Mapping histone modifications by nucleosome immunoprecipitation.
Studies of histone modification patterns and their role in gene regulation have led to the proposal that there is a "histone code." We have developed a method for nucleosome immunoprecipitation that can precisely identify the specific nucleosomes that carry a posttranslational modification of interest. The process involves the isolation and micrococcal nuclease digestion of minichromosomes to generate nucleosome core particles. These are then used in immunoprecipitation reactions with an antibody directed against the histone modification of interest. Subsequently, nucleosome core particle DNA is purified and end labeled. The original locations of the nucleosomes in the immunoprecipitate can be determined at low resolution (using a modified Southern blot hybridization procedure) or at maximal resolution (using the monomer extension method). Using the latter method, the positions of specific nucleosomes that carry the posttranslational modification of interest can be identified precisely. This method is sensitive, provides maximal resolution, and is inexpensive. The approach described here may serve as a paradigm for the study of histone-modifying patterns.
Clark
David J
DJ
Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland, USA.
Shen
Chang-Hui
CH
D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods
D006657:Histones; D009707:Nucleosomes