Multilayered specificity of transcription factor binding at cytokine promoters

Transcription factors (TFs) regulate gene expression through sequence-specific DNA binding, and their genomic occupancy is further influenced by TF expression, activation state, and protein-protein interactions. How these mechanisms determine context-specific gene regulation remains incompletely understood, particularly for tightly controlled immune genes such as cytokines. Here, we use paired yeast one-hybrid (pY1H) assays to systematically examine DNA binding of 236 TFs and 392 TF-pairs across 106 cytokine gene promoters. Of the 1,619 TF-promoter interactions identified, 555 required TF cooperativity and 410 were antagonized by at least one TF partner, suggesting that TF-DNA binding is highly dependent on TF partners. Usage of different partners can drastically alter a TF’s target repertoire and may result in the recruitment of different transcriptional cofactors. Integration with existing data on TF expression and activation further showed that cooperativity and antagonism provide additional, underappreciated layers of DNA-binding specificity.