Identification of YAP-TEAD gene signatures in metastatic melanoma
YAP1 (Yes-associated protein 1) is transcriptional co-activator that partners with the TEAD family of transcription factors to regulate gene expression. Increased YAP-TEAD activity is strongly implicated in the development, progression, and metastasis of several cancer types including melanoma, but the YAP-TEAD target genes that are responsible for YAP-TEAD-dependent melanoma progression and metastasis are largely unknown. To identify YAP-TEAD regulated genes in metastatic melanoma cells we used RNA-sequencing to compare gene expression in control A375 human melanoma cells to A375 cells expressing mutant forms of YAP with increased transcriptional activity due to the mutation of LATS inhibitory phosphorylation sites (YAPS127A or YAPS127A,S381A). To determine which YAP-dependent gene expression changes are mediated by TEADs we also included a mutant form of YAP that is unable to bind TEADS (YAPS94A,S127A). Overall design: A375 human melanoma cells were infected with the retrovirus that delivers either a control empty vector (MSCV-IRES-Hygro) or the indicated mutants of human YAP (YAPS127A, YAPS94A,S127A, or YAPS94A,S127A,S381A). Stably transduced cells were selected with hygromycin and then cells were plated tissue culture plastic in growth media (DMEM+ 10% fetal bovine serum, 2 mM L-glutamine) at ~60% confluence. Three individual biological replicates were plated for each cell line. Twenty-four hours later, cells were lysed with TRIzol and RNA was isolated according to the manufacturer's protocol. cDNA was generated as described and used for RNA-sequencing.