Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants.
Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.
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