Chromatin Immunoprecipitation Experiments to Investigate In Vivo Binding of Arabidopsis Transcription Factors to Target Sequences

For understanding the mechanism of transcriptional regulation, it is essential to know which transcription factor is bound in vivo to the promoter to be analysed. If transcription from a given promoter is regulated by developmental or environmental stimuli, the question of inducible versus constitutive binding has to be answered, particularly if the transcriptional regulator is expressed both under uninduced and induced conditions. Chromatin immunoprecipitation (ChIP) assays constitute the most adequate approach to address these issues as proteins are cross-linked to the DNA before disruption of the tissue. Thus, the DNA–protein interaction is stabilized during purification of the chromatin. The specific DNA–protein complex is immuno-enriched employing specific antibodies against the transcription factor to be analysed. After reversal of cross-links, the recovered DNA is amplified by PCR using specific primers that match sequences flanking the suspected binding site. The amount of PCR product is indicative of the relative abundance of the DNA–protein complex in vivo. A protocol for ChIP assays for Arabidopsis thaliana leaves is described.