Development of a TEAD-independent reporter system for specific and sensitive measurement of YAP/TAZ activity.

Accurate quantification of Yes-associated protein (YAP) and its paralog TAZ is critical for understanding their biological functions, however, existing reporter systems are constrained by their reliance on endogenous TEAD transcription factors. Here, we developed an endogenous TEAD-independent reporter system for YAP/TAZ activity, based on the GAL4-UAS system. The construct consists of the GAL4 DNA-binding domain (DBD) fused to the YAP-binding domain (YBD) of TEAD4, with the native TEAD DNA-binding domain (TEA) removed. Structural modeling of the G-TEAD4(217) fusion protein confirmed high similarity to experimentally resolved TEAD4-YAP or GAL4-DNA bound structures and revealed surface properties consistent with functional domain separation. Functional assays showed that the reporter reliably responded to both YAP knockdown and overexpression. Compared to the conventional 8 × GTIIC reporter, G-TEAD4(217) exhibited higher sensitivity and remained unaffected by TEAD4 silencing, confirming reporter specificity and TEAD independence. In addition, the system demonstrated its suitability for compound screening by validating known YAP inhibitors across a number of high-YAP cancer cell lines. A single-plasmid lentiviral version LV-G-TEAD4(217) was also developed to enable stable transduction and potential in vivo use. Together, these findings demonstrate that G-TEAD4(217) represents a sensitive, specific, and modular reporter platform for analyzing YAP/TAZ transcriptional activity in diverse biological settings.