In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in chromatin environment

Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination, Considera ble progress has recently been made in the identification of large, multisu bunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the posit ion of selectively modified or unique histone variants or nonhistone compon ents provides valuable insights into how these proteins (and their modifica tions) function in their normal chromatin context. Here we describe a versa tile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde crosslinking of intact cells followed by (2) selective immuno precipitation of protein-DNA complexes with specific antibodies. This metho d allows for detailed analyses of protein-DNA interactions in a native chro matin 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-d ependent replication and centromere-specific complexes with remarkable prec ision, and to map the in vivo position of reasonably rare transcription fac tors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation te chnique is remarkably versatile and has now been used in a wide range of ce ll types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and prot ein-DNA interactions in its native setting, will benefit from this techniqu e. 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 prese nted. (C) 1999 Academic Press.