Heterochromatic domains are complex structures composed of nucleosome arrays that are bound by silencing factors. This composition raises the possibility that certain configurations of nucleosome arrays facilitate heterochromatic silencing. We tested this possibility in by systematically altering the distance between heterochromatic nucleosome-depleted regions (NDRs), which is predicted to affect local nucleosome positioning by limiting how nucleosomes can be packed between NDRs. Consistent with this prediction, serial deletions that altered the distance between heterochromatic NDRs revealed a striking oscillatory relationship between inter-NDR distance and defects in nucleosome positioning. Furthermore, conditions that caused poor nucleosome positioning also led to defects in both heterochromatin stability and the ability of cells to generate and inherit epigenetic transcriptional states. These findings strongly suggest that nucleosome positioning can contribute to formation and maintenance of functional heterochromatin and point to previously unappreciated roles of NDR positioning within heterochromatic domains.
