘biological process’ terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including ‘cell cycle’ (cdc2, rik1, pas1, and leo1), ‘signaling’ (sck2, oga1, and cki3), and ‘vesicle-mediated transport’ (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the ‘signaling’ GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput

Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

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

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.

Fur4 mediated uracil-scavenging to screen for surface protein regulators

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

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 51 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 for chronological lifespan to facilitate medium- to high-throughput ageing studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay, validating 33 genes not previously associated with cellular ageing. 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.

Local chromatin context dictates the genetic determinants of the heterochromatin spreading reaction

Heterochromatin spreading, the expansion of gene-silencing structures from DNA-encoded nucleation sites, occurs in distinct chromatin contexts. Spreading re-establishes gene-poor constitutive heterochromatin every cell cycle, but also invades gene-rich euchromatin de novo to steer fate decisions. Unlike heterochromatin nucleation and assembly, the determinants of the spreading process remain poorly understood. Our heterochromatin spreading sensor separately records nucleation site-proximal, and distal, heterochromatin gene silencing. By screening a nuclear function gene deletion library in fission yeast, we identified regulators that alter the propensity, both positively and negatively, of a nucleation site to spread heterochromatin. Critically, the involvement of many regulators is conditioned by the chromatin context within which spreading occurs. We find spreading, but not nucleation, within constitutive heterochromatin, requires distinct Clr6 histone deacetylase complexes. However, spreading is universally antagonized by a suite of chromatin remodelers. Our results disentangle the machineries that control lateral heterochromatin spreading from those that instruct DNA-directed assembly.

Population dynamics analysis of Saccharomyces cerevisiae deletion library during fed-batch cultivation using Bar-seq

To understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance, we employed a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime. Our results demonstrate the utility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions. We find that mutant population diversity is maintained through multiple seed trains, enabling for large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant external stresses, such as the accumulation of the fermentative byproduct ethanol. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.