Teeatoscnrbtdeulyt hswork this to equally contributed authors *These Switzerland. Basel, 4056 n a,zxnepeso n pteilmsnhmltransition Diepenbruck epithelial–mesenchymal Maren Yap and of expression distribution zyxin Taz, subcellular and the control levels expression Tead2 ARTICLE RESEARCH eevd6Ags 03 cetd1 aur 2014 January 18 Accepted 2013; August 6 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` primary 2 the from 1 dissemination with cells permit tumor provides that only properties not invasive EMT Thiery Oncogenic can 2011; 2009). that Weinberg, cells al., mechanisms and et tumor the (Chaffer epithelial of spread one metastatic in as facilitate features regarded is these EMT) of (oncogenic a progression, some tumor and of solid During reactivation polarity, and 2011). (Kalluri Nieto, embryonic phenotype apical-basal 2009; invasive Weinberg, and and induces of migratory is a which adhesions cells by state, stages characterized mesenchymal cell–cell epithelial a into various trans-differentiation in of promotes at EMT loss of required a cell-biological Activation is a development. that is program (EMT) transition Epithelial–mesenchymal INTRODUCTION Zyxin Yap, Tead, Taz, metastasis, EMT, cancer, Breast WORDS: and KEY EMT for and critical Taz, genes and of Yap expression metastasis. of the distribution control cellular they by together the are evoked factors of transcription regulators phenotype Tead crucial that invasive demonstrate and results The migratory Tead2. the zyxin to these, Among EMT. contributes during Tead2 revealed of profiling targets complexes. expression transcriptional Tead2–Yap–Taz the co-factors gene with of combination generation its in next formation sequencing thereby and of the immunoprecipitation cells, chromatin localization via of Genome-wide Taz nucleus nuclear and the predominant Yap in previously cancer a increase breast Tead2 a and EMT.directing of cells during epithelial uncovered levels Tead2 gland cells, factor mammary have in transcription EMT the We During EMT of during role elusive. role unanticipated functional remained direct been their their expression and have have however, EMT; genes Taz gene an target and promoting transcriptional in Yap factors in co-activators implicated transcription changes previously transcriptional Tead global their factors. and transcription on by rely orchestrated transition largely epithelial–mesenchymal an (EMT) during changes cellular The ABSTRACT rkvnNimwegen van Erik uhrfrcrepnec ([email protected]) correspondence for Author iznrm nvriyo ae,adSisIsiueo Bioinformatics, of Institute Swiss Switzerland. and Basel, Basel, 4058 of Basel, University of Biozentrum, University Biomedicine, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,12–56doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 1, ,Lrn Waldmeier Lorenz *, n ehr Christofori Gerhard and 1, ,Rbr Ivanek Robert *, 1, ` oei etitn ra ieb naoiigteoncogenic signaling and adhesion the cell of network antagonizing complex 2012; A Guan, 2011). by and al., et (Hong Zhao size Taz and organ Yap co-activators transcriptional restricting in role al., et Moreno-Bueno 2009; 2011). al., Nieto, et 2008; (Acloque program EMT the coordinatorsof master pathways the are signaling factors transcription and EMT, during genes andactive many be Scheel the 2009; Weinberg, also Among and 2012). Polyak Weinberg, might 2012; al., and et Magee Weinberg, therapy 2011; and (Chaffer traits, organs cancer distant stem-cell-like at colonization for of for important acquisition implications the has in which results also but tumor, pteilclsi eurdfrteeclst neg TGF undergo mammary to murine cells EpH4 these in nuclear for the required Taz and is 2008b), and al., al., cells Yap et et epithelial Zhao (Lei of 2009; manner human al., accumulation Tead-dependent MCF10A et of a Zhang EMT in 2008; induce cells Moreover, to 2008b). epithelial sufficient al., breast are et and Taz Zhao (Ota 2009; and fibroblasts al., Yap et and Zhang cells 