Phase separation of DUX family proteins drives totipotent-like state via 3D genome reorganization and retrotransposon activation.

The acquisition of totipotency requires transcriptional activation of endogenous retroviruses (MERVL/HERVL) and zygotic genome activation (ZGA) related genes, yet the molecular mechanisms linking chromatin architecture to this process remain elusive. Here, we demonstrate that mouse Dux and human DUX4, double homeobox transcription factors essential for totipotency, form liquid-liquid phase-separated (LLPS) condensates through conserved arginine residues within intrinsically disordered regions (IDRs) in the Homeobox domain. These condensates recruit CBP/p300 and CTCF to establish super-enhancers (SEs) at MERVL/MT2 loci, enabling H3K27ac deposition and chromatin accessibility. Hi-C analysis revealed that DUX-driven phase separation facilitates 3D genome reorganization, including de novo formation of enhancer-promoter loops and TAD boundary shifts. Disruption of LLPS (DUXR70A) abolished SE assembly, transcriptional activation, and embryonic chimerism. Strikingly, human DUX4 required phase separation for both myotoxic gene activation and cytotoxicity in facioscapulohumeral muscular dystrophy (FSHD) models. Our study establishes a paradigm wherein phase separation integrates transcriptional control with 3D genome remodeling to license totipotency, with direct implications for developmental biology and disease therapy.