Project description:Endothelial cell is the major cell type that senses and transduces mechanosignal generated by shear stress. We have recently shown that Hippo/YAP pathway is a mechanosensitive pathway that is critical for maintaining endothelial cell homeostasis. However, the transcritpional targets and biological functions of YAP in endothelial cells remain largely unknown. To evaluate YAP-dependent gene expression in endothelial cells, we performed RNA-sequencing in YAP depleted (by transfection with by YAP siRNA) and overexpressed (by infection with YAP-S127A catalytically active adenovirus) human endothelial cells. We observed that YAP critically regulates endothelial function by modulating multiple atherosclerosis-related genes. Our study provides mechanistic insights into the question how YAP regulates endothelial function and atherosclerosis by modulating endothelial transcriptional profile.
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.
Project description:Epigenetic remodeling is essential for oncogene-induced cellular transformation and malignancy. In contrast to histone posttranslational modification, how oncogenic signaling remodels DNA methylation remains poorly understood. The oncoprotein YAP, a coactivator of the TEAD transcription factors mediating Hippo signaling, is widely activated in human cancer. Here we identify the 5-methylcytosine dioxygenase TET1 as a direct YAP target and a master regulator that coordinates the genome-wide epigenetic and transcriptional reprogramming of YAP target genes in the liver. YAP activation induces the expression of TET1, which physically interacts with TEAD to cause regional DNA demethylation, histone H3K27 acetylation and chromatin opening in YAP target genes to facilitate transcriptional activation. Loss of TET1 not only reverses YAP-induced epigenetic and transcriptional changes but also suppresses YAP-induced hepatomegaly and tumorigenesis. These findings exemplify how oncogenic signaling regulates site specificity of DNA demethylation to promote tumorigenesis and implicate TET1 as a potential target for modulating YAP signaling in physiology and diseases.
Project description:The renewing human epidermis constantly senses and adapts to a wide range of mechanical cues that are ubiquitous throughout life. The mechanisms of how mechanical forces are responded by interfollicular epidermal stem cells (IFESCs) and are transmitted directly into nucleus to modify gene expression was discovered to be mediated by transcriptional coactivators YAP. Interestingly, YAP, a critical modulator in controlling organ size by regulating both cell proliferation and apoptosis, was also found to induce self-renewal or amplification of epidermal progenitor/stem cells, and balance epidermal growth and differentiation. Because growth regulation imposed by different stimuli is achieved through distinct YAP target genes even in a single cell type. This study used chromatin immunoprecipitation (ChIP) coupled with human promoter tiling microarray analysis (ChIP-on-chip) to profile YAP at promoters genome-wide. The goal of the study was to identify the target genes occupied by YAP in IFESCs under the mechanical stretch condition.
Project description:Environmental cues, such as shear stress and heterotypic cell interactions play a critical role in endothelial cell function, yet their unique contributions to the endothelial cell transcriptome remain unclear. Using cell preparations from human umbilical cords (ex vivo), we performed individual sample analysis to assess transcriptional drifts associated with environmental changes but independent of sex or background. Global gene expression profiling by RNA-seq, ATACseq, and MS/MS directed proteomics distinguished freshly isolated endothelial cells from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Amongst several signatures, we observed that TGF-beta and BMP target genes were reduced. In contrast, cytoskeleton-based processes and proliferation-related genes were increased. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes including targets of BMP and Notch signaling known to be sensitive to flow. In contrast, co-culture of endothelial cells with smooth muscle cells normalized networks related to cell growth and differentiation, clathrin-vesicle related genes, and recovered targets downstream TGF-beta, recovering approximately 9% of the original in vivo signature. Our findings highlight specific genes, pathways and functional features of endothelial cells that require contextual information and exposure to physical forces. This transcriptional modulation is important to consider in all paradigms that are focused on understanding the ability of endothelial cells to maintain homeostasis and respond to disease processes.
Project description:ChIP-Seq profiling using human umbilical vein endothelial cells (HUVECs) to identify endogenous YAP, TAZ and TEAD1 DNA binding sites.