High-throughput data and modeling reveal insights into the mechanisms of cooperative DNA-binding by transcription factor proteins (ETS1 and RUNX1)

Cooperative DNA-binding by transcription factor (TF) proteins is critical for gene expression regulation in eukaryotes. In the human genome, many regulatory regions contain TF binding sites in close proximity to each other, which can facilitate cooperative interactions. However, binding site proximity does not necessarily imply cooperative binding, as two TFs could also bind independently to each of their neighboring target sites. Currently, the rules that drive cooperative versus independent binding of TFs to neighboring sites are not well understood. In addition, it is oftentimes difficult to infer direct cooperativity between TFs from existing DNA-binding data. Here, we show that in vitro binding assays using DNA libraries of a few thousand genomic sequences with putative cooperative TF binding events can be used to develop accurate models of cooperativity and to gain insights into cooperative binding mechanisms. Using human factors ETS1 and RUNX1 as our case study, we show that the distance and orientation between ETS1 sites are critical determinants of cooperative ETS1-ETS1 binding, while cooperative ETS1- RUNX1 interactions show more flexibility in distance and orientation and can be accurately predicted based on binding affinity and the sequence/shape features of the binding sites. The approach described here, combining custom experimental design with machine learning modeling, can be easily applied to study the cooperative DNA-binding patterns of any TFs. We performed cooperative gcPBM (genome-context protein binding microarray) experiments for recombinant, full-length, human transcription factors Ets1 and Runx1. Briefly, the PBMs involved binding of GST-tagged Ets1 and/or GST-tagged Runx1 to double-stranded 4x180k Agilent microarrays in order to identify Ets1-Runx1 cooperative binding events. Each genomic DNA sequence represented on the array is present in 8 replicate spots, four for each orientation. We report the PBM signal intensity of Ets1 and/or Runx1 for each spot.