A Repressive Type of TEAD4 Condensation and Engineering a Glue Peptide Therapy Against Cancer [RNA-Seq_TEAD4KO]. A Repressive Type of TEAD4 Condensation and Engineering a Glue Peptide Therapy Against Cancer [RNA-Seq_TEAD4KO]

Transcriptional factors (TFs) act as central determinants to coordinate cell death and survival by differentially modulating gene expression. Here, we systematically identified many TFs including TEAD4 forming condensates in stressed cells. In sharp contrast to YAP-induced transcription-activating condensation of TEAD4, we found co-factors such as VGLL4 and RFXANK could alternatively induce repressive condensation of TEAD4 to trigger cell death. Illustrated by VGLL4, we mechanistically revealed that the heterotypic interactions between TEAD4 with VGLL4 favors its oligomerization and assembly of large condensates to manifest a nonclassical inhibitory function of this transcription factor, i.e., causing DNA/chromatin to be aggregated and entangled, which eventually impede gene expression. Based on these findings, we engineered a linker peptide (GLUP) to selectively “glue” TEAD4 molecules together, therefore forcefully driving its repressive condensation against YAP-induced activation. This glue peptide displayed a strong antitumor effect via inhibition of TEAD4-related gene transcription. Collectively, this work revealed a new type of phase separation, i.e., repressive condensation of TFs, and revealed how cofactor-induced differential condensation orchestrate opposite functions of a given TF, and further developed a new class of antitumor strategy by artificially inducing repressive condensation. Overall design: To investigate the function of GLUP peptide in cancer, we perform RNA-Seq anlysis to find the altered pathway in this GLUP-treated group. To test the specificity of GLUP peptide, we also perform RNA-Seq anlysis in WT and TEAD4 knockout cell with or without GLUP treatment.

转录因子（Transcriptional factors, TFs）是通过差异化调控基因表达，协调细胞死亡与存活的核心调控因子。本研究系统鉴定出多种在应激细胞中形成凝聚体（condensates）的转录因子，其中包括TEAD4。与YAP诱导的TEAD4转录激活型凝聚体形成截然不同的是，我们发现VGLL4、RFXANK等辅因子可通过另一途径诱导TEAD4形成抑制型凝聚体，进而触发细胞死亡。以VGLL4为例，我们从机制上揭示了TEAD4与VGLL4之间的异源相互作用可促进其寡聚化与大型凝聚体的组装，从而展现该转录因子的非经典抑制功能——即引发DNA/染色质聚集与缠绕，最终阻碍基因表达。基于上述发现，我们设计了一种连接肽（GLUP），可选择性地将TEAD4分子‘粘合’在一起，从而强力驱动其形成抑制型凝聚体，拮抗YAP诱导的激活过程。该粘合肽通过抑制TEAD4相关基因的转录，展现出强劲的抗肿瘤活性。综上，本研究揭示了一种新型相分离现象——即转录因子的抑制型凝聚体形成，阐明了辅因子诱导的差异化凝聚体如何调控同一转录因子的相反功能，并通过人工诱导抑制型凝聚体开发了一类全新的抗肿瘤策略。整体实验设计：为探究GLUP肽在癌症中的功能，我们开展RNA测序（RNA-Seq）分析以筛选GLUP处理组中发生表达改变的信号通路；为验证GLUP肽的特异性，我们还分别在野生型（WT）与TEAD4敲除细胞中设置是否经GLUP处理的组别，进行RNA测序分析。