Nankai University team reveals new mechanism of TWIST1-YY1-p300 phase separation complex promoting malignant progression of liver cancer

The Nankai University team reveals a new mechanism by which the TWIST1-YY1-p300 phase separation complex promotes the malignant evolution of liver cancer. Research background and scientific significance. Hepatocellular carcinoma (HCC), as a highly prevalent malignant tumor type worldwide, has strong invasiveness, high metastasis rate, and easy postoperative recurrence, making the exploration of its malignant evolution mechanism an important direction in current oncology research. In recent years, the discovery of phase separation in biomolecules has provided a new perspective for understanding the spatiotemporal regulation of cellular activity. This phenomenon refers to the dynamic membraneless organelles formed by weak interactions between intracellular proteins, nucleic acids, and other biomolecules, which play important roles in key biological processes such as gene transcription regulation and signal transduction.
It is worth noting that abnormalities in the phase separation process are closely related to the occurrence and development of various diseases, especially in the fields of neurodegenerative diseases and tumors. Previous studies have shown that the intrinsic disordered regions (IDRs) of transcription factors (TFs) participate in the establishment of transcriptional regulatory networks by mediating phase separation. However, the specific mechanism of phase separation in the occurrence and development of liver cancer is still unknown. The latest research by the team of Professor Sun Tao, Professor Liu Huijuan, and Professor Yang Cheng from Nankai University fills this gap and reveals a novel molecular pathway in which the TWIST1-YY1-p300 transcription complex activates miR-9 expression through phase separation mechanism, thereby promoting malignant progression of liver cancer.
Research Design and Main Findings: Identification and Characterization of TWIST1-YY1-p300 Transcriptional Complex. The research team used systematic biochemical methods and first confirmed the physical interaction between TWIST1 and YY1 in liver cancer cells through immunoprecipitation (Co IP) technology. Further combining GST pull-down experiments and rapid protein liquid chromatography (FPLC) analysis, p300 was identified as the core component of the transcription complex. These experiments not only verified the direct binding relationship between the three, but more importantly, revealed the stable existence of the complex in liver cancer cells.
On the basis of confirming the composition of the composite, researchers innovatively focused on its phase separation characteristics. Through fluorescence bleaching recovery (FRAP) experiments, it was observed that the aggregates formed by TWIST1 and YY1 in the nucleus exhibit significant fluidity characteristics - a key indicator for determining phase separation. When treated with 1,6-hexanediol (a known phase separation inhibitor), these nuclear aggregates showed significant depolymerization, further confirming their phase separation nature. Of particular note is that the addition of p300 significantly enhances the co localization efficiency of TWIST1 and YY1, suggesting that p300 may act as a "molecular scaffold" to promote the phase separation and formation of transcriptional complexes.
The molecular basis and regulatory characteristics of phase separation are analyzed in depth to elucidate the molecular mechanism of phase separation. The research team conducted a detailed structural and functional analysis of TWIST1 and YY1 proteins. The phase separation droplet formation process was reconstructed in vitro by expressing and purifying protein fragments containing IDR. Experimental data shows that the phase separation behavior of these proteins exhibits a typical concentration dependence: when the concentration of TWIST1-IDR or YY1-IDR reaches a threshold, the solution spontaneously forms micrometer sized droplet structures. In addition, changes in ion strength in the buffer solution can significantly affect the degree of phase separation, reflecting the importance of electrostatic interactions in maintaining the phase separation structure. Through site directed mutagenesis strategy, researchers have identified the critical role of polar amino acid residues in phase separation. When the acidic amino acid cluster (D/E) or basic amino acid cluster (R/K) of TWIST1 undergoes mutations, its phase separation ability is significantly weakened; Similarly, the D/A mutation of YY1 also disrupted the formation of phase separation. These findings not only elucidate the molecular basis of transcription factor phase separation, but also provide theoretical basis for subsequent targeted interventions.
Clinical association and functional validation
Clinical prognostic value of TWIST1 complex

Through in-depth analysis of liver cancer samples in the TCGA database, the research team established a clear association between TWIST1YY1-p300 complex and clinical prognosis.
. The data shows that about 68% of liver cancer tissues exhibit co overexpression of TWIST1 and YY1, and this co expression pattern is significantly correlated with the overall survival of patients. It is worth noting that when all three components are highly expressed simultaneously, the median survival time of patients is shortened by about 40% compared to the single gene high expression group, which strongly suggests that this complex may serve as a novel molecular marker for liver cancer prognosis.
Gene co expression network analysis further revealed that the mRNA levels of TWIST1, YY1, and p300 were significantly positively correlated in liver cancer tissues (correlation coefficient r>0.7, p<0.001). This collaborative expression pattern suggests that the three may be regulated by a common upstream signaling pathway or have a mutually reinforcing positive feedback regulatory mechanism.
At the cellular functional level, the research team designed a series of rigorous experiments to validate the biological effects of the TWIST1-YY1p300 complex in promoting metastasis. The scratch healing experiment showed that the migration speed of liver cancer cells overexpressing this complex was 2-3 times higher than that of the control group; The Transwell invasion experiment confirmed that its ability to penetrate the matrix gel was significantly enhanced. More importantly, gelatinase spectrum analysis revealed that overexpression of the complex led to a significant upregulation of MMP2/9 activity, providing an enzymatic basis for explaining its pro invasion effect. Epithelial mesenchymal transition (EMT) phenotype analysis showed that the TWIST1-YY1-p300 complex can induce downregulation of E-cadherin expression and upregulation of N-cadherin and vimentin expression, which is highly consistent with its pro metastatic function. By constructing a mutant complex (losing phase separation ability but retaining DNA binding activity), researchers further confirmed that phase separation characteristics are a necessary condition for its ability to promote metastasis.