2008; Taz-mediated epithelial Sasaki, they or example, in (Mahoney For Yap- proliferation functions. Upon Taz control multiple 2001). exert or al., can Teads et Yap association activation, Vassilev 2005; physical co-activators al., their 2008; transcriptional et activity requires al., Transcriptional the Teads 2007). et with al., DNA-binding et Sawada the Yagi 2004; of 2005; whose al., and al., et notochord crest, et Sawada 2009), al., neural (Milewski et development including (Thiery trophectoderm EMT the tissues, on regulating depends and embryonic formation in 1998) various DePamphilis, roles and of redundant Kaneko partially 1996; expressed exert anti- al., ubiquitously et are and They (Jacquemin (Tead1–Tead4). proliferative members four of transcription expression DNA-binding the Tead genes. nucleus apoptotic signaling, the with activate into Hippo together imported factors, activated are they, of Taz where absence and by the Yap in phosphorylated retained In unphosphorylated are are Taz cytoplasm. and Taz the Yap the and phosphorylated When and Yap Taz. Lats2, and and active, Lats1 Yap is and co-factors the Lats1 pathway kinases transcriptional Hippo from protein the neurofibromin-2/ the leading to via cascade, Mst2 Lats2 suppressor and kinase Mst1 Hippo kinases tumor protein the the regulates Merlin, including molecules, nmdaigETidcinadcne rgeso,the progression, Taz transcriptional Tead cancer and of Yap and regulation the Teads, and induction in for cancer involved EMT role mechanisms breast studies mediating pro-metastatic crucial these in though a in a Even Tead demonstrate 2012). and al., clearly to et growth (Lamar cells binding melanoma tumor through of levels increased elevated phenotype Finally, trigger 2010b). al., Yap et (Varelas EMT induced 1 h ip uo upesrsgaigptwypasacritical a plays pathway signaling suppressor tumor Hippo The nmmas edtasrpinfcoscmrs aiyof family a comprise factors transcription Tead mammals, In hlp Berninger Philipp , 2 hlArnold Phil , 2 , 1523 b -

Journal of Cell Science edfml ebr surgltddrn M,concomitant EMT, during upregulated of expression is the members that report family We of cells. Tead cells cancer systems target breast epithelial murine model gland transcriptional in cellular mammary and murine their utilized non-transformed have identify in we EMT to EMT, and during genes Teads of be activities to remain EMT an during genes target identified. direct the and activity ARTICLE RESEARCH 1524 (MT gene E-cadherin the of ablation genetic mediated the ( of alleles gene MMTV- (floxed) a conditional E-cadherin of carrying tumor mouse mammary transgenic a MMTV- Neu from which Py2T established of of been and both have tumor TGF cells 2012), cells with a al., treatment upon et NMuMG from EMT underwent (Waldmeier derived utilized mice cells transgenic we PyMT cancer process, breast EMT murine the in S1A). an Fig. species- during material a (supplementary upregulated cells contain are NMuMG that of motif EMT genes MCAT motif that Fig. Tead-binding a suggested material conserved analysis (supplementary including factors This dynamics, predicted transcription S1A). important expression Tead timecourse EMT were by bound EMT the motifs of the transcription-factor-binding regulators from several gene derived of that 2009) al., data et (Suzuki expression (MARA) a Motif Analysis acquired 2008). Response al., and Activity et EMT with (Lehembre progressive morphology treatment mesenchymal underwent by complete cells 2005) the course, al., time et mammary (Maeda murine NMuMG normal TGF line untransformed cell the EMT, in gland an of EMT stages induced multiple the we underlying genes crucial identify To EMT its during and activity complex Tead2–Yap–Taz transcriptional nuclear a of Formation RESULTS an invasion. and EMT-related and adhesions for migration required focal is of protein, remodeling component cytoskeleton a actin zyxin, these, as Among encoding EMT. gene genes during the Tead2 EMT-relevant of targets several