The stress-responsive chaperone Hsp90 controls the phenotypic consequences of genetic variation primarily through cis-regulatory variants rather than coding mutations, with most effects arising in genes encoding direct Hsp90/Hsp70 clients or targets of client kinases, where the chaperone modulates sigmoidal activity-fitness relationships to buffer variant effects under normal conditions and release cryptic phenotypic diversity under stress, with particularly strong effects on evolutionarily young genes that drive adaptive novelty.

The essential, stress-responsive protein chaperone Hsp90 impacts adaptation from microbes to humans. Yet the molecular mechanisms involved remain unclear and are often presumed to be driven by variation in opening reading frames. Here, we identify over 1,000 natural genotype-to-phenotype associations governed by Hsp90 at single-nucleotide resolution in Saccharomyces cerevisiae. Strikingly, cis-regulatory variants contributed to the chaperone’s effect on heredity more strongly than coding variation. Most of these mutations nonetheless impacted clients of Hsp90 or targets of its direct binding partners. The chaperone’s influence was especially potent on evolutionarily young genes, highlighting its influence on variation central to novelty. Synthetic reconstructions and genome editing revealed that Hsp90 may regulate the relationship between activity and phenotype for many dosage-sensitive genes. The unexpected role of the chaperone in buffering the effects of regulatory variants and releasing their consequences under stress suggests a mechanism for its widespread effects on evolution and development.