Identification of DNA-Binding of Tumor Suppressor Genes by Chromatin Immunoprecipitation

Abstract

The processes of DNA replication and transcription are intimately affected by the status of chromatin structure. Proteins bind and dissociate at rapid rates in order to control the levels of activity at the genome. Much work has been done over the years to determine the function of transcription factors and proteins that control the speed and fidelity of DNA replication. However, the bulk of these studies have attempted simply to correlate the expression levels of a DNA-binding protein with the function attributed to it, and not necessarily the dynamics of actual association with DNA. Indeed in the field of transcriptional control, proteins have been shown to bind a consensus sequence in the promoter region of a particular gene, but these experiments are performed entirely in vitro, out of context with the genomic structure of the actual promoter region of the gene (1). In the last few years, with the advent of highly specific antibodies, the development of the chromatin immunoprecipitation assay has been a major step forward in understanding the true in-vivo nature of and dynamics of DNA protein binding (2–4). This technique allows the covalent cross-linking of DNA-binding proteins to chromatin and coincident precipitation of the DNA fragment with the protein upon antibody binding. Here we describe a protocol for the immunoprecipitation of transcription factors bound to promoter sequences in cells, and use the p53 tumor suppressor as an example (Fig. 1).