transcriptional direct identified gene profiling with combination expression in (ChIP-Seq) and and sequencing immunoprecipitation generation Taz EMT next chromatin and Genome-wide an Yap EMT. undergoing of blocks redistribution cells subcellular in efficient Teads prevents phenotype. of tumor malignant activity knockdown a transcriptional increased and Conversely, Tead2 EMT complex of to increased induction a result, the form lead provokes a they We As where Tead2 Taz, Tead2. activity. and of with transcriptional Yap levels of Tead localization elevated nuclear in that increase demonstrate overall an with soitdicesdTa2epeso a oeyosre in observed EMT- solely 1B). was (Fig. expression Tead2 induction Tead2 EMT that increased upon revealed associated increase immunoblotting three all levels by in protein systems levels expression model antibodies Tead2 polyclonal EMT S1C– endogenous Fig. of generated Analysis material we and E). (supplementary Tead2, Tead2 Tead2 of detecting on expression specifically focus reproducible to and S1B). robust after Fig. chose the material and on (supplementary during across analyzed Based be upregulated before, systems were could levels model systems mRNA members all Tead2 model family only that Tead three and EMT, four the all in of detected transcripts the that 1A). (Fig. 2008) al., et (Lehembre odlnaetemcaim neligtetranscriptional the underlying mechanisms the delineate To odlnaetergltr oeo edtasrpinfactors transcription Tead of role regulatory the delineate To eeepeso rfln n uniaieR-C revealed RT-PCR quantitative and profiling expression Gene b o ,1 ,7 0ad2 as(aantson.Drn this During shown). not (data days 20 and 10 7, 4, 1, 0, for Cdh1 .TeeclsudroETuo Cre- upon EMT undergo cells These ). b Fg A.MTflECad 1A). (Fig. D ECad) h nrae eeso ed oYpadTzi h nucleus the whether activity in investigated first Taz transcriptional we hypothesis, and Tead2-mediated this test Yap To activate EMT. to during to Tead2 of required binding of that is levels hypothesized we increased above, the presented data the From and EMT Tead2 by for mediated required is is Taz and Yap of localization Nuclear a n a,hgl iia ota fTa2(i.1C). (Fig. Tead2 of that to similar highly Taz, Py2T and and Yap NMuMG Conversely, MT TGF 1C). (Fig. undergoing 2010) of cells nuclei al., the cells et proliferating growing, in (Zhao sparsely in and expected cytoplasm as cells, the epithelial they throughout that revealed distributed Taz were of and Yap process co-activators transcriptional the Tead undergone MT in had 1C, or were (Fig. Py2T) that and EMT NMuMG cells 1C, (Fig. in EMT observed nuclearundergoing stronger be much a could yet cells, staining epithelial in present were Tead2 S1H). Fig. is material TGF (supplementary expression cells during Tead2 Py2T activity and that NMuMG Sox4 revealed on 2013), regulator dependent al., crucial strictly et a (Tiwari be Sox4 EMT to of known of factor ablation transcription siRNA-mediated a TGF Notably, expression, S1G). canonical Fig. of material independent is TGF that suggesting levels, expression expression TGF Smad4 during of ablation (siRNA)-mediated small RNA Furthermore, interfering Tead2 S1F). induce Fig. to able material not (supplementary were expression IL-6 or PDGF IGF, HGF, FGF, EGF, TGF with stimulated cells Py2T ed–a–a ope ntences nacrac with accordance increases In EMT. activity transcriptional during Sox4- nucleus. Tead overall a the the observations, of these in in formation increased complex enhanced with Tead2–Yap–Taz are concomitant levels manner dependent expression Tead2 induction, S2D). Fig. material complete (supplementary a activity in reporter resulted the site of Tead-binding loss cells, the NMuMG of MCAT of EMT mutation during Tead-responsive induced described whereas was 2011) a previously al., In et containing (Lai a 2008). motif reporter of Sasaki, promoter activity and Cyr61 transcriptional (Ota the S2B) (ACATTCCAC; motifs addition, Fig. Fig. core GTIIC material material or 1996) supplementary supplementary either al., et (CATTCCT; bearing (Larkin S2A) motifs constructs core reporter by MCAT assessed luciferase was Tead EMT undergoing Tead-responsive S2A–C). cells Fig. in activity material Sox4-transcriptional (supplementary a in fashion induction dependent EMT EMT upon upon increased activity decrease transcriptional Taz S1I). Fig. and material Yap supplementary of 1D; (Fig. EMT, levels in induction during total increased Tead2 formation is though that complex to conclude even Tead2–Yap–Taz we nuclear bound data these a only From of MT). 