Single molecule imaging reveals the collective and independent search mechanisms of cFos and cJun on DNA

AP-1 proteins are members of the basic leucine zipper (bZIP) family of dimeric transcription factors, which facilitate a multitude of cellular processes, but are primarily known for their oncogenic potential in several cancer types. The oncogenic transcription factor AP-1 binds a specific DNA target site (5TCA[G/C]TGA), however the physical mechanism of how this is achieved has not been determined. The archetypal AP-1 complex is formed by cFos and cJun, which heterodimerize via their leucine zipper domains. We investigated the DNA-binding bZIP domains of AP-1 interacting with DNA tightropes using real-time single molecule fluorescence imaging in vitro. We find that AP-1 bZIP domains comprising cFos:cJun and cJun:cJun rapidly scan DNA using a 1D diffusional search with average diffusion constants of 0.14 m2s-1 and 0.26 m2s-1 respectively. We also report for the first time that cFos is able to bind to and diffuse on DNA (0.29 m2s-1) as a mixed population of monomers and homodimers, despite previous studies suggesting that it is incapable of independent DNA binding. Additionally, we note increased pause lifetimes for the cFos:cJun heterodimer compared to the cJun:cJun homodimer, and were able to detect distinct pausing behaviours within diffusion data. Understanding how cFos:cJun and other transcription factors identify their targets is highly relevant to the development of new therapeutics which target DNA binding proteins using these search mechanisms.