title: In vitro reconstitution of in vivo-like nucleosome positioning on yeast DNA.
abstract: Genome-wide nucleosome mapping in vivo highlighted the extensive degree of well-defined nucleosome positioning. Such positioned nucleosomes, especially in promoter regions, control access to DNA and constitute an important level of genome regulation. However, the molecular mechanisms that lead to nucleosome positioning are far from understood. In order to dissect this mechanism in detail with biochemical tools, an in vitro system is necessary that can generate proper nucleosome positioning de novo. We present a protocol that allows the assembly of nucleosomes with very much in vivo-like positioning on budding yeast DNA, either of single loci or of the whole-genome. Our method combines salt gradient dialysis and incubation with yeast extract in the presence of ATP. It provides an invaluable tool for the study of nucleosome positioning mechanisms, and can be used to assess the relative stability of properly positioned nucleosomes. It may also generate more physiological templates for in vitro studies of, e.g., nucleosome remodeling or transcription through chromatin.
authors.affiliation: Molecular Biology Unit, Adolf-Butenandt-Institut, University of Munich, Munich, Germany.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Wippo
authors.forename: Philipp
authors.initials: P
authors.lastname: Korber
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D042002:Chromatin Assembly and Disassembly; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004331:Drosophila melanogaster; D056426:Genetic Loci / Q000235:genetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D009707:Nucleosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D012492:Salts
chemical_list: D004271:DNA, Fungal; D009707:Nucleosomes; D012492:Salts; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease