Two-Hybrid Systems

José A. Jarillo, Dorota N. Komar, Manuel Piñeiro, Jarillo, José A., Komar, Dorota N., Piñeiro, Manuel

Two-hybrid systems allow for the identification of proteins that physically interact in the context of biological processes. In the cases where these proteins interact with DNA it is essential to define their binding properties with specific regions of the genome to shed light on the intricate gene regulatory networks that modulate the biological response of interest. The chromatin immunoprecipitation (ChIP) protocol described here provides a powerful means to identify the DNA-binding sites of transcription factors, proteins involved in chromatin remodeling processes, or histone marks that modulate gene expression in eukaryotes and specifically in plants like the model species Arabidopsis thaliana. This procedure involves the in vivo fixation of protein-DNA complexes, the physical fragmentation of chromatin with ultrasounds, the specific immunoprecipitation of protein-DNA complexes, and the use of quantitative PCR techniques for the relative quantification of the DNA sequences associated with the proteins of study. This valuable methodology has contributed significantly to a better understanding of the gene expression regulatory mechanisms underlying the control of a variety of biological processes in Arabidopsis.