title: Genomic Nucleosome Organization Reconstituted with Pure Proteins.
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.
authors: Molecular Biology Division, Biomedical Center, LMU Munich, 82152 Planegg-Martinsried near Munich, Germany.
authors: Nils
authors: N
authors: Krietenstein
authors: Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors: Megha
authors: M
authors: Wal
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Shinya
authors: S
authors: Watanabe
authors: Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors: Bongsoo
authors: B
authors: Park
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Craig L
authors: CL
authors: Peterson
authors: Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. Electronic address: bfp2@psu.edu.
authors: B Franklin
authors: BF
authors: Pugh
authors: Molecular Biology Division, Biomedical Center, LMU Munich, 82152 Planegg-Martinsried near Munich, Germany. Electronic address: pkorber@lmu.de.
authors: Philipp
authors: P
authors: Korber
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D042002:Chromatin Assembly and Disassembly; D004271:DNA, Fungal / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D016681:Genome, Fungal; D006657:Histones / Q000737:chemistry / Q000235:genetics; D009707:Nucleosomes / Q000737:chemistry* / Q000235:genetics*; D011067:Poly dA-dT / Q000737:chemistry; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
chemical_list: C061886:ABF1 protein, S cerevisiae; C090297:AFT1 protein, S cerevisiae; D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D006657:Histones; C483404:INO80 complex, S cerevisiae; C086468:ISWI protein; D009707:Nucleosomes; C065681:REB1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011067:Poly dA-dT; D000251:Adenosine Triphosphatases
keywords: Abf1; INO80; Isw; RSC; Reb1; Saccharomyces cerevisiae; chromatin; general regulatory factors (GRFs); in vitro reconstitution; nucleosome positioning and remodeling