High throughput measurements of direct activation domain-coactivator interactions.

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Human gene expression is regulated by over two thousand transcription factors and chromatin regulator proteins. Activation domains (ADs) within these proteins recruit shared co-activators and Pol II to genes to activate transcription. However, for many human ADs we do not know which co-activators they recruit, if ADs bind single co-activators specifically or many promiscuously, and why some ADs are stronger at activating gene expression than others. In previous work, we have measured the effect on gene expression for thousands of human ADs and mutants inside cells. Here, we developed a microfluidic platform for expression and purification of hundreds of these ADs in parallel followed by direct measurement of AD/co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). STAMMPPING can reliably quantify interaction strengths ranging across two orders of magnitude (Kds from 0.5 to 50 μM). To date, we used STAMMPPING to measure over 3000 interactions between 204 ADs and 15 co-activators, identifying both specific and promiscuous interactions. Affinities between ADs and individual domains of the co-activator P300 identified ADs that bind multiple domains of the same co-activator, suggesting longer lived total interactions can be achieved with individual short-lived interactions. Complementary mutagenesis of ADs revealed that decreasing their strength of binding to co-activators proportionally decreases their ability to activate gene expression in cells. Our systematic quantification of AD/co-activator interactions advances our understanding of how human ADs activate gene expression. Moreover, direct affinity data produced here will be a rich resource for mapping gene regulatory networks and building interpretable predictive models of AD/co-activator binding.