Mechanism of nucleosome positioning, spacing and regularity in Saccharomyces cerevisiae

Numerous nucleosome remodeling enzymes tightly regulate nucleosome positions in eukaryotic cells. Transcription and statistical positioning of nucleosomes may also contribute to proper nucleosome organization. Individual contributions remain controversial due to strong redundancy of processes acting on the nucleosome landscape. By incisive yeast genome engineering we radically decreased their redundancy. We find the transcriptional machinery to be disruptive of evenly spaced nucleosomes, and proper nucleosome density critical for their biogenesis. INO80 spaces nucleosomes in vivo and positions the first nucleosome covering genes. It requires its Arp8 and Ies2 subunits, but unexpectedly not the Nhp10 module, for spacing. Whereas H2A.Z stimulates INO80 in vitro, its presence is dispensable for INO80 +1 positioning function in vivo. DNA damage, recombination and transposon integration assays suggest that evenly spaced nucleosomes protect cells against genotoxic stress. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome. MNase digestion of yeast nuclei followed by paired-end sequencing on following strains: 1. RNA Polymerase II and INO80 depletion in all 3 possible combinations in W303 wild type yeast and a mutant yeast lacking Isw1, Isw2 and Chd1 (TKO) for different time points; 2. Deletion of several subunits of yeast INO80 complex in WT, TKO and TKO depleted with RNA Polymerase II; 3. Histone depletion via Gal1-10 promoter in WT and TKO; ATAC-seq of WT, TKO and arp8Δ strains; 4. Depletion of TBP in TKO cells; 5. Depletion of RNA Polymerase II in TKO cells after G1 arrest.