1D, Taz (Fig. and cells to Taz Yap MT and TGF both mesenchymal Yap upon Similarly, nucleus of the binding tion. in the material exclusively indeed, increased (supplementary that, Tead2 in extracts revealed or S1I) cell NMuMG) Fig. Py2T, nuclear 1D, and (Fig. Co-immunoprecipitation lysates cytoplasmic EMT. cell an total in during experiments change to subject was muoloecneaayi hwdta o eeso nuclear of levels low that showed analysis Immunofluorescence ae oehr hs aadmntaeta,uo EMT upon that, demonstrate data these together, Taken pan-Tead that found we observations, these with Consistent Taz and/or Yap to binding Tead2 whether investigated next We D cdclsdslydpeoiatyancerlclzto of localization nuclear a predominantly displayed cells Ecad ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal b idcdETi MM el i o fetTead2 affect not did cells NMuMG in EMT -induced D D cdclsadnti pteilMTflEcad epithelial in not and cells Ecad cd.Imnfursec tiigo the of staining Immunofluorescence Ecad). b idcdETo tbymesenchymal stably or EMT -induced b hra te rwhfcoslike factors growth other whereas , b b idcdTa2expression Tead2 -induced inln (supplementary signaling b idcdETin EMT -induced b stimula-

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Journal of Cell Science pteilMfEa el yCercmiaemdae ncoto h E- the 50 of bars: Scale knockout gene. Cre-recombinase-mediated cadherin by cells MTflEcad MT TGF epithelial mesenchymal with Stably treated EMT. were induce cells to Py2T systems and model NMuMG EMT Epithelial cellular different used. three the of redistribution counterparts subcellular mesenchymal Taz and Yap EMT. and during upregulation Tead2 1. Fig. ARTICLE RESEARCH 1526 (supplementary cells with TGF these expected, or expression in As whose (siCtr) S1B). EMT (siTead1-3) Fig. siRNA 3 during material and upregulated control 2 were with Tead1, against transfected NMuMG siRNAs EMT. for were and redistribution Taz cells and Yap for required is (supplementary Y440H Tead2 mutant but S3A). of Tead2 Fig. expression wild-type material of the expression by the not by transcriptional increased Tead was observations, activity direct these by the with distribution subcellular in Consistent Taz binding. Taz and and Yap controls suggesting Yap Taz, Tead2 and of that Yap cytoplasmic concentration of levels NMuMG marked Li reduced and in a nucleus 2007; (Tead2-WT) in the Kitagawa, Tead2 Conversely, resulted 2008b). 2010; wild-type cells al., et al., of Tead2 Zhao expression et 2010; a al., forced (Chen et express Tian 2A) 2010; to al., (Fig. to binding et Taz of transfected incapable or is cells which Yap cells Y440H), evenly in Tead2 control 2A, were or in (Fig. mutant nucleus Taz a Vector) and and 2A, provoke cytoplasm (Fig. Yap the can that between Immunofluorescence distributed cells revealed Taz. analysis and epithelial Yap microscopy in of redistribution Tead2 subcellular of overexpression D neato fTa2wt a n a.Clswr rae ihTGF 25 with bar: treated Scale were Cells nuclei. Taz. visualize and Yap to with Tead2 used of was Interaction Taz staining DAPI (D) E-cadherin and control, proteins. Yap a EMT. both detects as of that served induction antibody after an and and with Tead2 stained before of Taz were Actin staining and cells. Immunofluorescence Yap MTflEcad (C) co-factors in control. its deletion loading a genetic as by served and cells, TGF Py2T after and and NMuMG during before, levels