Transcription activation is enhanced by multivalent interactions independent of phase separation

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation. [Display omitted] •Strongly activating TFs have a high propensity to engage in multivalent interactions•Formation of liquid-like TF droplets does not enhance transcription activation•Longer TF residence time increases transcription independent of site occupancy•High TF multivalency promotes recruitment of transcription coactivators Phase separation of TFs into condensates has been proposed to enhance transcription activation. Trojanowski et al. show that a high propensity of TFs to engage in multivalent interactions is needed for their full activation capacity independent of TF assembly into phase-separated liquid-like droplets.