No abstract

No 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.

Antisense oligonucleotide therapy targeting ATXN2, a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis, has entered clinical trials in humans. Additional methods to lower ataxin 2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin 2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v ATPase) as regulators of ataxin 2 levels. We demonstrate that Etidronate, a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v ATPase, lowers ataxin 2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.

No abstract