Cancer histone H2A.Z missense mutations disrupt function through distinct local and allosteric effects.

H2A.Z is a conserved variant of histone H2A that functions in a wide range of processes including transcriptional, chromatin organization, and genome stability, but whose exact role in the cell remains incompletely understood. A growing body of literature highlights the utility of cancer histone missense mutations in revealing fundamental aspects of histone structure and function not captured by previous efforts including comprehensive alanine scans. Motivated by this work, we systematically examined the impact of cancer missense mutations affecting H2A.Z using Saccharomyces cerevisiae as a model system. This work led us to identify amino acids critical for normal H2A.Z function and the mechanisms by which mutations at those sites disrupt H2A.Z activity. Combining approaches from the fields of genetics, molecular biology, biochemistry, and biophysics, we found that cancer H2A.Z mutations show decreased genome-wide occupancy, disrupt interactions with DNA, other histones, and nucleosome-binding proteins, and decrease nucleosome stability. Importantly, we show that these effects are recapitulated in human cell lines carrying histone H2A.Z mutations, highlighting yeast as a powerful system to begin to understand the impact of cancer histone mutations. Collectively, our work identified previously unappreciated amino acids that are critical for normal H2A.Z function, revealing functional consequences of altered nucleosome structure and dynamics.