expression E-cadherin ramn ihdxcciefr3dy e oaheterogeneous a to led system. days doxycycline-inducible 3 the for to doxycycline of sufficient wild-type with control expressed Treatment is the that cells cells under Py2T Tead2 epithelial generated We in the EMT. induce activity regulating transcriptional for and EMT. for required crucial EMT is during process regulatory are Taz this and that Teads and Yap of These that TGF redistribution S3D,E). cytoplasmic-nuclear in Fig. indicate material process (supplementary EMT results cells the transcriptional Py2T attenuated treated Tead (Tead2-EnR) Tead2 blocking version dominant-negative of a of Similarly, expression inducible mesenchymal the S3C). by activity the Fig. (supplementary of was delayed material were E-cadherin upregulation N-cadherin and of fibronectin conversely, markers downregulation and, ablation: inhibited Tead of attenuation upon an revealed EMT expression marker EMT of immunoblot- analysis Moreover, ting S3B). Fig. disassembly material during (supplementary the observed normally EMT junctions averted Yap 2B). adherens and also (Fig. of junctions tight failure redistribution of redistribution resulting subcellular the Taz subcellular and and Teads this of depletion prevented Importantly, Teads localized TGF to of nuclear response in predominantly Taz and displayed Yap siRNA control with oto.Lvl fYp a n ed eedtrie yimmunoblotting control. by loading determined negative as were a served Tead2 as Gapdh and IgG Taz analysis. Yap, irrelevant of or Tead2 Levels was against control. (IP) antibody Co-immunoprecipitation an (Py2T). with days performed 7 or (NMuMG) days enx sesdwehrurglto fTasdrn EMT during Teads of upregulation whether assessed next We enx se hte xeietlyicesn ed levels Tead2 increasing experimentally whether asked next We A opooia ifrne ewe pteiland epithelial between differences Morphological (A) m .()Imnbotn nlsso ed and Tead2 of analysis Immunoblotting (B) m. b tetet hra knockdown whereas -treatment, D cdclswr eie from derived were cells Ecad b tetdclstransfected cells -treated b idcdETin EMT -induced b o 3days 13 for b m o 4 for m. b - eatss(i.3,) ihahge niec e os as mouse per 3E). incidence six (Fig. higher macroscopic cells of control a out developed to with five compared cells and 3C,D), Tead2-VP16 (Fig. cells metastases with Tead2-WT half with injected contrast, injected found mice In mice were 3C,D). the (Fig. nodules vessels of these mice blood Notably, six within macroscopically clusters. of encapsulated displayed cell out cells tumor one by control visible only epithelial staining the that with and revealed injected and lungs eosin injection. mice, after and paraffin-embedded days hematoxylin of immune-deficient 33 scored sectioning nu/nu was Serial metastasis Balb/c lung of of expressing formation the stably into veins injected cells were vector tail Py2T control a cells. in or Tead2-VP16 capability cancer Tead2-WT, metastatic breast higher murine a into Py2T translated invasion cell and that, invasion. indicate and promotes migration results also cell Tead2 These cancer activities, EMT-inducing 3B). its (Fig. cells, with consistent matrix into control filopodia extracellular to projected the contrast and cells Matrigel Tead2-VP16 In there-dimensional and 3A). Tead2-WT invaded (Fig. spheres, smooth invade to formed which capability to of increased and and acquisition an Yilmaz migrate exhibited the Tead2-WT 2011; expressing is phenotype, (Nieto, mesenchymal cells their EMT with properties Consistent 2009). undergoing invasive Christofori, cells and of migratory hallmark A and invasion migration, metastasis cell tumor promotes Tead2 akr -ahrnadZ-,icesdepeso fthe of expression increased ZO-1, epithelial the of and loss the E-cadherin and [i.e. EMT morpholo- markers Tead2 with the consistent in wild-type changes resulted gical both also Tead2-VP16 of active nuclear constitutively expression S3G,I). Fig. increased forced material no (supplementary Furthermore, Taz