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 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, we show that this distinct phase behavior can arise from TF-chromatin interactions, using engineered fusion proteins with tunable IDR-DBD architectures. 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 on overexpression forms large, spherical condensates in cells, its fusion with FOXA1 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.