TEAD mediates YAP-dependent gene induction and growth control

Abstract

The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

Figure 1.

TEAD is required for YAP-induced gene expression. (A) YAP potently activates TEAD family transcription factors. The indicated Gal4-fused transcription factors were cotransfected with a 5× UAS-Luc reporter and a CMV-β-gal construct into 293T cells in the presence or absence of YAP. The β-galactosidase activity normalized luciferase activity in the absence of YAP (Gal4-TEAD1 in the absence of YAP in the left panel) was set to 1. Flag-YAP Western blot shows that the YAP expression level was not decreased by ErbB4. (B) YAP-S94A cannot activate TEAD4. The indicated plasmids were cotransfected with a 5× UAS-luciferase reporter for Gal4-TEAD4 or a 6× OSE2-luciferase reporter for RUNX2 into 293T cells. Luciferase activity was measured and normalized to cotransfected β-galactosidase. (C) Serine 94 of YAP is required for its interaction with TEAD4. The indicated plasmids were transfected into HEK293 cells. Flag-YAP (left panel) or Myc-TEAD4 (right panel) was immunoprecipitated, and the immunoprecipitates were probed as indicated. D. YAP-S94A is defective in gene expression regulation. The left panel shows cluster analysis of gene expression profiles in YAP-WT, 5SA, or S94A-overexpressing MCF10A cells. The group of genes presented was chosen by the following standard: a P call in all samples and up-regulated more than fivefold or down-regulated more than fourfold by YAP-wild-type overexpression. Cluster analysis was done with Eisen Lab Cluster software using average linkage clustering. (Right panel) The same data sets were drawn into boxplots using the R program. Red and green indicate up-regulated and down-regulated genes, respectively. (E) TEAD is required for YAP-induced expression of CTGF and ITGB2. The indicated shRNAs were infected into native or YAP-5SA-expressing MCF10A cells. Expression of CTGF and ITGB2 was determined by quantitative RT–PCR and compared to vector control cells. (C) Scramble shRNA control; (#1 and #2) two different shRNAs targeting TEAD1/3/4. (F) YAP and TEAD1 occupy common promoters. ChIP-on-chip was performed with YAP or TEAD1 antibody against endogenous proteins in MCF10A cells. Genome-wide location analysis was performed. AR ChIP was included as a negative control.

Figure 2.

TEAD is required for YAP activity in growth promotion and EMT. (A) YAP-S94A is defective in promoting cell growth. The growth curve of NIH-3T3 stable cells with expression of Vector, YAP, YAP-S94A, TEAD1, or TEAD1-YAP-S94A was determined. (B) Fusion of YAP-S94A with TEAD1 rescued YAP target gene expression. (Right panel) NIH-3T3 stable cells with expression of YAP-S94A, TEAD1, and TEAD1-YAP-S94A fusion protein were generated, and the expression of these proteins was shown by anti-Myc-tag Western blot. The expression of Ctgf and Inhba, two YAP target genes in NIH-3T3 cells, was measured by quantitative PCR. The induction of these two genes by YAP-WT was also shown for comparison. (C) YAP-5SA-S94A is compromised in eliciting EMT-like morphology. Indicated MCF10A stable cells were cultured in monolayer or in 3D on reconstituted basement membrane for 16 d before pictures were taken. (D) YAP-5SA-S94A is defective in reducing membrane E-cadherin and cortical actin. The indicated MCF10A stable cells were stained by anti-E-cadherin (green), rhodamine-phalloidin (red), and DAPI (blue). (E) The TEAD-binding-defective YAP is compromised in altering EMT marker expression. Western blot of epithelial and mesenchymal markers was performed using lysates from indicated MCF10A stable cells. (F) TEAD1/3/4 shRNAs blocked YAP induced EMT-like morphology and acinar overgrowth. YAP-5SA-expressing MCF10A cells were infected with indicated shRNA lentiviruses. The morphology in 2D and 3D culture was documented as in C. (G) TEAD1/3/4 shRNAs blocked YAP-induced anchorage-independent growth in soft agar. The indicated MCF10A stable cells were plated in soft agar and allowed to grow for 3 wk, after which colonies were stained with crystal violet and counted.

