Project description:To understand the direct target genes YAP transcription co-activator invovled with in gonome-wide levels, next generation sequencing was conducted in Flag-YAP-5SA overexpressing MCF10A cells using Flag (sigma) antibody.
Project description:YAP is an oncogene and an inducer of Epithelial-to-Mesenchymal Transition (EMT). We used microarrays to detail the global program of gene expression to identify YAP target genes. PUBLICATION ABSTRACT:; The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. We have recently shown that the human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and that it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. Here we show that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Since this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition (EMT), suggesting that this is a key feature of YAP signaling in mammalian cells. Experiment Overall Design: MCF10A cells were infected with retrovirus constructs (vector or YAP) and puromycin was used to select for transduced cells. Cells were split and grown to ~60-75%% confluency at which point they were harvested for RNA. Vector vs. YAP comparison was done in duplicate.