RESEARCH ARTICLE ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors Brian D Alford1†, Eduardo Tassoni-Tsuchida1,2†, Danish Khan1, Jeremy J Work1, Gregory Valiant3, Onn Brandman1* 1Department of Biochemistry, Stanford University, Stanford, United States; 2Department of Biology, Stanford University, Stanford, United States; 3Department of Computer Science, Stanford University, Stanford, United States 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. *For correspondence:
[email protected] †These authors contributed equally to this work Introduction Competing interests: The The heat shock response (HSR) is a conserved stress response that shields cells from cytoplasmic authors declare that no proteotoxicity by increasing the expression of protective proteins (Lindquist, 1986; Mori- competing interests exist.