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Background  Establishing transcriptional regulatory networks that include protein-protein and protein-DNA interactions has become a key component to better understanding many fundamental biological processes. Although a variety of techniques are available to expose protein-protein and protein-DNA interactions, unequivocally establishing whether two proteins are targeted together to the same promoter or DNA molecule poses a very challenging endeavour. Yet, the recruitment of multiple regulatory proteins simultaneously to the same promoter provides the basis for combinatorial transcriptional regulation, central to the transcriptional regulatory network of eukaryotes. The serial ChIP (sChIP) technology was developed to fill this gap in our knowledge, and we illustrate here its application in plants.    Results  Here we describe a modified sChIP protocol that provides robust and quantitative information on the co-association or exclusion of DNA-binding proteins on particular promoters. As a proof of principle, we investigated the association of histone H3 protein variants with modified tails (H3K9ac and H3K9me2) with  Arabidopsis  RNA polymerase II (RNPII) on the promoter of the constitutively expressed  actin  gene (At5g09810), and the trichome-expressed  GLABRA3  ( GL3 ) gene. As anticipated, our results show a strong positive correlation between H3K9ac and RNPII and a negative correlation between H3K9me2 and RNPII on the actin gene promoter. Our findings also establish a weak positive correlation between both H3K9ac and H3K9me2 and RNPII on the  GL3  gene promoter, whose expression is restricted to a discrete number of cell types. We also describe mathematical tools that allow the easy interpretation of sChIP results.    Conclusion  The sChIP method described here provides a reliable tool to determine whether the tethering of two proteins to the same DNA molecule is positively or negatively correlated. With the increasing need for establishing transcriptional regulatory networks, this modified sChIP method is anticipated to provide an excellent way to explore combinatorial gene regulation in eukaryotes.

Serial ChIP as a tool to investigate the co-localization or exclusion of proteins on plant genes