Insights into Homodimerization of the Transcription Factor ETS1

Abstract

ETS1 is the archetype of the ETS transcription factor (TF) family. ETS TFs share a DNA binding domain, the ETS domain. Because of this all ETS TFs have a core GGA binding site, and thus ETS TFs are found to redundantly regulate the same genes. However, each ETS TF has unique targets as well. One of the prevailing hypotheses explaining this duality is protein-protein interactions, including homodimerization, that allows each TF distinct behavior. The goal of this work was to study the effect of homodimerization on ETS1 specificity. We made mutations in the promoter of MMP3, a gene tightly regulated by ETS1. This promoter has two head-to-head ETS1 binding sites. Binding assays show that both sites are needed for DNA-binding; ETS1 is unable to bind as a monomer in this context. In a crosslinking assay, we also show that ETS1's ability to parse binding sites is based on its free monomeric state, as a mutation made in ETS1's DNA binding helix perturbs its ability to differentiate specific and non-specific targets, apparently by preventing direct contact with the binding site, but also by disrupting autoinhibition, allowing ETS1 to dimerize in the absence of DNA. Here we show that, on the MMP3 site, ETS1 must relieve its autoinhibitory state to bind DNA and must bind as a dimer. As non-specific DNA can induce dimerization, our results indicate how ETS1 finds binding sites on a larger scale. We have also shown that autoinhibition, which was previously thought to only prevent off-target DNA binding, also functions to prevent inappropriate dimerization.