Figure 2: Antagonistic function of the two TEAD4 isoforms.
From: A splicing isoform of TEAD4 attenuates the Hippo–YAP signalling to inhibit tumour proliferation
(a) A luciferase reporter driven by CTGF promoter was co-transfected in the presence or absence of YAP as indicated with TEAD4-FL, TEAD4-S or RBM4. The relative luciferase activities were determined by calculating the ratio of firefly luciferase activities over Renilla luciferase activities. Three independent experiments were conducted, with the mean±s.d. of relative luciferase activities were shown. (b) Expression vector of HA-YAP was co-transfected into 293T cells with Flag-TEAD4-FL, Flag-TEAD-S or Flag-TEAD4-FL in the presence of untagged TEAD4-S, and the binding of HA-YAP was determined by co-immunoprecipitation (co-IP) experiment using anti-Flag antibody. (c) 293T cells were co-transfected with Flag-TEAD4-FL, HA-YAP and increasing amounts of untagged TEAD4-S. The interaction between HA-YAP and Flag-TEAD4-FL was determined by co-IP assay. The relative levels of two TEAD4 isoforms in these cells were determined by western blot using anti-TEAD4 antibody. (d) H157 cells stably expressing TEAD4-FL or TEAD4-S were generated. Chromatin immunoprecipitation (ChIP) from these cells was performed with control IgG and TEAD4 antibody. The precipitation of CTGF promoter was examined by PCR. (e,f) H157 and A549 cells stably expressing YAP, YAP/TEAD4-FL, YAP/TEAD4-S, YAP/RBM4 and control were generated. The expression of two YAP target genes, Ctgf and Itgb, was measured by real-time PCR. The mean±s.d. of relative mRNA levels from triplicate experiments were plotted. (g) The transcriptomes of H157 cells expressing YAP, YAP/TEAD4-FL, YAP/TEAD4-S or YAP/RBM4 were determined by RNA-seq. Using hierarchical clustering of all samples, we identified a subcluster of 429 genes that are activated by YAP/TEAD4-FL, but the activation is eliminated by co-expression of YAP with TEAD4-S or RBM4. Relative expression levels are displayed using Java TreeView. (h) The protein interaction networks of 429 genes were identified using the STRING database, and the highly connected groups were defined by MCODE and displayed using Cytoscape. The hub proteins interacting with multiple clusters were coded with multiple colours, and the enriched functions/gene ontology terms were labelled.
