It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation. Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes. We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae . Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation.

Removal could occur by nucleosome disassembly or by sliding of nucleosomes away from the promoter. We activated the PHO5 promoter on chromatin circles following excision from the chromosomal locus. Whereas sliding would conserve the number of nucleosomes on the circle, we found that the number was diminished, demonstrating chromatin remodeling by nucleosome disassembly.

Our characterization of the chromatin transition at the PHO5 promoter led naturally to ask if the same nucleosome dynamics are important in other chromatin contexts. In budding yeast, genes at the silent mating loci are constitutively repressed and it has been shown that proteins required for silencing interact directly with histones, motivating the hypothesis that silencing is accomplished through the assembly and maintenance of a special chromatin structure that is refractory to transcription. To study the mechanism of chromatin-mediated silencing we placed the euchromatic PHO5 gene under the influence of silencers and asked if the well-characterized nucleosomal arrangement of the PHO5 promoter was modified. We report nuclease digestion, topology and ChIP analyses that demonstrate that an additional nucleosome is stabilized on the silenced PHO5 promoter, interfering with activator binding, chromatin remodeling and transcriptional activation. The additional nucleosome is unstable and its loss restores the capacity to remodel chromatin and activate the gene.