but and Yap domain), of the localization transactivation by that VP16 (mediated activity Tead virus on transcriptional a dependent a lacking highly showed was a site (Tead2-VP16) Taz-binding of Tead2 and overexpression of Yap- although version S3G,H) Notably, active 2008). Fig. constitutively Sasaki, material wild- transcriptional and expressing (supplementary stably (Ota (Tead2-WT) cells Tead Py2T Tead2 in heightened type observed also and was activity complexes EMT. Taz–Tead2 induce to elevation sufficient this the was that levels expression; Tead2-expressing indicating Tead2 E-cadherin of S3F), treatment Fig. doxycycline induction, lose prolonged material a to (supplementary doxycycline with enforced started was after already phenotype had days 2C). (Fig. cells nucleus 3 the in Taz nucleus. and By the Tead2 Yap nuclear in of of as concentration levels in well resulted elevated Non- as the cytoplasm 2C). cells, the doxycycline-treated (Fig. in In showed staining cells Tead2 Taz of these and levels Yap endogenous in low expressing expression cells induced Tead2 of induction htTa2i bet nuea M i h omto of formation the via EMT an Taz. induce and Taz- Yap to with or complexes able Yap- active transcriptionally is morphology in demonstrate Tead2 results E-cadherin epithelial these that Collectively, of an 2E,F). (Fig. expression mesenchymal-to-epithelial cells by deficient increased a indicated the to as and led (MET), Transient cells transition and S3J)]. (siYap respectively) Tead2-WT in Fig. Taz siTaz, or stress material Yap actin of supplementary shift ablation to siRNA-mediated a actin 2D; cortical (Fig. and displaying Slug, fibers from and cytoskeleton the Zeb1/2 of vimentin, markers mesenchymal enx sesdwehrTa2idcdET elmigration cell EMT, Tead2-induced whether assessed next We a n a ula oaiain nrae omto fYap– of formation increased localization, nuclear Taz and Yap ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal epssimplex Herpes

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Journal of Cell Science nue oudroETb TGF by were Tead1-3 EMT targeting undergo pools to siRNA induced with EMT. not during Taz or transfected and Yap cells NMuMG of levels cytoplasmic in reduction the 20 prevents bar: Scale nuclei. visualize to was used and DAPI Taz staining. immunofluorescence and by Yap assessed and was cells, localization Tead2 NMuMG binding in Taz expressed and stably Yap were in Y440H) defective (Tead2 mutant wild-type (Vector), point Tead2 control vector a A or (Tead2-WT) Tead2. Tead2 to binding direct their on dependent EMT. induce and Taz of localization nuclear and predominant Yap a induce levels Tead2 Elevated 2. Fig. ARTICLE RESEARCH 1528 (invasion) Matrigel mean with coated as or shown (migration) coated are not Data were quantified. that were inserts cells culture cell utilizing performed were metastasis. assays Transwell and control. invasion vector migration, cell promotes Tead2 3. Fig. 15 bar: Scale shown. are (F) expression Taz and Yap E-cadherin, immunoblotting of analysis and (E) microscopy phase-contrast by morphology (siCtr). cell siRNA control Overall a with or Taz and Yap against pools siRNA with transfected were Tead2 overexpressing stably cells Py2T Tead2-induced EMT. prevents 15 bars: of Scale staining. view enlarged F-actin an show insets The staining). (phalloidin cytoskeleton and actin vimentin the ZO-1, E-cadherin, against staining immunofluorescence by microscopyand phase-contrast by visualized were control. changes morphological vector empty Overall an or (Tead2-VP16) Tead2 of a (Tead2-WT), version Tead2 active constitutively for coding cells constructs Py2T with EMT. transduced and stably morphology were cell on gain-of-function Tead2 of 20 Effect bar: (D) Scale staining. E-cadherin immunofluorescence and an Taz by and visualized Yap Tead2, were of localization the (Dox)- doxycycline and fashion, a inducible (Py2T-iTead2) in cells Py2T in expressed was differentiation.Tead2 epithelial and localization