title: Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions.
abstract: The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic-nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Agarinis
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: V
authors.initials: V
authors.lastname: Orsini
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Megel
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: Y
authors.initials: Y
authors.lastname: Abraham
authors.affiliation: Novartis Institutes for Biomedical Research, Novartis, Cambridge, MA, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.affiliation: Novartis Institutes for Biomedical Research, Novartis, Cambridge, MA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Mickanin
authors.affiliation: Novartis Institutes for Biomedical Research, Novartis, Cambridge, MA, USA.
authors.forename: V
authors.initials: V
authors.lastname: Myer
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Bouwmeester
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: J S
authors.initials: JS
authors.lastname: Tchorz
authors.affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland Christian.Parker@novartis.com.
authors.forename: C N
authors.initials: CN
authors.lastname: Parker
mesh_terms: D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D055808:Drug Discovery; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C456659:YY1AP1 protein, human
keywords: Hippo; YAP; frequent hitters; siRNA screening