Chromatin immunoprecipitation on native chromatin from cells and tissues (PROT22)

In cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications (Luger & Richmond, 1998), such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organisation and gene expression in eukaryotes (Hebbes et al., 1994; O'Neill & Turner, 1995; Grunstein, 1998; Turner, 2000; Jenuwein & Allis, 2001). Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immuno-fluorescence studies on cultured cells have been pivotal in unravelling the roles of histone modifications. These studies have been highly informative on the functions of specific histone modifications in, for instance, pericentric chromatin condensation (Peters, et al., 2001; Maison, et al., 2002) and X-chromosome inactivation (Heard et al., 2001, Boggs et al., 2002, Peters et al., 2002) in mammals (H3 and H4 deacetylation, and H3-K9 methylation). However, particularly in mammalian model systems, it remains poorly understood how histone modifications are organised at specific chromosomal regions and genes. To address in detail what happens at specific sites in vivo, chromatin immuno-precipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on native chromatin extracted from cells, or tissues, to analyse histone methylation and acetylation at specific chromosomal sites. In addition, we present different PCR-based methods that allow the analysis of a locus of interest in chromatin precipitated with antibodies to specific histone marks (see figure 1 for an overview of the described procedures). Should you require a literature reference, please, quote an earlier paper by our group, where these methodologies were originally described (Umlauf et al., 2003).