Cell
Volume 167, Issue 3, 20 October 2016, Pages 709-721.e12
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Article
Genomic Nucleosome Organization Reconstituted with Pure Proteins

https://doi.org/10.1016/j.cell.2016.09.045Get rights and content
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Highlights

  • Genome-wide reconstitution of promoter nucleosome organization with purified factors

  • DNA alone guides RSC and INO80 remodelers to create nucleosome-free regions (NFRs)

  • GRFs (“barriers”), DNA sequence/shape, and remodelers position −1/+1 nucleosomes

  • Remodelers create arrays with characteristic spacing

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.

Keywords

chromatin
nucleosome positioning and remodeling
in vitro reconstitution
Saccharomyces cerevisiae
Isw
INO80
RSC
general regulatory factors (GRFs)
Abf1
Reb1

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