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A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of aging yeast.

ABSTRACT: Replicative aging of Saccharomyces cerevisiae is an established model system for eukaryotic cellular aging. A limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities for genomic and biochemical characterization of aging yeast populations. Using MAD, we measured DNA accessibility and gene expression changes in aging cells. Our data highlight an intimate connection between aging, growth rate, and stress. Stress-independent genes that change with age are highly enriched for targets of the signal recognition particle (SRP). Combining MAD with an improved ATAC-seq method, we find that increasing proteasome activity reduces rDNA instability usually observed in aging cells and, contrary to published findings, provide evidence that global nucleosome occupancy does not change significantly with age.

SUBMITTER: Hendrickson DG

PROVIDER: S-EPMC6261268 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of aging yeast.

Hendrickson David G DG Soifer Ilya I Wranik Bernd J BJ Kim Griffin G Robles Michael M Gibney Patrick A PA McIsaac R Scott RS

eLife 20181019

Replicative aging of <i>Saccharomyces cerevisiae</i> is an established model system for eukaryotic cellular aging. A limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities for genomic and biochemical characterization of aging yeast populations. Using MAD, we measured DNA accessibility and gen ...[more]

PMID: 30334737

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Project description:Previous studies have described a transcriptional "memory effect," whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TATA-binding protein and Pol II decreases after the loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that although transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast, the promoter regions of memory effect targets show no change in abf1 ts mutants, maintaining an open chromatin conformation even after Abf1 eviction. This contrasting behavior reflects different inherent propensity for nucleosome formation between the two classes, driven by the presence of A/T-rich sequences upstream of the Abf1 site in memory effect gene promoters. These sequence-based differences show conservation in closely related fungi and also correlate with different gene expression noise, suggesting a physiological basis for greater access to "memory effect" promoter regions. Thus, our results establish a conserved mechanism underlying a transcriptional memory effect whereby sequences surrounding Abf1 binding sequences affect local nucleosome occupancy following loss of Abf1 binding. Furthermore, these findings demonstrate that sequence-based differences in the propensity for nucleosome occupancy can influence the transcriptional response of genes to an altered regulatory signal.

Project description:BackgroundRNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties.ResultsUsing high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at -140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes.ConclusionsBudding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5' NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC. The RSC-regulated nucleosome dynamics at the 3' gene-end serves as a novel regulatory mechanism for pol III transcription in vivo, probably by controlling terminator-dependent facilitated recycling of pol III. Salient features of yeast tDNA chromatin structure reported in this study can explain the basis of the novel non-transcriptional roles ascribed to tDNAs.

Dataset Information

A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of aging yeast.

Publications

A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of aging yeast.

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