Molecular mechanism analysis: The research team on the transcriptional activation mechanism of miR-9 adopted a multi omics integrated analysis strategy. Firstly, the common target gene group of TWIST1 and YY1 was identified through whole transcriptome sequencing. Based on the liver cancer specific miRNA expression profile, four significantly upregulated miRNA candidate molecules were screened. After survival analysis and functional validation, miR-9 was ultimately identified as a key effector molecule in the regulatory network of this complex. In depth mechanistic studies have shown that the TWIST1-YY1-p300 complex activates transcription by binding to a super enhancer region approximately 66kb upstream of the miR-9 gene. The chromosome conformation capture (3C) experiment confirmed the spatial interaction between the enhancer and the miR-9 promoter. When the enhancer region is specifically knocked out using CRISPR technology, even overexpression of the TWIST1 complex cannot induce upregulation of miR-9, fully demonstrating the central role of enhancers in transcriptional regulation. The transcriptional activation mode dependent on phase separation is different from the traditional transcription factor binding mode. This study found that the TWIST1-YY1-p300 complex is enriched in the super enhancer region in the form of phase separation condensates. Through single-molecule fluorescence in situ hybridization (smFISH), clear co localization signal points were observed between transcription factors and enhancer DNA probes in the nucleus. When phase separation inhibitors or mutant proteins are introduced, this co localization is significantly weakened, accompanied by a decrease in miR-9 expression levels. This discovery provides a new perspective for understanding the working mechanism of super enhancers: transcription factors form local high concentration microenvironments through phase separation, which may promote the formation or maintenance of enhancer promoter circularization, thereby achieving specific activation of target genes. This phase separation dependent transcriptional regulation mode has higher efficiency and specificity compared to traditional models.

Based on a deep understanding of the complex structure, the research team conducted targeted drug screening to investigate the phase separation inhibitory effect of metformin in therapeutic intervention and new drug discovery. Through molecular docking simulations, it was found that the molecular structure of the hypoglycemic drug metformin can form specific interactions with polar amino acid residues of TWIST1/YY1. In vitro recombination experiments have confirmed that metformin can dose dependently inhibit the formation of TWIST1-YY1 phase separation droplets (IC50 ≈ 5mM). At the cellular level, metformin treatment resulted in a reduction of approximately 60% in the number of nuclear transcription factor aggregates and a significant decrease in their fluidity. This disturbance directly affects the transcriptional activity of the complex: Chromatin immunoprecipitation (ChIP) quantification showed that after treatment with metformin, the enrichment level of the complex in the miR-9 enhancer region decreased by 70-80%, and correspondingly, the expression level of miR-9 also returned to near normal range. Functional validation of anti-tumor effects showed that in animal models, the growth rate of liver cancer transplanted tumors in the metformin treatment group was slowed down by about 50% compared to the control group, and the number of lung metastases was reduced by more than 80%. Molecular level analysis showed that the expression of EMT markers was reversed in the tumor tissues of the treatment group, accompanied by downregulation of miR-9 levels. It is worth noting that when using phase separation deficient mutants to establish models, the anti-tumor effect of metformin is significantly weakened, which directly proves that its mechanism of action is closely related to interfering with the phase separation process. These findings not only expand our understanding of the anti-tumor mechanism of metformin, but more importantly, provide new targeted strategies for the treatment of liver cancer. By specifically interfering with the phase separation process of oncogenic transcription complexes, effective inhibition of tumor metastasis may be achieved while reducing toxic side effects on normal cells.
Research prospects and clinical translation: The findings of this study have opened up multiple directions worth exploring in depth. At the level of basic research, it is necessary to further elucidate the fine structure and dynamic assembly rules of TWIST1-YY1-p300 phase separation condensates. How super level enhancers selectively recruit specific transcription factors to form phase separated microdomains, and how this physical state affects the three-dimensional structure of chromatin, are all highly scientifically valuable questions.
In the field of translational medicine, the development of specific inhibitors based on phase separation principles has broad prospects. In addition to metformin, more efficient phase separation regulators may be discovered through virtual screening and structural optimization. In addition, associating the separation characteristics of TWIST1 complex with patient prognosis is expected to establish a more accurate molecular subtyping system for liver cancer.
The significance of this study lies not only in revealing new mechanisms of liver cancer metastasis, but also in proposing a novel anti-cancer strategy of "targeted transcription factor phase separation". With the deepening understanding of biological molecular aggregates, phase separation regulation is expected to become the third largest tumor treatment target field after gene mutation and epigenetics. Future research needs to integrate interdisciplinary approaches such as structural biology, biophysics, and clinical medicine to promote the translation of this innovative concept into clinical applications.