ATAC-seq profiling of open chromatin in PLC-PRF cells

Epithelial–mesenchymal transition (EMT) is an important mechanism of metastasis and malignant progression of hepatocellular carcinoma (HCC). However, the transcriptional regulation mechanism of EMT remains poorly understood. Some transcriptional co-activators can form phase-separated condensates at super-enhancers that compartmentalize and concentrate the transcription apparatus to drive robust gene expression. Here, we demonstrate that Twist1 and YY1, interact with p300 and form phase-separated local high-concentration interaction hubs at super-enhancers of miRNA-9 and activate its expression to induce EMT and promote the malignant evolution of HCC. Phase separation disrupted by metformin perturbs Twist–YY1 condensates, thereby inhibiting EMT. To explore how the Twist1 complex regulates miR-9 expression by binding SE region, we performed Twist1 ChIP-seq combined with H3K4me3, H3K4me1, H3K27ac, DNAse, p300, and YY1 ChIP-seq data to detect the SEs of miR-9. After metformin treatment, the peak of Twist1/YY1 on miR-9 SE decreased. Metformin specifically affects the binding of Twist1/YY1 to the miR-9 SE region. 1,6-hexanediol, as an aliphatic alcohol, can weaken hydrophobic interactions and inhibit liquid–liquid phase separation by disrupting hydrophobic interactions. In order to prove Twist1/YY1 interaction at these sites requires phase separation, we detect the accessibility of miR-9 SE regions in 1,6-hexanediol-treated cells. The result showed a general decrease in the accessibility of miR-9 super-enhancer regions in 1,6-hexanediol-treated cells and Twist1 / YY1 combines the SE region of miR-9 in the form of phase separation. Overall design: The assay for transposase-accessible chromatin combined with high-throughput sequencing (ATAC-seq) to detect the accessibility of miR-9 super-enhancer regions in 1,6-hexanediol-treated cells.

上皮间质转化（Epithelial–mesenchymal transition, EMT）是肝细胞癌（hepatocellular carcinoma, HCC）转移与恶性进展的关键机制，但其转录调控机制至今仍未被充分解析。部分转录共激活因子（transcriptional co-activators）可在超级增强子（super-enhancers, SE）处形成相分离凝聚体（phase-separated condensates），通过区室化并浓缩转录装置（transcription apparatus）以驱动高效的基因表达。本研究证实，Twist1与YY1可与p300相互作用，在微小RNA-9（microRNA-9, miR-9）的超级增强子处形成相分离的局部高浓度互作枢纽，进而激活miR-9的表达，诱导上皮间质转化进程并促进肝细胞癌的恶性演化。二甲双胍（metformin）可破坏相分离过程，扰乱Twist1-YY1凝聚体，从而抑制上皮间质转化。
为探究Twist1复合物如何通过结合超级增强子区域调控miR-9的表达，我们整合Twist1染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）与H3K4me3、H3K4me1、H3K27ac、DNAse、p300及YY1的ChIP-seq数据，以鉴定miR-9的超级增强子区域。经二甲双胍处理后，Twist1/YY1在miR-9超级增强子处的结合峰信号显著下调，表明二甲双胍可特异性影响Twist1/YY1与miR-9超级增强子区域的结合。
1,6-己二醇（1,6-hexanediol）作为一种脂肪族醇（aliphatic alcohol），可通过破坏疏水相互作用（hydrophobic interactions）削弱疏水结合力，进而抑制液-液相分离（liquid–liquid phase separation）。为验证Twist1/YY1在上述位点的互作依赖于相分离过程，我们检测了经1,6-己二醇处理的细胞中miR-9超级增强子区域的染色质可及性。结果显示，经1,6-己二醇处理的细胞中，miR-9超级增强子区域的可及性普遍下降，证实Twist1/YY1以相分离的形式结合miR-9的超级增强子区域。
实验整体设计：采用转座酶可及性染色质测序（assay for transposase-accessible chromatin with high-throughput sequencing, ATAC-seq）技术，检测经1,6-己二醇处理的细胞中miR-9超级增强子区域的染色质可及性。