A Zinc finger (ZnF) transcription factor exerts its multifaceted function through ZnF-regulated phase separation

Zinc finger (ZnF) transcription factors (TFs) consist of ZnF-containing DNA-binding domains (DBDs) and intrinsically disordered region (IDR)-containing activation domains (ADs). Recent studies have suggested that liquid-liquid phase separation (LLPS) is the fundamental mechanism underlying human health and disease, with ZnF TFs activating gene expression through the LLPS capacity of their IDR-containing ADs. However, little is known about how the well-folded DBD of ZnF TFs is involved in their LLPS mechanism. GATA3 is one of the most frequently mutated genes in breast cancer, and its encoded protein GATA3, which contains two ZnFs (ZnF1 and ZnF2) in its DBD, is a master regulator of immunity. Here, we show that GATA3 undergoes LLPS in cells and in vitro, and its DBD plays an important regulatory role. Mechanistically, ZnF2 in the DBD contains two arginine amino acids (R329 and R330) that provide critical charges to regulate GATA3 LLPS and DNA binding by generating multivalent electrostatic interactions. Functionally, we demonstrated that ZnF2-regulated GATA3 LLPS is the mechanism underlying the multifaceted function of GATA3 in breast cancer development and immune regulation, where aberrant GATA3 LLPS caused by artificial or breast cancer-associated ZnF2-defective mutations by reducing Suv39H1 protein stability showed significantly reduced potential in promoting breast cancer development and exhibited remarkably enhanced capacities for activating type I interferon signaling. Since ZnF is a common feature in the DBDs of ZnF TFs, by describing GATA3 as a proof-of-principle, our data suggest that ZnF-regulated LLPS may be a general mechanism underlying the multifaceted function of ZnF TFs in human health and disease.