Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally-shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR we validate TEAD1 trans occupancy at accessibility sites within EGFR, AQP4, and CDH4. To further characterize TEAD's functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1-AQP4 in GBM migration. Overall design: Open chromatin areas were analyzed by ATAC-seq in stem cells from fetal germinal matrix (GM) and from human glioblastoma (GBM) in biological replicates (n = 3). Changes in transcriptome after ablation of TEAD1 and TEAD4 was analyzed by RNA-seq in TEAD1 and TEAD4 CRISPR-KO different GBM patient-derived cell lines (n = 4), as well as in TEAD1 KO gliomaspheres and compared to their corresponding SHAM lines.胶质母细胞瘤（glioblastoma, GBM）迁移的内在驱动机制仍不甚明晰。为更精准地捕捉胶质母细胞瘤的天然分子印记及其发育背景，我们从胶质母细胞瘤组织（胶质瘤干细胞，glioblastoma stem cell, GSC）与生发基质（germinal matrix, GM）组织中分离人类干细胞群体，并通过转座酶可及性染色质测序（ATAC-seq）绘制其染色质可及性图谱。本研究揭示了两类截然不同的胶质瘤干细胞调控特征：一类为发育共有的/增殖相关特征，另一类为肿瘤特异性/迁移相关特征，其中TEAD1/4基序在后者中呈现独特的富集状态。我们通过染色质免疫沉淀PCR（ChIP-PCR）验证了TEAD1在EGFR、AQP4及CDH4基因座内可及位点的转录结合活性。为进一步阐明TEAD在胶质母细胞瘤中的功能角色，我们利用CRISPR-Cas9技术在患者来源的胶质母细胞瘤细胞系中敲除TEAD1或TEAD4。实验结果显示，TEAD1敲除可显著抑制体外及体内的细胞迁移能力，并改变迁移相关与上皮间质转化（epithelial-mesenchymal transition, EMT）的转录组特征，同时其靶基因AQP4呈现一致性下调。过表达TEAD1可恢复AQP4的表达水平，且TEAD1与AQP4的过表达均能挽救TEAD1敲除细胞的迁移缺陷，这表明TEAD1-AQP4调控轴在胶质母细胞瘤迁移中发挥直接调控作用。实验总体设计：我们对胎儿生发基质与人类胶质母细胞瘤来源的干细胞群体中的开放染色质区域进行了ATAC-seq分析，设置生物学重复（n=3）。我们通过RNA-seq分析了TEAD1与TEAD4 CRISPR敲除的4株不同患者来源胶质母细胞瘤细胞系、TEAD1敲除胶质瘤球的转录组变化，并与对应的假手术对照（SHAM）细胞系进行比较。