Genomic nucleosome organization reconstituted with pure proteins

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. In this study, nucleosomes were assembled using Salt Gradient Dialysis (SGD) on yeast genomic DNA library. Assembled nucleosomes were either left untreated (labelled as "SGD", control), treated with whole cell extract (WCE), mutant extracts (rsc3ts WCE, isw1 isw2 chd1 WCE), purified remodelers; singly or in combinations (RSC, ISW1a, ISW1b, ISW2, INO80, CHD1, SWI/SNF), combinations of mutant extracts and chromatin remodelers or combination of General Regulatory Factors (Abf1, Reb1) and chromatin remodelers. The resulting nucleosome positions were mapped genome-wide using MNase-(anti-H3-ChIP)-Seq.