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Quantitative Real-Time PCR to Measure YAP/TAZ Activity in Human Cells.

Research paper by Xiaolei X Cao, Bin B Zhao

Indexed on: 20 Dec '18Published on: 20 Dec '18Published in: Methods in molecular biology (Clifton, N.J.)



Abstract

Transcription coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also known as WWTR1) are homologs of the Drosophila Yorkie (Yki) protein and are major downstream effectors of the evolutionarily conserved Hippo pathway. YAP/TAZ play critical roles in regulation of cell proliferation, apoptosis, and stemness, thus mediate functions of the Hippo pathway in organ size control and tumorigenesis. The Hippo pathway inhibits YAP/TAZ through phosphorylation, which leads to YAP/TAZ cytoplasmic retention and degradation. Dephosphorylated and nuclear-localized YAP/TAZ bind to transcription factors, especially the TEAD family proteins, thus transactivate the expression of specific genes. Therefore, measuring the expression level of YAP/TAZ target genes is a critical approach to assess Hippo pathway activity. Through gene expression profiling in different tissues and cells using techniques such as microarray and RNA-seq, many target genes of YAP/TAZ have been identified. Some of these genes were confirmed to be direct YAP/TAZ targets by chromatin immunoprecipitation (ChIP)-PCR or ChIP-seq. These works made it possible to quickly determine YAP/TAZ activity by measuring the mRNA levels of several YAP/TAZ target genes, such as CTGF, CYR61, and miR-130a by quantitative real-time PCR (qPCR). In this chapter, we demonstrate the use of qPCR to measure YAP/TAZ activity in MCF10A cells.