Biochemical assay for histone H2A.Z replacement by the yeast SWR1 chromatin remodeling complex.
The evolutionarily conserved histone variant H2A.Z has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. Swr1 is required for the deposition of histone H2A.Z at stereotypical promoter locations in vivo, and Swr1 and H2A.Z commonly regulate a subset of yeast genes. Here, we describe an integrated nucleosome assembly-histone replacement system whereby histone exchange by chromatin remodeling activities may be analyzed in vitro. The system demonstrates ATP- and SWR1-complex-dependent replacement of histone H2A for histone H2A.Z on a preassembled nucleosome array. This system may also be adapted to analyze dynamic interactions between chromatin remodeling and modifying enzymes, histone chaperones, and nucleosome substrates containing canonical, variant, or covalently modified histones.
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA. mizuguchim@janelia.hhmi.org
Gaku
G
Mizuguchi
Wei-Hua
WH
Wu
Samar
S
Alami
Ed
E
Luk
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification; D061925:Immobilized Nucleic Acids / Q000737:chemistry; D056490:Nucleosome Assembly Protein 1 / Q000737:chemistry / Q000302:isolation & purification; D009707:Nucleosomes / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D006657:Histones; C493530:Htz1 protein, S cerevisiae; D061925:Immobilized Nucleic Acids; C533556:NAP1 protein, S cerevisiae; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; C470546:ISW1 protein, S cerevisiae; C481261:Swr1 protein, S cerevisiae