Genomic Nucleosome Organization Reconstituted with Pure Proteins

Nils Krietenstein, Megha Wal, Shinya Watanabe, Bongsoo Park, Craig L. Peterson, B. Franklin Pugh, Philipp Korber

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)709-721.e12
JournalCell
Volume167
Issue number3
DOIs
StatePublished - Oct 20 2016

Keywords

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

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Krietenstein, N., Wal, M., Watanabe, S., Park, B., Peterson, C. L., Pugh, B. F., & Korber, P. (2016). Genomic Nucleosome Organization Reconstituted with Pure Proteins. Cell, 167(3), 709-721.e12. https://doi.org/10.1016/j.cell.2016.09.045