Genome-wide in vitro reconstitution of yeast chromatin with in vivo-like nucleosome positioning

Methods Enzymol. 2012;513:205-32. doi: 10.1016/B978-0-12-391938-0.00009-4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Chromatin / chemistry
  • Chromatin / genetics*
  • Chromatin Assembly and Disassembly
  • DNA Restriction Enzymes / chemistry
  • DNA, Fungal / chemistry*
  • DNA, Fungal / genetics
  • Dialysis / methods
  • Drosophila / chemistry
  • Drosophila / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Electroporation / methods
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Genome, Fungal*
  • Genomic Library
  • Histones / chemistry
  • Histones / genetics
  • Micrococcal Nuclease / chemistry
  • Nucleic Acid Conformation
  • Nucleosomes / chemistry
  • Nucleosomes / genetics*
  • Plasmids / chemistry
  • Plasmids / genetics
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics*
  • Sodium Chloride / chemistry
  • Titrimetry / methods

Substances

  • Chromatin
  • DNA, Fungal
  • Histones
  • Nucleosomes
  • Sodium Chloride
  • Adenosine Triphosphate
  • DNA Restriction Enzymes
  • Micrococcal Nuclease