Transcriptional kinetic synergy: a complex landscape revealed by integrating modelling and synthetic biology

Eukaryotic genes are combinatorially regulated by a diversity of factors, including specific DNA-binding proteins called transcription factors (TFs). Physical interactions between regulatory factors have long been known to mediate synergistic behaviour, commonly defined as deviation from additivity when TFs or sites act in combination. Beyond binding-based interactions, the possibility of synergy emerging from functional interactions between TFs was theoretically proposed, but its governing principles have remained largely unexplored. Theoretically, the interplay between the binding of TFs and their effects over transcription has been challenging to integrate. Experimentally, probing kinetic synergy is easily confounded by physical interactions. Here we circumvent both of these limitations by focusing on a scenario where only one TF can be specifically bound at any given time, which we build using a synthetic biology approach in a mammalian cell line. We develop and analyze a mathematical model that explicitly incorporates the details of the binding of the TFs and their effects over transcription. The model reveals that synergy depends not only on the biochemical activities of the TFs, but also on their binding kinetics. We find experimental evidence for this result in a reporter-based system where fusions of mammalian TFs with engineered zinc fingers bind to a single, shared site. A complex synergy landscape emerges where TF activity, concentration and binding affinity shape the expression response. Our results highlight the relevance of an integrated understanding of TF function in eukaryotic transcriptional control. ### Competing Interest Statement The authors have declared no competing interest.