Figure 3.

CTGF is a direct target of YAP and TEAD. (A) Both YAP and TEAD1 bind to the CTGF promoter. ChIP from MCF10A cells was performed with control IgG, YAP, or TEAD1 antibody as indicated. The presence of CTGF promoter was detected by PCR. (B) Activation of CTGF reporter by YAP and TEAD1. A luciferase reporter driven by CTGF promoter was cotransfected with YAP wild type or S94A mutant as indicated with or without TEAD1 cotransfection. Luciferase activity was measured and normalized to cotransfected β-galactosidase. (C) Dominant-negative TEAD1 blocks the YAP stimulation of the CTGF reporter. The indicated plasmids were cotransfected, and luciferase activity was determined as in B. (D) The human CTGF promoter region contains three putative TEAD-binding sites. The putative TEAD-binding sites (TB1–TB3) are shown in red. (E) The putative TEAD-binding sites are important for CTGF promoter activity. The putative TEAD-binding sites (TB) were mutated individually or in combination. The luciferase activity of each reporter was measured in the presence or absence of YAP and TEAD1. The activation folds by YAP and TEAD1 are shown. (F) YAP and TEAD are required for CTGF expression. ACHN cells were infected with the indicated shRNA lentiviruses, and CTGF mRNA levels were determined by quantitative RT–PCR. (G) Knockdown of YAP or TEAD1/3/4 decreases CTGF protein levels. Experiments were similar to F except Western blotting was performed with the indicated antibodies. (H) YAP, TEAD, and CTGF are important for the AHCN cell growth. YAP, TEAD1/3/4, and CTGF were knocked down by shRNAs. Cell growth rate was determined. (I) CTGF is required for YAP-induced growth and morphological change in 3D culture. MCF10A cells expressing YAP-5SA were infected with indicated shRNA lentiviruses. The morphology in 2D and 3D culture was documented as in Figure 2C. (J) CTGF knockdown attenuates YAP induced anchorage-independent growth in soft agar. The indicated MCF10A stable cells were plated in soft agar and allowed to grow for 3 wk, after which colonies were stained with crystal violet and counted. Pictures of the stained colonies were presented in higher magnification to show the colony size reduction by CTGF shRNAs.

Figure 4.

yki and scalloped genetically interact to control tissue growth and organ size. (A) The TEAD/Sd-binding-defective YAP and Yki are compromised in inducing extra interommatidial cells. Mid-pupal eye discs were stained with Discs large (Dlg) antibody to outline cells. The genotypes of the fly tissues are Wild-type (Canton S) (panel a), GMR-Gal4/UAS-yki-V5 (panel b), GMR-Gal4/UAS- yki^S97A-V5 (panel c), GMR-Gal4/UAS-Flag-YAP^S127A (panel d), and GMR-Gal4/UAS-Flag-YAP^S94A/S127A (panel e). (B) The TEAD/Sd-binding-defective YAP and Yki are compromised in inducing clone expansion. Wing imaginal discs containing 72-h-old control (panel a) or various YAP/Yki-overexpressing clones (panels b–e) were generated by flip-out and positively marked by GFP. The genotypes of the fly tissues are hsFLP/+; act>y+>Gal4, UAS-GFP^S65T/+ (panel a), hsFLP/+; act>y+>Gal4, UAS-GFP^S65T/UAS-yki-V5 (panel b), hsFLP/+; act>y+>Gal4, UAS-GFP^S65T/UAS-yki^S97A-V5 (panel c), hsFLP/+; act>y+>Gal4, UAS-GFP^S65T/UAS-Flag-YAP^S127A (panel d), and hsFLP/+; act>y+>Gal4, UAS-GFP^S65T/UAS-Flag-YAP^S94A/S127A (panel e). (C) yki and scalloped genetically interact to control tissue growth and organ size. Genotypes of the fly tissues are indicated. SEM (scanning electron microscopy) images of adult eyes are shown in panels a–e, g, and h. A late pupal head is shown in panel f. The arrow in panel f indicates where a retina is normally expected to grow.