Near the 5′ end of most eukaryotic genes, nucleosomes form highly regular arrays that begin at canonical distances from the transcriptional start site. Determinants of this and other aspects of genomic nucleosome organization have been ascribed to statistical positioning, intrinsically DNA-encoded positioning, or some aspect of transcription initiation. Here, we provide evidence for a different explanation. Biochemical reconstitution of proper nucleosome positioning, spacing, and occupancy levels was achieved across the 5′ ends of most yeast genes by adenosine triphosphate–dependent trans-acting factors. These transcription-independent activities override DNA-intrinsic positioning and maintain uniform spacing at the 5′ ends of genes even at low nucleosome densities. Thus, an active, nonstatistical nucleosome packing mechanism creates chromatin organizing centers at the 5′ ends of genes where important regulatory elements reside.

We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength.

Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo‐like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo‐like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome‐wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex.

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Genome-wide nucleosome mapping in vivo highlighted the extensive degree of well-defined nucleosome positioning. Such positioned nucleosomes, especially in promoter regions, control access to DNA and constitute an important level of genome regulation. However, the molecular mechanisms that lead to nucleosome positioning are far from understood. In order to dissect this mechanism in detail with biochemical tools, an in vitro system is necessary that can generate proper nucleosome positioning de novo. We present a protocol that allows the assembly of nucleosomes with very much in vivo-like positioning on budding yeast DNA, either of single loci or of the whole-genome. Our method combines salt gradient dialysis and incubation with yeast extract in the presence of ATP. It provides an invaluable tool for the study of nucleosome positioning mechanisms, and can be used to assess the relative stability of properly positioned nucleosomes. It may also generate more physiological templates for in vitro studies of, e.g., nucleosome remodeling or transcription through chromatin.