
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.19.608698
preprint
1
Article
High-throughput affinity measurements of direct interactions between activation domains and co-activators
DelRosso Nicole http://orcid.org/0000-0003-4339-3353

Suzuki Peter H. http://orcid.org/0000-0002-6108-0273

Griffith Daniel http://orcid.org/0000-0002-9633-9601

Lotthammer Jeffrey M. http://orcid.org/0000-0002-5022-7006

Novak Borna
Kocalar Selin http://orcid.org/0000-0003-4521-8647

Sheth Maya U.
Holehouse Alex S. http://orcid.org/0000-0002-4155-5729

Bintu Lacramioara http://orcid.org/0000-0001-5443-6633

Fordyce Polly http://orcid.org/0000-0002-9505-0638

20 8 2024
2024.08.19.608698https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.19.608698
nihpp-2024.08.19.608698.pdf
Abstract

Sequence-specific activation by transcription factors is essential for gene regulation 1,2 . Key to this are activation domains, which often fall within disordered regions of transcription factors 3,4 and recruit co-activators to initiate transcription 5 . These interactions are difficult to characterize via most experimental techniques because they are typically weak and transient 6,7 . Consequently, we know very little about whether these interactions are promiscuous or specific, the mechanisms of binding, and how these interactions tune the strength of gene activation. To address these questions, we developed a microfluidic platform for expression and purification of hundreds of activation domains in parallel followed by direct measurement of co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). By applying STAMMPPING to quantify direct interactions between eight co-activators and 204 human activation domains (>1,500 K d s), we provide the first quantitative map of these interactions and reveal 334 novel binding pairs. We find that the metazoan-specific co-activator P300 directly binds >100 activation domains, potentially explaining its widespread recruitment across the genome to influence transcriptional activation. Despite sharing similar molecular properties ( e.g. enrichment of negative and hydrophobic residues), activation domains utilize distinct biophysical properties to recruit certain co-activator domains. Co-activator domain affinity and occupancy are well-predicted by analytical models that account for multivalency, and in vitro affinities quantitatively predict activation in cells with an ultrasensitive response. Not only do our results demonstrate the ability to measure affinities between even weak protein-protein interactions in high throughput, but they also provide a necessary resource of over 1,500 activation domain/co-activator affinities which lays the foundation for understanding the molecular basis of transcriptional activation.
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pmc
