Elevated Tead2 levels induce a predominant nuclear localization of Yap and Taz and induce EMT. (A) Yap and Taz cellular localization is dependent on their direct binding to Tead2. A vector control (Vector), wild-type Tead2 (Tead2-WT) or a Tead2 point mutant defective in Yap and Taz binding (Tead2 Y440H) were stably expressed in NMuMG cells, and Yap and Taz and Tead2 localization was assessed by immunofluorescence staining. DAPI was used to visualize nuclei. Scale bar: 20 µm. (B) Depletion of Tead expression prevents the reduction in cytoplasmic levels of Yap and Taz during EMT. NMuMG cells transfected or not with siRNA pools targeting Tead1-3 were induced to undergo EMT by TGFβ-treatment for 4 days. Yap and Taz, and Tead2 were visualized as described in A. Scale bar: 25 µm. (C) Effect of acute Tead2 overexpression on Yap and Taz localization and epithelial differentiation. Tead2 was expressed in Py2T cells (Py2T-iTead2) in a doxycycline (Dox)-inducible fashion, and the localization of Tead2, Yap and Taz and E-cadherin were visualized by an immunofluorescence staining. Scale bar: 20 µm. (D) Effect of Tead2 gain-of-function on cell morphology and EMT. Py2T cells were stably transduced with constructs coding for Tead2 (Tead2-WT), a constitutively active version of Tead2 (Tead2-VP16) or an empty vector control. Overall morphological changes were visualized by phase-contrast microscopy and by immunofluorescence staining against E-cadherin, ZO-1, vimentin and the actin cytoskeleton (phalloidin staining). The insets show an enlarged view of F-actin staining. Scale bars: 15 µm. (E,F) Depletion of Yap or Taz expression prevents Tead2-induced EMT. Py2T cells stably overexpressing Tead2 were transfected with siRNA pools against Yap and Taz or with a control siRNA (siCtr). Overall cell morphology by phase-contrast microscopy (E) and immunoblotting analysis of E-cadherin, Yap and Taz expression (F) are shown. Scale bar: 15 µm.

Tead2 promotes cell migration, invasion and metastasis. (A) Chemotactic migration and invasion of Py2T cells stably expressing Tead2-WT or an empty vector control. Transwell assays were performed utilizing cell culture inserts that were not coated (migration) or coated with Matrigel (invasion). Migrated and invaded cells were quantified. Data are shown as mean ± s.e.m. (n = 3; **P<0.01). (B) Cell invasion in a 3D extracellular matrix. Py2T cells stably expressing Tead2-WT or Tead2-VP16 were embedded in Matrigel and allowed to grow for 5 days. Empty-vector-transduced Py2T cells served as a control. Scale bars: 50 µm. (C) Experimental metastasis. Py2T cells as described in B were injected into the tail veins of female Balb/c nu/nu mice. Mice were killed 33 days post-injection and lungs were sectioned and stained by hematoxilin and eosin (H&E). Higher magnifications are also shown (right). Scale bars: 100 µm. (D,E) Quantification of lung metastasis incidence and number of lung metastasis per mouse as determined by serial sectioning and microscopic analysis of lungs as described in C. The metastatic incidence was calculated as mice harboring metastases/total number of mice per group. Data are shown as mean±s.e.m. (n = 6 mice per group; *P<0.05).

Identification of direct Tead2 transcriptional target genes during EMT. (A) DNA-binding motifs that are overrepresented in Tead2-binding regions. Py2T cells treated with TGFβ for 5 days were subjected to ChIP using an antibody for Tead2 followed by next generation sequencing (ChIP-Seq; n = 2). The sequencing data were subjected to Hypergeometric Optimization of Motif EnRichment analysis (HOMER). Shown are the motifs that are significantly enriched. (B) De novo generation of sequence motifs overrepresented in Tead2-binding regions using PhyloGibbs. Shown is the most significant Tead2-binding motif. (C) Determination of potential direct target genes of Tead2 during EMT. The Venn diagram depicts the number of genes from the Tead2 ChIP-Seq analysis, the number of genes that were regulated differently in Py2T cells before and after 5 days of TGFβ treatment and the number of overlapping genes. (D) Gene ontology analysis was performed on overlapping genes described in C using GOstats. The table shows the functional annotation clustering analysis for the top five cellular compartments, the associated genes per group and their P-values within the groups. (E) Validation of genes directly bound by Tead2 by quantitative PCR. Chromatin from the cells treated as described in A was subjected to qPCR using primer pairs spanning the Tead2-binding regions determined by ChIP-Seq. The data are presented as fold enrichment above background (IP over input) and were normalized to control IgG. An intergenic region was used as negative control. Data are represented as means±s.e.m. (n = 2). (F) Expression of Tead2-bound genes during EMT as determined by RT-qPCR. Py2T and NMuMG cells were treated with TGFβ for 5 or 4 days, respectively. Fold changes in mRNA expression are presented as means±s.e.m. (n = 3). *P<0.05, **P<0.01, ****P<0.0001.