Abstract
The essential stress-responsive chaperone Hsp90 impacts development and adaptation from microbes to humans. Yet despite evidence of its role in evolution, pathogenesis, and oncogenic transformation, the molecular mechanisms by which Hsp90 alters the consequences of mutations remain vigorously debated. Here we exploit the power of nucleotide-resolution genetic mapping in Saccharomyces cerevisiae to uncover more than 1,000 natural variant-to-phenotype associations governed by this molecular chaperone. Strikingly, Hsp90 more frequently modified the phenotypic effects of cis-regulatory variation than variants that altered protein sequence. Moreover, these interactions made the largest contribution to Hsp90-dependent heredity. Nearly all interacting variants-both regulatory and protein-coding-fell within clients of Hsp90 or targets of its direct binding partners. Hsp90 activity affected mutations in evolutionarily young genes, segmental deletions, and heterozygotes, highlighting its influence on variation central to evolutionary novelty. Reconciling the diverse epistatic effects of this chaperone, synthetic transcriptional regulation and reconstructions of natural alleles by genome editing revealed a central role for Hsp90 in regulating the fundamental relationship between activity and phenotype. Our findings establish that non-coding variation is a core driver of Hsp90's influence on heredity, offering a mechanistic explanation for the chaperone's strong effects on evolution and development across species.