Single-cell RNA sequencing reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress

Abstract

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single Saccharomyces cerevisiae cells treated with and without salt stress to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells appears to decouple expression of ribosomal protein genes from the environmental stress response in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results suggest that cells maintain a cellular filter against decoupled bursts of transcription factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

Author summary

Genetically identical cells growing in the same environment can vary in their cellular state and behavior. Such heterogeneity may explain why some cells in an isogenic population can survive sudden severe environmental stress whereas other cells succumb. Cell-to-cell variation in gene expression has been linked to variable stress survival, but how and why transcript levels vary across the transcriptome in single cells is only beginning to emerge. Here, we used single-cell RNA sequencing (scRNA-seq) to measure cell-to-cell heterogeneity in the transcriptome of budding yeast ( Saccharomyces cerevisiae). We find surprising patterns of variation across known sets of transcription factor targets, indicating that cells vary in their transcriptome profile both before and after stress exposure. scRNA-seq analysis combined with live-cell imaging of transcription factor activation dynamics revealed some cells in which the stress response was coordinately activated and other cells in which the traditional response was decoupled, suggesting unrecognized regulatory nuances that expand our understanding of stress response and survival.