Improvement of the chromatin immunoprecipitation (ChIP) assay by DNA fragment size fractionation

摘要

The chromatin immunoprecipitation (CHIP) assay is a valuable technique that allows researchers to establish what proteins (e.g., transcription factors) bind in vivo to a specific region of DNA, usually a gene promoter (1). In vitro proteinDNA binding assays, such as the electrophoretic mobility shift assay, are useful in identifying potential transcription factors that bind to a specific DNA sequence and regulate transcription of a gene. However, it is not unusual to identify several different transcription factors, or isoforms of a transcription factor, that can bind, at least in vitro, to a particular sequence. Thus, determining which transcription factor is physiologically relevant, or determining whether the transcription factor bound to a promoter region changes under different physiological or pathophysiological conditions, can be difficult if not impossible without some in vivo analysis. The ChIP assay, which involves (i) crosslinking proteins to DNA with formaldehyde, (ii) shearing of DNA in small fragments, (iii) immunoprecipitation of fragments with an antibody directed against a known transcription factor, and (iv) PCR amplification of the DNA region containing the sequence of interest, offers the ability to obtain a "snapshot" of which proteins are bound to specific cis-elements in vivo.