The Hippo pathway effector proteins YAP and TAZ have both distinct and overlapping functions in the cell

Abstract

The Hippo pathway plays an important role in regulating tissue homeostasis, and its effectors, the transcriptional co-activators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1 or TAZ), are responsible for mediating the vast majority of its physiological functions. Although YAP and TAZ are thought to be largely redundant and similarly regulated by Hippo signaling, they have developmental, structural, and physiological differences that suggest they may differ in their regulation and downstream functions. To better understand the functions of YAP and TAZ in the Hippo pathway, using CRISPR/Cas9, we generated YAP KO, TAZ KO, and YAP/TAZ KO cell lines in HEK293A cells. We evaluated them in response to many environmental conditions and stimuli and used RNA-Seq to compare their transcriptional profiles. We found that YAP inactivation has a greater effect on cellular physiology (namely, cell spreading, volume, granularity, glucose uptake, proliferation, and migration) than TAZ inactivation. However, functional redundancy between YAP and TAZ was also observed. In summary, our findings confirm that the Hippo pathway effectors YAP and TAZ are master regulators for multiple cellular processes but also reveal that YAP has a stronger influence than TAZ.

Keywords: CRISPR/Cas; Hippo pathway; TAZ; Yes-associated protein (YAP); cell biology; gene knockout.

Figure 1.

Comparison of YAP and TAZ knockout cells. A, schematic of the structural differences between YAP and TAZ. The domains are labeled proline-rich region (PRL), TEAD binding domain (TBD), WW domain (WW), SH3 binding domain (SH3), transcriptional activation domain (TAD), PXXOP site (PXXOP), 14-3-3 binding site (14-3-3), coiled-coil region (CC), PDZ binding domain (PDZ), and phosphodegron (PD). B, Western blots showing loss of LATS1/2, YAP, and/or TAZ. Cells were serum-starved overnight and either harvested or subsequently stimulated with serum for 60 min. Total cell lysates were used for Western blotting with the indicated antibodies. C, immunofluorescence showing YAP/TAZ (red), TEAD1–4 (green), and 4′,6-diamidino-2-phenylindole (DAPI, a DNA stain, blue) localization following either overnight serum starvation or overnight serum starvation followed by 60 min of serum stimulation. D–F, qPCR of CTGF, CYR61, and LGR5 following either overnight serum starvation (white columns) or overnight serum starvation followed by 60 min of serum stimulation (gray columns). Data are represented as ± S.D. **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 2.

Loss of YAP results in decreased cell spreading, volume, and granularity. A, bright-field images showing cell spreading on a dish. B, quantification of A. Data are represented as ± S.D. C, immunofluorescence showing F-actin (red), YAP/TAZ (green), or 4′,6-diamidino-2-phenylindole (DAPI, a DNA stain, blue). D and E, FACS data showing differences in cell volume (forward scatter (FSC)) and granularity (side scatter (SSC)) for double knockout cells (D) and single knockout cells (E). F, quantification of D and E. G, PCR quantification of the relative mitochondrial DNA content for each cell line. Data are represented as ± S.D. **, p ≤ 0.01; ***, p ≤ 0.001.

Figure 3.

Loss of YAP results in decreased glucose uptake, proliferation, and migration. A, glucose levels remaining in the culture medium following 6 h of incubation. Data are represented as ± S.D. B, cell proliferation curves for each of the cell lines under normal growth conditions. Data are represented as ± S.D. C, cell cycle analysis of each of the cell lines under normal growth conditions. Data are represented as ± S.D. D, bright-field images showing migration through an 8-μm PET pore membrane after 8 h. E, quantification of D. Data are represented as ± S.D. F, bright-field images showing migration following a scratch assay. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 4.

Comparing the kinetics of YAP and TAZ activation. A, immunofluorescence showing a time course of YAP/TAZ (red) localization in response to serum stimulation following overnight serum starvation. B, Western blots showing YAP and TAZ phosphorylation in response to serum starvation and λ phosphatase (LPP) treatment.

Figure 5.

Comparing YAP and TAZ protein expression. A, mRNA expression levels of TAZ and YAP as detected by RNA-seq. Data are represented as ± S.D. B, Western blots showing changes in YAP and TAZ phosphorylation and protein levels following serum starvation for the indicated times. (l.e. indicates longer exposure.) C, Western blots comparing detection of HA-YAP and HA-TAZ using either an HA antibody or the YAP/TAZ antibody and detection of endogenous YAP and TAZ using the YAP/TAZ antibody. D, Western blots showing HA-YAP and HA-TAZ expression following transfection along with a CTGF-luciferase reporter in YAP/TAZ KO cells. E, luciferase activity following transfection of a CTGF-luciferase reporter with varying amounts of either HA-YAP or HA-TAZ in YAP/TAZ KO cells. Data are represented as mean ± S.D. F, qPCR of CTGF expression in various cell lines as indicated following either overnight starvation (white columns) or overnight starvation followed by serum stimulation for 90 min (gray columns). Data are represented as mean ± S.D. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 6.

Using RNA-seq to examine transcriptional differences between YAP and TAZ. A, qPCR time course of CTGF expression following overnight serum starvation and treatment with LPA for the indicated times in WT, YAP KO, TAZ KO, and YAP/TAZ KO cells. Data are represented as ± S.D. B, schematic representing genes that were statistically significant and showed more than a 2-fold change following LPA stimulation. Genes in green represent a 2-fold induction, and genes in red represent a 2-fold decrease in expression following stimulation. C, relative expression from RNA-seq of genes that are YAP/TAZ-dependent following serum starvation (red columns) or LPA stimulation (blue columns). Data are represented as ± S.D. D, relative expression from RNA-seq of genes that are YAP-dependent following serum starvation (red columns) or LPA stimulation (blue columns). Data are represented as ± S.D. E, heatmap summarizing induction of genes in the WT, YAP KO, TAZ KO, and YAP/TAZ KO cell lines in response to LPA stimulation by RNA-seq. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.