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Common and Distinctive Functions of the Hippo Effectors Taz and Yap In

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Common and Distinctive Functions of the Hippo Effectors Taz and Yap In TISSUE-SPECIFIC STEM CELLS aRandall Division of Cell and Common and Distinctive Functions of the Hippo Molecular Biophysics, King’s College London, London, UK; Effectors Taz and Yap in Skeletal Muscle Stem Cell bSchool of Medicine, Medical Sciences & Nutrition, Function University of Aberdeen, Foresterhill, Aberdeen, a b b a Scotland, UK; cSystems CONGSHAN SUN, VANESSA DE MELLO, ABDALLA MOHAMED, HUASCAR P. O RTUSTE QUIROGA, c b d,e,f Biology Ireland, Conway AMAYA GARCIA-MUNOZ, ABDULLAH AL BLOSHI, ANNIE M. TREMBLAY, c g b c Institute, Dublin, Ireland; ALEXANDER VON KRIEGSHEIM, ELAINA COLLIE-DUGUID, NEIL VARGESSON, DAVID MATALLANAS, d b,h a Stem Cell Program, HENNING WACKERHAGE, PETER S. ZAMMIT Children’s Hospital, Boston, Massachusetts, USA; Key Words. Taz • Yap • Tead • Satellite cells • Muscle stem cells eDepartment of Stem Cell and Regenerative Biology, Harvard University, ABSTRACT Cambridge, Massachusetts, USA; fHarvard Stem Cell Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regen- Institute, Cambridge, eration, regulating gene expression especially via Tead transcription factors. To investigate their Massachusetts, USA; gCentre role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. for Genome Enabled Biology Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or con- and Medicine, School of stitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a func- Medicine, Medical Sciences tion shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic and Nutrition, University of differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while mus- Aberdeen, Foresterhill, cle growth was mildly affected in Taz (gene Wwtr1–/–) knockout mice, there were no overt Aberdeen, Scotland, UK; effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/1: h Faculty of Sport and Health Yapfl8x/fl8x:Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, Sciences, Technical University microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some of Munich, Munich, Germany genes independently of YAP, including myogenic genes such as Pax7, Myf5,andMyod1 (ArrayEx- Correspondence: Peter S. press–E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in Zammit Ph.D., King’s College myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in London, Randall Division of Cell myogenesis and aspects of cytoskeleton organization (ProteomeXchange–PXD005751). Neither TAZ and Molecular Biophysics, nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt London SE1 1UL, UK. Telephone: and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 144-207-848-8217; Fax: 144- to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in pro- 207-848-6435; moting myoblast proliferation but Taz then switches to enhance myogenic differentiation. STEM e-mail: [email protected] CELLS 2017;35:1958–1972 Received June 14, 2016; accepted for publication January SIGNIFICANCE STATEMENT 7, 2017; first published online in STEM CELLS EXPRESS June 7, Hippo pathway effectors Yap and Taz play key roles in cell proliferation and tissue growth. We 2017. analyzed Taz in comparison with Yap in muscle stem cells. Taz promoted proliferation, a func- tion shared with Yap. However, Taz also enhanced differentiation into myotubes, unlike Yap. VC AlphaMed Press –/– 1066-5099/2017/$30.00/0 Muscle growth was affected in Taz (Wwtr1 ) knockout mice, while satellite cell-specific condi- tional knockout of Yap produced a regeneration deficit. Taz regulates some genes independently http://dx.doi.org/ of Yap, and Taz also interacts with proteins distinct from Yap, mainly involved in myogenesis/ 10.1002/stem.2652 cytoskeleton. In particular, Taz operates through Tead4 to enhance differentiation. In summary, This article was published online Taz and Yap have overlapping functions in promoting myoblast proliferation but later, Taz also on 28 June 2017. However, enhances myogenic differentiation. proof typesetting was subse- quently noticed to be incom- plete. This notice is included in the online and print versions to Many other signaling modules also regulate indicate that these have been INTRODUCTION Yap and Taz activity, such as mechanotransduc- finalized 5 July 2017. Transcriptional cofactors Yap (Yap1) and Taz tion [4], glucose-signaling [5], G-protein cou- pled receptors (GPCR) [6], Wnt [7–9], Smad This is an open access article (Wwtr1) mainly regulate gene expression by under the terms of the Creative binding Tead1–4 transcription factors. Together [10, 11], Notch [12], Pten-Akt-mechanistic Commons Attribution License, Yap, Taz, and Teads are the nexus of the Hippo target of rapamycin (mTOR) [13–16], and Lkb1- which permits use, distribution signal transduction network that includes the Ampk [17–20]. Yap and Taz are inhibited by and reproduction in any Hippo kinase cascade, comprising kinases Mst1 phosphorylation of multiple HXRSS motifs by medium, provided the original work is properly cited. (Stk4), Mst2 (Stk3), Lats1, and Lats2 [1–3]. Lats1/2, which promotes localization to the STEM CELLS 2017;35:1958–1972 www.StemCells.com VC 2017 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2017 Sun, De Mello, Mohamed et al. 1959 cytosol, 14–3-3 binding, and degradation [3, 21]. Yap Ser127 mice were donated by Fernando Camargo. Breeding/experi- and Taz Ser89 are key phosphorylation sites, and the mutation mental procedures were approved by the Ethical Review Pro- of Ser127 or Ser89 to alanine in Yap S127A and Taz S89A cess Committee of King’s College London, and performed respectively prevents phosphorylation at these residues and under the Animals (Scientific Procedures) Act 1986. results in constitutive activity. Signaling modules involving Yap/Taz control skeletal mus- Cell Culture cle myogenesis and adaptation to exercise. Tead transcription Satellite cells/myofibers were isolated as described previously factors bind CATTCC/GGAATG (MCAT or GTIIC motifs) found [41]. Briefly, murine extensor digitorum longus (EDL) myofib- in/near promoters and enhancers of cardiac and skeletal mus- ers were isolated by 0.05% type I collagenase for 2 hours. cle genes [3, 22–24]. However, Tead1 binds muscle genes Myofibers were washed in GlutaMax Dulbecco’s modified repressed in rhabdomyosarcoma [25], consistent with obser- Eagle’s medium (DMEM) (ThermoFisher Scientific (http://cor- vation that Yap and Taz can also repress gene expression [26]. porate.thermofisher.com/en/home.html), Waltham, Massachu- Satellite cells are responsible for postnatal skeletal muscle setts, USA) and plated on matrigel-coated plates in growth growth, hypertrophy, and repair/regeneration [27], and we medium (GlutaMax DMEM, 30% fetal bovine serum [FBS], 1% have shown that Yap promotes proliferation of myoblasts [28, chick embryo extract [CEE], 10 ng/mL basic fibroblast growth 29] but inhibits myogenic differentiation. YAP1 S127A expres- factor, 1% penicillin-streptomycin (Sigma-Aldrich (http://www. sion in activated, but not quiescent, satellite cells causes sigmaaldrich.com/united-kingdom.html), St. Louis, Missouri, embryonal rhabdomyosarcoma-like tumours with short latency USA.) at 378C with 5% CO2. After 72 hours, myofibers were and 100% penetrance [25]. Expression of YAP1 S127A in mus- removed, and satellite cells passaged and preplated for 30 cle fibers causes myopathy [30], whereas other types of Yap minutes to remove fibroblasts, before being plated on delivery or Yap mutants can cause hypertrophy [31, 32], with matrigel-coated plates. Differentiation was induced in differ- outcome likely dependent on Yap levels. entiation medium (GlutaMax DMEM, 2% horse serum, 1% Taz harbors the same functionally important WW and penicillin–streptomycin). Nonadherent cultures: myofibers Tead-binding domains as Yap and often acts as a paralogue, were cultured in GlutaMax DMEM, 10% horse serum, 0.5% but not always [2, 3]. This is demonstrated by Yap or Taz CEE, 1% penicillin–streptomycin. knockout mice: while Yap knockout causes early embryonic lethality [33], 50% of Taz-knockout (Wwtr1–/–) mice are viable Muscle Injury but develop glomerulocystic kidney disease [34]. Earlier To recombine via the loxP sites flanking Yap exons 1 and 2, reports suggest that Yap and Taz also have divergent functions 200 mg of Tamoxifen/gram body weight (Sigma T5648) was in the skeletal muscle lineage: both Yap and Taz promote skel- injected intraperitoneally in sunflower oil/5% ethanol for 3 etal muscle fiber hypertrophy and regeneration [35, 36], but consecutive days, followed by maintenance on a tamoxifen- only Taz promotes fusion into multinucleated myotubes [29, containing diet (Tekland). Injury was induced in tibialis ante- 37, 38]. rior (TA) by 30 mL intramuscular injection of 20 mM cardio- The distinct functions of YAP and TAZ are poorly under- toxin (CTX)/saline. stood, so we investigated their regulation, function, target Retroviral Expression and Small Interfering RNA genes, and binding partners in murine myoblasts. Taz and Yap have overlapping functions in promoting satellite cell prolifer- Wild-type (WT) TAZ, TAZ S89A, YAP S127A, or WT YAP was ation. However, later in myogenesis,
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