The biogenesis of nucleosome arrays and their decline during cellular aging

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Nucleosomes and nucleosome remodeling enzymes are crucial to human health. Aging, for instance, disrupts the nucleosome landscape, destabilizing the genome, and mutations in nucleosome remodelers are drivers of cancers. Large fractions of the genome assemble into nucleosomes. Most nucleosomes are thought to arrange on DNA like beads on a string, forming arrays of evenly spaced nucleosomes. ISWI, Chd1 and -as we recently proposed- INO80 nucleosome remodeling enzymes appear to catalyze even spacing.Even spacing between nucleosomes is surprisingly difficult to measure because most assays, incl. MNase-seq, must destroy arrays to detect nucleosomes, so that only indirect evidence for even spacing is provided. Most techniques also cannot detect the fraction of DNA at a certain genomic location that is bound by a nucleosome, termed the nucleosome occupancy. We and others have developed long-read nucleosome mapping techniques as they offer a solution to these problems. They natively detect nucleosome occupancy and carry the potential to reveal even spacing between nucleosomes on individual DNA molecules. We employ enzymes that methylate the DNA in between nucleosomes. Long-read sequencing then directly detects the positions of methylated and unmethylated bases, and thereby reveals the footprints of nucleosomes.This footprinting technology will be instrumental throughout this proposal. In Aim 1, we exploit our engineered yeast strain, in which INO80’s activity can be cleanly isolated. By methylation footprinting, we will directly confirm our hypothesis that INO80 evenly spaces nucleosomes in vivo. We will then engage in a structure-function analysis of INO80’s remodeling activity in vivo.Aim 2 dissects transcription, a process we find to be deeply disruptive to nucleosome arrays. We will critically test our model that transcription destroys even spacing between nucleosomes, and assess decade-old hypotheses that transcription partially or even fully evicts nucleosomes. We do so by rapid depletion of Polymerase II and by visualizing the changes to the nucleosome landscape that take place during rapid, stress-induced reprogramming of the transcriptome.In Aim 3 we reveal how cells cope with drastic nucleosome loss, which is proposed to occur during aging. We will directly visualize changes to the nucleosome landscape during aging, including concomitant nucleosome loss, in both yeast and human primary cells. We also test our hypothesis that nucleosome remodelers act as guardians of the yeast genome against premature aging, and follow changes to the nucleosome landscape upon rejuvenation of aged human cells. By contrasting results from yeast and human, we will derive conserved principles of aging across eukaryotes.Our dissections of enzymes generating and destroying nucleosome arrays may serve as a blueprint for studying related enzymes in the future. The results carry the potential to better understand and treat age-related symptoms and diseases.