macroH2A1 drives nucleosome dephasing and genome instability in histone humanized yeast.

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects. [Display omitted] • Expanded toolset for swapping histones in S. cerevisiae • Complementation tests between human replicative histones and their variants • Amino acid swapping experiments between replicative human H2A and macroH2A1.2 • Human macroH2A1.2 alters the structure of yeast chromatin fiber locally and at long range Haase et al. used a genetic swapping assay to replace replicative human histones with non-replicative variants in yeast. H2A.J, TsH2B, and H3.5 complemented their replicative counterparts, while macroH2A1.2 proved toxic, disrupting chromatin organization, increasing nucleosome repeat length, and—when associated with chromosome instability—leading to fitness defects in yeast. [ABSTRACT FROM AUTHOR]