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 suggest 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 might be involved in their LLPS mechanism. GATA3 has been identified as 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 , with its DBD playing an important regulatory role. Mechanistically, ZnF2 in the DBD contains two arginine amino acids (R329 and R330) to provide the critical charges to regulate GATA3 LLPS 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 showed significantly reduced potentials in promoting breast cancer development and exhibited remarkably enhanced capacities in 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. Significance Statement Zinc finger (ZnF) transcription factors (TFs), the most prominent TFs in humans, play critical roles in human health and disease. The ZnF TF GATA3 is important for breast cancer development and immune regulation. Here, we show that GATA3 undergoes liquid-liquid phase separation (LLPS), which is regulated by its well-structured DNA-binding domain, specifically ZnF2. We demonstrate that ZnF2-regulated LLPS is a phenotype-associated mechanism underlying the multifaceted functions of GATA3 in breast cancer development and immune regulation. This study reports GATA3 as a proof-of-principle to reveal a previously unrecognized mechanism that can potentially be generalized to improve the mechanistic understanding of the multifaceted function of ZnF TFs in various human health and disease settings, including the complex diseases such as cancer. ### Competing Interest Statement The authors have declared no competing interest.