Genomic Nucleosome Organization Reconstituted with Pure Proteins

Cell. 2016 Oct 20;167(3):709-721.e12. doi: 10.1016/j.cell.2016.09.045.


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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Chromatin / chemistry
  • Chromatin / genetics
  • Chromatin Assembly and Disassembly*
  • DNA, Fungal / chemistry
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • Genome, Fungal
  • Histones / chemistry
  • Histones / genetics
  • Nucleosomes / chemistry*
  • Nucleosomes / genetics*
  • Poly dA-dT / chemistry
  • Protein Biosynthesis
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Transcription Factors / chemistry
  • Transcription Factors / genetics


  • ABF1 protein, S cerevisiae
  • AFT1 protein, S cerevisiae
  • Chromatin
  • DNA, Fungal
  • DNA-Binding Proteins
  • Histones
  • INO80 complex, S cerevisiae
  • ISWI protein
  • Nucleosomes
  • REB1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Poly dA-dT
  • Adenosine Triphosphatases