title: Assaying chromatin structure and remodeling by restriction enzyme accessibility.
abstract: The packaging of eukaryotic DNA into nucleosomes, the fundamental unit of chromatin, creates a barrier to nuclear processes, such as transcription, DNA replication, recombination, and repair. This obstructive nature of chromatin can be overcome by the enzymatic activity of chromatin remodeling complexes, which create a more favorable environment for the association of essential factors and regulators to sequences within target genes. Here, we describe a detailed approach for analyzing chromatin architecture and remodeling by restriction endonuclease hypersensitivity assay. This procedure uses restriction endonucleases to characterize changes in chromatin that accompany nucleosome remodeling. The specific experimental example described in this article is the BRG1 complex-dependent chromatin remodeling of the steroid hormone-responsive mouse mammary tumor virus promoter. Through the use of these methodologies one is able to quantify changes at specific nucleosomes in response to regulatory signals.
authors.affiliation: Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Trotter
authors.forename: Trevor K
authors.initials: TK
authors.lastname: Archer
mesh_terms: D000818:Animals; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry*; D042002:Chromatin Assembly and Disassembly; D004265:DNA Helicases / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D013194:Staining and Labeling; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D002843:Chromatin; D017931:DNA Primers; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D009841:Oligonucleotides; D014157:Transcription Factors; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; C084108:SMARCA4 protein, human; D004265:DNA Helicases