ABSTRACT
Gene-environment interactions have long been theorized to influence molecular evolution. However, the environmental dependence of most mutations remains unknown. Using deep mutational scanning, we engineered yeast with all 44,604 single codon changes encoding 14,160 amino acid variants in Hsp90 and quantified growth effects under standard conditions and under five stress conditions. To our knowledge, these are the largest determined comprehensive fitness maps of point mutants. The growth of many variants differed between conditions, indicating that environment can have a large impact on Hsp90 evolution. Multiple variants provided growth advantages under individual conditions; however, these variants tended to exhibit growth defects in other environments. The diversity of Hsp90 sequences observed in extant eukaryotes preferentially contains variants that supported robust growth under all tested conditions. Rather than favoring substitutions in individual conditions, the long-term selective pressure on Hsp90 may have been that of fluctuating environments, leading to robustness under a variety of conditions.
Subject(s)
Gene Expression Regulation, Fungal/physiology , Gene-Environment Interaction , HSP90 Heat-Shock Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Adaptation, Physiological , Epistasis, Genetic , Genetic Fitness , HSP90 Heat-Shock Proteins/genetics , Models, Molecular , Mutation , Protein Conformation , Saccharomyces cerevisiae Proteins/genetics , Stress, PhysiologicalABSTRACT
Biology has, and continues to be, shaped by evolutionary mechanisms. Within the past decade, local fitness landscapes have become experimentally tractable and are providing new perspectives on evolutionary mechanisms. Powered by next-generation sequencing, the impacts of all individual amino acid substitutions on function have been quantified for dozens of proteins. These fitness maps have been utilized to investigate the biophysical underpinnings of existing protein function as well as the appearance and enhancement of new protein functions. This review highlights emerging trends from this rapidly growing area of research, including an expanded understanding of the biophysical mechanisms underlying existing and new protein function, the roles epistasis and adaptation play in shaping evolution, and the prediction of disease-causing alleles in humans.
Subject(s)
Adaptation, Physiological/genetics , Epistasis, Genetic , Evolution, Molecular , Genetic Fitness , Mitogen-Activated Protein Kinase 1/genetics , Amino Acid Substitution , Drug Resistance/genetics , High-Throughput Nucleotide Sequencing , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mutation , Protein Conformation , Protein Folding , Protein StabilityABSTRACT
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.
