Here, we identify TF pairs that bind cooperatively to DNA, and characterize their spacing and orientation preferences using a novel method, consecutive affinity purification SELEX (CAP-SELEX). The analysis of 9,400 potential interactions revealed 315 co-operative TF–TF pairs that recognized 618 heterodimeric motifs, most of which had not been previously described. Importantly, binding of two TFs in close proximity resulted in changes in their binding specificity, which could not be predicted from their individual motifs, suggesting that the gene regulatory code is more complex than what previously have been appreciated.
