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.

Summary Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here we reconstitute four stages of nucleosome architecture using purified components: Yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.

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Eukaryotic DNA is packaged into chromatin where nucleosomes form the basic building unit. Knowing the precise positions of nucleosomes is important because they determine the accessibility of underlying regulatory DNA sequences. Here we describe a detailed method to map on a genomic scale the locations of nucleosomes with very high resolution. Micrococcal nuclease (MNase) digestion followed by chromatin immunoprecipitation and facilitated library construction for deep sequencing provides a simple and accurate map of nucleosome positions.

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