In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.
Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination. Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context. Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde cross-linking of intact cells followed by (2) selective immunoprecipitation of protein-DNA complexes with specific antibodies. This method allows for detailed analyses of protein-DNA interactions in a native chromatin environment. Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and protein-DNA interactions in its native setting, will benefit from this technique. In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided. Procedures for protein recovery as well as limitations and extensions of the method are also presented.
Kuo
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MH
Department of Biochemistry and Molecular Genetics and Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA. kuom@pilot.msu.edu
Allis
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CD
D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification; D011233:Precipitin Tests / Q000379:methods*; D015003:Yeasts / Q000235:genetics
D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins