Abstract
Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.
Highlights
The heat shock response (HSR) is a conserved stress response that shields cells from cytoplasmic proteotoxicity by increasing the expression of protective proteins (Lindquist, 1986; Morimoto, 2011)
Though ReporterSeq can be implemented with any encodable genetic perturbation (e.g. RNAi, CRISPR knockouts), we used CRISPRi (Gilbert et al, 2013) as a perturbation to lower expression of each gene and paired it with a barcoded mRNA encoding GFP downstream of an Hsf1-responsive synthetic promoter built upon a ‘crippled’ CYC1 promoter sequence derived from a 225 nucleotide fragment of the CYC1 promoter (Brandman et al, 2012; Guarente and Mason, 1983)
We dissected the genetic modulators of the HSR using ReporterSeq, a pooled, high-throughput screening method that measures the effect of genome-wide genetic perturbations on the expression of a single-reporter-driven RNA
Summary
The heat shock response (HSR) is a conserved stress response that shields cells from cytoplasmic proteotoxicity by increasing the expression of protective proteins (Lindquist, 1986; Morimoto, 2011). A diverse array of stressors activate the HSR, including heat, ethanol, oxidative stressors, and amino acid analogs (Morano et al, 2012). In these conditions, HSR activity can follow diverse trajectories, with both transient or prolonged activity observed (Sorger, 1990). The complexity of the HSR has led to a multitude of models for how the HSR is regulated (Anckar and Sistonen, 2011) These include negative regulation in which the chaperones Hsp (Krakowiak et al, 2018; Zheng et al, 2016), Hsp (Ali et al, 1998; Zou et al, 1998), and Hsp (Neef et al, 2014) bind and inhibit Hsf directly.
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