Abstract

Brown adipose tissue (BAT) dissipates energy through non-shivering thermogenesis and holds promise as a therapeutic target for obesity. The transcription factor Early B Cell Factor 2 (EBF2) is a key regulator of the thermogenic gene program, yet its underlying transcriptional regulatory mechanisms and potential for pharmacological targeting remain incompletely defined. Here, we identify a low-complexity C-terminal domain (CTD) within EBF2 that drives biomolecular condensation critical for thermogenic activation. Deletion of the CTD (ΔCTD) or mutation of conserved proline residues disrupts EBF2 phase separation without affecting genomic occupancy, thereby impairing brown adipocyte differentiation and thermogenic capacity both in vitro and in male mice. Remarkably, fusion of the intrinsically disordered region (IDR) of FUS to the EBF2 ΔCTD mutant fully rescues its function, whereas fusion with the MED1-IDR produces distinct transcriptional and phenotypic outcomes. Mechanistically, EBF2 condensates sequester the transcriptional repressor ZFP423 while excluding HDAC1, creating a permissive chromatin environment that promotes thermogenic gene expression. Finally, a phenotypic small-molecule screen targeting phase separation identifies compounds that modulate EBF2 condensate dynamics and thermogenic programming. Together, our findings reveal phase separation as an emerging regulatory mechanism underlying brown fat determination and suggest that targeting biomolecular condensation offers a potential therapeutic strategy for obesity.