title: Genome-wide in vitro reconstitution of yeast chromatin with in vivo-like nucleosome positioning.
abstract: Recent genome-wide mapping of nucleosome positions revealed that well-positioned nucleosomes are pervasive across eukaryotic genomes, especially in important regulatory regions such as promoters or origins of replication. As nucleosomes impede access to DNA, their positioning is a primary mode of genome regulation. In vivo studies, especially in yeast, shed some light on factors involved in nucleosome positioning, but there is an urgent need for a complementary biochemical approach in order to confirm their direct roles, identify missing factors, and study their mechanisms. Here we describe a method that allows the genome-wide in vitro reconstitution of nucleosomes with very in vivo-like positions by a combination of salt gradient dialysis reconstitution, yeast whole cell extracts, and ATP. This system provides a starting point and positive control for the biochemical dissection of nucleosome positioning mechanisms.
authors.affiliation: Adolf-Butenandt-Institut, Molecular Biology Unit, University of Munich, Munich, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Krietenstein
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Wippo
authors.forename: Corinna
authors.initials: C
authors.lastname: Lieleg
authors.forename: Philipp
authors.initials: P
authors.lastname: Korber
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000235:genetics*; D042002:Chromatin Assembly and Disassembly; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics; D003956:Dialysis / Q000379:methods; D004330:Drosophila / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D016681:Genome, Fungal; D015698:Genomic Library; D006657:Histones / Q000737:chemistry / Q000235:genetics; D008836:Micrococcal Nuclease / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D012965:Sodium Chloride / Q000737:chemistry; D017186:Titrimetry / Q000379:methods
chemical_list: D002843:Chromatin; D004271:DNA, Fungal; D006657:Histones; D009707:Nucleosomes; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease