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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.GATA3has 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 andin 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 StatementZinc 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.

Bmal1 knockdown decreases paclitaxel sensitivity in SCC9 cells

Supplementary Material and Methods

Circadian clock genes regulate cancer development and chemotherapy susceptibility. Accordingly, chronotherapy based on circadian phenotypes might be applied to improve therapeutic efficacy. In this study, we investigated whether the circadian clock gene Bmal1 inhibited tumor development and increased paclitaxel sensitivity in tongue squamous cell carcinoma (TSCC). Bmal1 expression was downregulated and its rhythmic pattern of expression was affected in TSCC samples and cell lines. Ectopic Bmal1 inhibited cell proliferation, migration and invasion in vitro, and tumor growth in mouse xenograft models of TSCC. After exposure to paclitaxel, Bmal1-overexpressing cells displayed a relative increase in apoptosis and were more susceptible to paclitaxel treatment in vivo. Mechanistic investigations suggested a regulatory connection between Bmal1, TERT, and the oncogenic transcriptional repressor EZH2 (enhancer of zeste homolog 2), the recruitment of which to the TERT promoter increased paclitaxel-induced apoptosis and cell growth inhibition. Clinically, paclitaxel efficacy correlated positively with Bmal1 expression levels in TSCC. Overall, our results identified Bmal1 as a novel tumor suppressor gene that elevates the sensitivity of cancer cells to paclitaxel, with potential implications as a chronotherapy timing biomarker in TSCC. Cancer Res; 77(2); 532–44. ©2016 AACR.

Supplementary Figure Legends

The clinical characteristics subjects in patient tissue samples