Date of this Version
Bio-protocol Vol 5, Iss 21, Nov 05, 2015.
Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae.