Chromatin Binding Regulates Phase Behavior and Morphology of Condensates Formed by Prion-like Domains

Many transcription factors (TFs) contain intrinsically disordered regions (IDRs) and are thought to form biomolecular condensates in the cell nucleus. These proteins can be conceptualized as block co-polymers, with the IDRs driving both homotypic and heterotypic protein–protein interactions and the DNA-binding domain (DBD) mediating heterotypic interactions with chromatin. While in vitro studies have predominantly reported micron-scale, spherical condensates in the absence of chromatin, TF condensates in live cells exhibit strikingly different behavior, adopting diverse, nanoscale, often aspherical morphologies and displaying sub-diffusive dynamics. Here, using engineered fusion proteins with tunable IDR-DBD architectures, we show that this distinct phase behavior can arise from TF-chromatin interactions. Specifically, we fused the prion-like domain (PLD) of the SS18 subunit from the mammalian SWI/SNF complex, a domain known to drive homotypic phase separation, to the DBD of the pioneer factor FOXA1. While SS18^PLD on overexpression forms large, spherical condensates in cells, its fusion with FOXA1^DBD leads to condensates that re-localize to chromatin, adopt aspherical morphologies, and exhibit chromatin-wetting behavior. Disruption of DBD-chromatin binding shifts condensate morphology toward a mixed or spherical state, implicating chromatin affinity as a key regulator of condensate coarsening and spatial organization. Coarse-grained simulations recapitulate these observations, revealing a finely balanced interplay between PLD-PLD and DBD-DNA interactions that collectively determine condensate dynamics and structure. Together, our findings demonstrate that chromatin binding is a critical modulator of transcriptional condensate behavior in vivo.