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(vestigial-like, coactivator and al., Vgll1-4 (transcriptional et and Oka motif) Taz 2011; binding al., paralogue et its (Chan angiomotin Yap, pathway or 2011) Hippo al., the et of independently 2011). regulated al., by et be Zhao also auxiliary 2010; can and Harvey, Mst1/2 Yap and Sudol by which 2010; regulated Lats1/2 (Pan, and kinase proteins phosphorylated upstream is its turn by at in residues phosphorylated other is and Yap pathway. Ser127 transduction within signal co-factor Hippo transcriptional a the is (Yap) protein Yes-associated Introduction words: Key Yap decisions. fate cell satellite Pax7 self-renewal of versus the maintains regulator differentiation maintain Yap novel the to after a of until self-renew as expression expressed or Yap highly Constitutive fibres, remains co-factor proliferate made. Yap muscle transcriptional to and activated is regenerating the activation be or cell decision identify can satellite hypertrophying they we during muscle, to Here, increases mature myonuclei expression pool. in quiescent further cell Mitotically supply stem muscle. to resident skeletal of myoblasts cells generate stem and resident the are cells Satellite Summary work this to equally ` contributed authors *These 6 5 4 3 2 1 o:10.1242/jcs.109546 doi: 6009–6019 125, Science Cell of Journal 2012 September 5 Accepted raeepesdi emnlydfeetae kltl ada or cardiac skeletal, Mrf4), differentiated terminally Myf5, in MyoD, expressed that are (myogenin) genes (e.g. differentiation or key terminal D1), myogenesis of cyclin (e.g. regions early proliferation enhancer regulate or promoter either the in located ee .Zammit S. Peter uhrfrcrepnec ( correspondence for Author igsCleeLno,RnalDvso fCl n oeua ipyis e utsHue u’ aps odnS11L UK 1UL, SE1 London Campus, Guy’s Australia House, 6027, Hunt’s WA New Joondalup, Biophysics, University, Molecular Cowan and Edith Cell (ParkC), USA of Centre 02138, Division USA Parkinson’s MA Randall 02138, Cambridge, London, MA University, College Cambridge, Harvard King’s Institute, Biology, USA Cell Regenerative 02115, Stem and MA Harvard Cell Boston, Stem Boston, of Hospital Department Children’s Program, Cell and Stem Sciences, Medical of School 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. a ctnn(clglic ta. 2011), al., et (Schlegelmilch -catenin aelt el,Seea uce e-soitdpoen(a) ip pathway Hippo (Yap), protein Yes-associated muscle, Skeletal cells, Satellite < 0.Cnitn ihteclua hntp,mcorasso htYpicessepeso fgnsascae with associated genes of expression increases Yap that show microarrays phenotype, cellular the with Consistent 40%. 5 ennoD Camargo D. 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Robert , 0 ftecss(hoe l,20)sgetn htYpco- Yap that suggesting 2008) al., et (Zhao cases the of 80% h lmnsadfnto fteHpoptwywr first were pathway Hippo the of function and elements The + aelt el n aelt eldrvdmyoblasts, cell-derived satellite and cells satellite 1, ` 5,6 oyUrcia Roby , hr ncoto h inhibitory the of knockout where , < -odlvrhypertrophy liver 4-fold 1 oioD Bari De Cosimo , 6009 7 , Journal of Cell Science eoeatvtdadr-ne h elcce yDi expressed is MyoD cycle, cell the (Pax7 re-enter and activated become ossetwt hs bevtos irary identify microarrays differentiation. observations, their these prevents with but satellite Consistent and myoblasts self- cells satellite pool or MyoD-positive the cell-derived and expands differentiate Pax7 activity Yap either activated, constitutive of cells that report satellite elevated We remains renew. increases cell activated Yap expression satellite and after the Yap activation in until that cell that role satellite time show key during first We greatly a fate. the plays and for Yap proliferation demonstrate member Mourikis we pathway 2008; Here, al., Hippo et 2012). 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Yap in that biogenesis that MCAT-elements co-activate which found genes and by ribosomal factors we mechanism as molecular works, cycle, the differentiation Yap Exploring Yap. cell myogenic by targeted of the modulators of regulators 00.Atvtdstlieclste rlfrt n either and proliferate then cells (Pax7 satellite and differentiate al., et Activated genes (Cao of factors the 2010). transcription thousands up sequence-specific open to for presumably chromatin which binds modifications MyoD chromatin induces 2012). 