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

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Book cover The Hippo Pathway

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1893))

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.

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Acknowledgments

This work was supported by grants to B. Zhao from the National Natural Science Foundation of China Key Project (81730069), General Project (31471316) and the National Key R&D Program of China (2017YFA0504502), the National Natural Science Foundation of China International Collaboration Project (31661130150), the Fundamental Research Funds for the Central Universities, and the Qianjiang Scholar Plan of Hangzhou, the Thousand Young Talents Plan of China, and the Newton Advanced Fellowship from the Academy of Medical Sciences, UK.

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  1. Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, China

    Xiaolei Cao & Bin Zhao

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  1. Xiaolei Cao
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Correspondence to Bin Zhao .

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  1. Cancer Institute, University College London, London, UK

    Alexander Hergovich

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Cao, X., Zhao, B. (2019). Quantitative Real-Time PCR to Measure YAP/TAZ Activity in Human Cells. In: Hergovich, A. (eds) The Hippo Pathway. Methods in Molecular Biology, vol 1893. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8910-2_12

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