A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells

Abstract

AbstractThe activation of yes‐associated protein 1 (YAP1) and transcriptional co‐activator with PDZ‐binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double‐edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD‐responsive DNA element (YRE) and incorporated it into a lentivirus‐based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF‐ and NF‐κB‐binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β‐catenin/TCF‐ and IL‐1β/NF‐κB‐mediated signaling, respectively. The practicality of this system was evaluated by performing cell‐based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter‐introduced cells capable of quantifying YAP1/TAZ‐ and β‐catenin‐mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet‐derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.