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Journal of Cell Science eosre significant, to in a medium. triggered mRNA differentiation observed were mitogen-low Yap in We that measured culture myoblasts by we differentiate cell-derived differentiation satellite downregulation during plated the for Yap self-renewing evidence further and of that obtain differentiating To implies both cells. fibres in satellite muscle in downregulated and is cells Yap satellite quiescent in Yap oprbefnto nstlieclsw are u ohgi-and gain- both out a carried of has we cells Yap proliferation satellite whether in the investigate function To comparable regulate cells. stem to adult of shown types previously several been has myoblasts cell-derived Yap satellite of and proliferation cells phosphorylation promotes satellite activity and Ser127 expression Yap. 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Funding joint Peter are paper. Wackerhage this shRNA- Henning for construct. Yap and authors the senior Camargo a providing (MCAT-reporter) for D. for providing Fernando lentivirus Piccolo Zammit, for 8xGTIIC-luciferase Stefano a Dr. Sudol and for the Marius Yap of Guan Dr knockdown Kun-Liang mediated thank Dr to antibody, like would We Acknowledgements instructions. Dual-Luciferase manufacturer’s a the quantified following activity using hours luciferase luminescence Firefly/Renilla Twenty-four and by instructions. lysed were with manufacturer’s cells transfection, co-transfected the Lipofectamine post then following using hYAP1 were (Invitrogen) control, construct with Renilla Cells LTX transduced and retroviruses. and (MCAT-reporter) S127A plates 8xGTIIC-luciferase well hYAP1 6 and into wild-type seeded were cells C2C12 assay reporter luciferase MCAT 13,000 cocktail at supplemented inhibitor spun protease X-100) and 4 buffer and Triton homogenised at EDTA were 1% lysis lysates mM NaF, cell 0.5 mM (IP) Total orthovanadate, 50 (Sigma). sodium immunoprecipitation NaCl, mM mM in 1 150 with lysed HCl, Tris were mM (50 myoblasts C2C12 Plated assays Co-immunoprecipitation rprdfrwsenbo nlssa above. as needle analysis G 30 26 blot in a western resuspended using for then removed were was prepared Beads supernatant loss. 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Vector (4,6-diamidino-2-phenylindole; DAPI of fpoenlstswr nuae ih15 with incubated were lysates protein of g ˚ ormv rtista idnnseiial.Ec protein Each non-specifically. bind that proteins remove to C m fete d rIUadclswr usdfr2– for pulsed were cells and IdU or EdU either of M < 3 H m d i Ivtoe)wsue olwn the following used was (Invitrogen) kit EdU fYpatbd gf rmMrsSdl or Sudol) Maris from (gift antibody Yap of g a nstliecls6017 cells satellite in Yap H eotrAsySse (Promega) System Assay Reporter ˚ n osse frbi anti-Yap rabbit of consisted and C m l2 ˚ m :rbi niYp(a anti-Yap rabbit C: 6 rti G-agarose protein l 9 a apebfe and buffer sample doyrdn)or -deoxyuridine) tbln(Sigma, -tubulin g o 5minutes 15 for ˚ C. Journal of Cell Science uot . ost . rgn,M,Ez,E,Guit,S,Creos,M., Cordenonsi, S., Giulitti, E., Enzo, M., Aragona, L., Morsut, S., Dupont, M. Gayyed, A., S. Comerford, N., Zhang, S., Wu, J., Huang, G., Feldmann, J., Dong, a hkr . eg . aanta,R,Spa .J,Set,J .and B. J. Skeath, J., M. Seppa, R., Jagannathan, Y., Feng, M., Thakur, Das oln,C . le,I,Zmi,P . elp . ere . atig,T .and A. T. Partridge, A., Petrie, L., Heslop, S., P. Zammit, I., Olsen, A., C. Collins, S. P. Zammit, and A. C. Collins, hn .W,Lm .J,Cog .F,Pbai .V,Hag .adHn,W. Hong, and C. Huang, V., A. Pobbati, F., Y. Chong, J., C. Lim, W., S. Chan, Goodyear, L., J. Shadrach, F., M. Hirshman, A., C. Witczak, S., Jurga, M., Cerletti, ah .N,Agra,E . atmr,C,Nln . ho . ii,Y., Sidis, H., Zhao, K., Nolan, C., Kattamuri, B., E. Angerman, N., J. Cash, a,Y,Yo . akr . arne . ace,G . akr .H., M. Parker, J., G. Sanchez, M., Lawrence, D., Sarkar, Z., Yao, Y., Cao, a,X,Paf .L n ae .H. F. Gage, and L. S. and Pfaff, R. Jaenisch, X., W., Cao, G. Bell, D., Fu, B., J. Johnnidis, S., Gokhale, D., F. Camargo, and G. Mengus, B., Jost, I., Michel, A., Morlon, T., Ye, C., Keime, A., Benhaddou, ele,T,Zag . ag . oil-lui,M,Kyi,E,Jhsn .L. R. 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