Project description:The Hippo pathway downstream effectors, Yap and Taz, play key roles in cell proliferation and tissue growth, regulating gene expression especially via interaction with Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analysed gene expression changes driven by Taz and compared these to Yap mediated changes to the transcriptome by measurement of gene expression on Affymetrix microarrays. To interrogate overlapping and unique transcriptional changes driven by these Hippo effectors, satellite cell-derived myoblasts were transduced with constitutively active TAZ S89A or YAP S127A retrovirus for 24h or 48h, with empty retrovirus as control. Triplicate microarray analyses of empty vector controls, hYAP1 S127A and TAZ S89A transgenic primary myoblasts were conducted.
Project description:Abstract Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via interaction with Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analysed Taz in vivo and ex vivo in comparison to Yap. Taz was expressed in activated satellite cells. siRNA knockdown or constitutive expression of wildtype or constitutively active TAZ mutants showed that TAZ promoted proliferation, a function that was shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene symbol Wwtr1-/-) knockout mice, there were no overt effect on regeneration. However, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapflox/flox : Rosa26Lacz mice produced a marked regeneration deficit. To identify potential mechanisms, microarray analysis showed many common Taz/Yap targets, but Taz also regulates some genes independently of Yap, including myogenic genes such as Pax7, Myf5 and Myod1. Proteomic analysis of Yap/Taz revealed many common binding partners, but Taz also interacts with proteins distinct from Yap, that are mainly involved in myogenesis and aspects of cytoskeleton organization. Neither TAZ nor YAP bind members of the Wnt destruction complex but both extensively changed expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to promote myogenic differentiation.
Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma. 60 pooled eyes from 36 hpf wild type or vsx2:Gal4/dsRed:14xUAS:YapS87A embryos were pooled for one sample. Three wild type and three vsx2:Gal4/dsRed:14xUAS:YapS87A pools were analyzed for RNA.
Project description:The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ overcomes PLX4032 resistance, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and cytoskeletal tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a promising synthetic lethal strategy to suppress resistance in BRAF inhibitor therapies.
Project description:Human cancer is often caused by dysfunctional developmental pathways, but such mechanisms do not always present clear opportunities for therapeutic intervention. This is exemplified by the Hippo tumor suppressor pathway, which is comprised of a kinase module that restrains the function of YAP/TAZ transcriptional coactivators; a pathway that becomes dysregulated in a wide array of human cancers. Hence, YAP/TAZ hyperactivation is a tumorigenic mechanism and a validated therapeutic target in oncology. In this study, we used a paralog co-targeting genetic screening strategy to identify the kinases MARK2/3 as co-dependencies of YAP/TAZ in diverse cancer contexts. We use biochemical and epistasis experiments to show that MARK2/3 phosphorylate and inhibit the activity of Hippo pathway components NF2, MST1/2, and MAP4Ks, which leads to indirect upstream control of LATS1/2 activity. In addition, MARK2/3 directly phosphorylate YAP/TAZ to shield these coactivators from LATS1/2-mediated inhibition. The net consequence of this multi-level regulation is that YAP/TAZ-dependent human cancers have an absolute requirement for MARK2/3 catalytic activity to sustain tumor cell proliferation and viability. To simulate therapeutic targeting of MARK2/3 in vivo, we adapted the EPIYA-repeat region of the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show exerts potent anti-tumor activity via on-target mechanisms. Together, these findings reveal MARK2/3 as an obligate catalytic requirement for YAP/TAZ function in human cancer; kinase targets that may allow for novel pharmacology that restores Hippo-mediated tumor suppression.
Project description:The two effector proteins of the Hippo signaling pathway, YAP and TAZ, play a pivotal role in the cellular homeostasis of podocytes and in the pathogenesis of focal segmental glomerulosclerosis (FSGS). We aim to unravel the unique and redundant functions of YAP and TAZ in the podocyte by identifying podocyte-specific interactors. We generated stable heat sensitive mouse podocytes (hsMPs) carrying a single copy integration of a transgenic construct expressing a flagged version of mouse Yap (3XFLAG.YAP), Taz (3XFLAG.TAZ) or Ruby (3XFLAG.RUBY) in the Rosa26 locus. To explore the interactome of YAP and TAZ in podocytes we immunoprecipitated the tagged proteins and characterized the co-immunoprecipitated protein complexes by mass spectrometry. Within the interactome analyses of the hsMPs, we identified shared and non-shared interacting proteins between YAP and TAZ. Among these identified proteins many well established interactors of YAP and TAZ were included, like proteins of the Tead family, different angiomotins or large tumor suppressor kinase 1 (Lats1). Strikingly, among the shared proteins were numerous proteins of the nuclear shuttling machinery, like importins (Ipo), exportins (Xpo), transportins (Tnpo) and nucleoporins (Nup) that form the nuclear pore complex (NPC), such as NUP107, NUP133, NUP205 and XPO5.
Project description:The uptake of macromolecules and cellular debris through macropinocytosis has emerged as an important nutrient acquisition strategy of cancer cells. Genetic alterations commonly found in human cancers (e.g. mutations in KRAS or loss of PTEN) have been shown to increase macropinocytosis. To identify additional effectors that enable cell growth dependent on the uptake of extracellular proteins, pancreatic ductal adenocarcinoma (PDA) cells were selected for growth in medium where extracellular albumin was the obligate source of the essential amino acid leucine. Analysis of global changes in chromatin availability and gene expression revealed that PDA cells selected under these conditions exhibited elevated activity of the transcriptional activators Yap/Taz. Knockout of Yap/Taz prevented growth of PDA cells in leucine-deficient medium, but not in complete medium. Furthermore, constitutively active forms of Yap or Taz were sufficient to stimulate macropinocytosis of extracellular protein. Together, these studies suggest that the Hippo pathway effectors Yap and Taz are important transcriptional regulators of endocytic nutrient uptake.