Yes-associated Protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized YAP’s transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced transcription of YAP-specific proliferation genes. Super-resolution imaging using Assay for Transposase Accessible Chromatin with photoactivated localization microscopy (ATAC-PALM) revealed that YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA Polymerase II (RNAPII), the accessible chromatin domains later acquired RNAPII, transcribing RNA. Removal of YAP’s intrinsically-disordered transcription activation domain (TAD) prevented YAP condensate formation and diminished downstream YAP signaling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

Weakly bound protein complexes play a crucial role in metabolic, regulatory, and signaling pathways, due in part to the high tunability of their bound and unbound populations. This tunability makes weak binding (micromolar to millimolar dissociation constants) difficult to quantify under biologically relevant conditions. Here, we use rapid perturbation of cell volume to modulate the concentration of weakly bound protein complexes, allowing us to detect their dissociation constant and stoichiometry directly inside the cell. We control cell volume by modulating media osmotic pressure and observe the resulting complex association and dissociation by FRET microscopy. We quantitatively examine the interaction between GAPDH and PGK, two sequential enzymes in the glycolysis catalytic cycle. GAPDH and PGK have been shown to interact weakly, but the interaction has not been quantified in vivo. A quantitative model fits our experimental results with log K d = −9.7 ± 0.3 and a 2:1 prevalent stoichiometry of the GAPDH:PGK complex. Cellular volume perturbation is a widely applicable tool to detect transient protein interactions and other biomolecular interactions in situ. Our results also suggest that cells could use volume change (e.g., as occurs upon entry to mitosis) to regulate function by altering biomolecular complex concentrations.quinary interactions | protein-protein interactions | cell volume | FRET | live-cell microscopy F rom forming active enzymatic complexes to facilitating signal transduction in regulatory networks, protein interactions are pivotal to cell function. Strong interactions, with dissociation constants (K d ) of nanomolar and lower (1, 2), can be measured accurately both in vitro and in vivo (3, 4). These interactions are ideal in cases where the complex should form with high fidelity and persist. The downside of strong binding is that its regulation in the cellular environment is limited: Once a complex is formed, it seldom dissociates.Another type of protein complexes relies on weak, transient interactions, collectively termed quinary interactions (5-7). Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9). Their transient nature is key to their function: Unlike tightly bound complexes, quinary interactions are highly sensitive to variations in their environment and respond rapidly to changes in temperature, pressure, pH, or the local concentration of surrounding molecules. The sensitivity of quinary interactions makes them important in fine-tuning cellular processes. For example, it has been proposed that sequential metabolic enzymes could associate to improve substrate transfer between catalytic enzymes (10), that weak protein association can mediate cytokine release (11), or that phase-separated protein droplets held together by quinary interactions could serve for cellular storage or stress response (12, 13).Despite growing interest, quantification of quinary inte...

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Intrinsically disordered protein-regions (IDRs) make up roughly 30% of the human proteome and are central to a wide range of biological processes. Given a lack of persistent tertiary structure, all residues in IDRs are, to some extent, solvent exposed. This extensive surface area, coupled with the absence of strong intramolecular contacts, makes IDRs inherently sensitive to their chemical environment. We report a combined experimental, computational, and analytical framework for high-throughput characterization of IDR sensitivity. Our framework reveals that IDRs can expand or compact in response to changes in their solution environment. Importantly, the direction and magnitude of conformational change depend on both protein sequence and cosolute identity. For example, some solutes such as short polyethylene glycol chains exert an expanding effect on some IDRs and a compacting effect on others. Despite this complex behavior, we can rationally interpret IDR responsiveness to solution composition changes using relatively simple polymer models. Our results imply that solution-responsive IDRs are ubiquitous and can provide an additional layer of regulation to biological systems.