Taz 25 and Yap bar: on Scale overexpression Tead2 A. in described as visualized were Tead2 To ed-P6wr meddi argladalwdt rwfr5dy.Epyvco-rndcdP2 el evda oto.Saebr:50 bars: Scale control. a as served cells Empty-vector-transduced Py2T days. 5 for grow to allowed and Matrigel in embedded were Tead2-VP16 or WT eescindadsandb eaoii n oi HE.Hge anfctosaeas hw rgt.Saebr:100 bars: Scale (right). post-i shown days also 33 killed are magnifications were Higher Mice (H&E). mice. eosin nu/nu and Balb/c hematoxilin female by of stained veins tail and sectioned the were into injected were B in described as Py2Tcells metastasis. Experimental (C) niec n ubro ugmtsai e os sdtrie ysra etoigadmcocpcaayi flnsa ecie nC h metastat The mean C. as in shown described are as Data lungs group. of per analysis microscopic mice and of sectioning number metastases/total serial harboring by mice determined as as calculated mouse was per incidence metastasis lung of number and incidence m A a n a ellrlclzto is localization cellular Taz and Yap (A) .(,)Dpeino a rTzexpression Taz or Yap of Depletion (E,F) m. b tetetfr4dy.YpadTz and Taz, and Yap days. 4 for -treatment m .()Dpeino edexpression Tead of Depletion (B) m. 6 ...( s.e.m. m .()Efc facute of Effect (C) m. n 5 ;** 3; P , m m. A hmtci irto n naino yTclssal xrsigTa2W ra empty an Tead2-WT or expressing stably cells Py2T of invasion and migration Chemotactic (A) .1.()Cl naini Detaellrmti.P2 el tbyepesn Tead2- expressing stably cells Py2T matrix. extracellular 3D a in invasion Cell (B) 0.01). m m. vltoaiycnevdsqec oisfo hPSqdata, ChIP-Seq from motifs sequence 4A). conserved (Fig. Tead2 evolutionarily Jun-AP1 in by that found bound algorithm an motif motif (supplementary PhyloGibbs, a significant by motif most followed regions, the Tead-binding bound is (HOMER) S1A) MCAT of Fig. the material EnRichment by Detection that motifs Motif S2A). revealed binding cells Optimization factor Fig. Py2T Hypergeometric transcription 5 in material overrepresented increased at known supplementary robustly (ChIP-Seq) was 1B; (Fig. activity sequencing our TGF and Using generation expression of EMT. next immunoprecipitation of days chromatin by process performed the followed we during antibody downstream Tead2 Tead2 transcriptionally corresponding are by that is the genes regulated identify function elucidate to sought cancer Tead2 we To mechanisms malignant a that phenotype. to suggest leading cell EMT, above during differently presented regulated EMT results during genes The target Tead2 transcriptional The nrae acrcl irto/nainadmetastatic and migration/invasion lung. the in cell cells Py2T of outgrowth cancer increased ae oehr hs eut niaeta ed promotes Tead2 that indicate results these together, Taken ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal b tetet iepita hc Tead2 which at point time a -treatment, 6 ...( s.e.m. m enovo de .(,)Qatfcto fln metastasis lung of Quantification (D,E) m. n 5 ieprgop * group; per mice 6 nesoerpeetdand overrepresented infers irtdadinvaded and Migrated . jcinadlungs and njection P , 0.05). m m. ic

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Journal of Cell Science xlsvl rncitoa ciao u loarpesro gene S4B). of Fig. repressor material a supplementary also 4E,F; (Fig. but expression activator transcriptional a exclusively MT TGF mesenchymal during decreased was genes these of expression The candidates. gene target Tead2 were migration, cell and protein, complex agtgnso ed uigET h endarmdpcstenme of number the depicts diagram Venn The direct EMT. during potential Tead2 of of Determination genes the target (C) is motif. Tead2-binding Shown significant PhyloGibbs. most using regions Tead2-binding motifs in (B) the overrepresented are enriched. Shown significantly (HOMER). are analysis that EnRichment Motif (ChIP- of sequencing Optimization generation next by followed Tead2 Seq; for antibody an TGF using with treated cells Py2T during regions. genes target transcriptional Tead2 direct EMT. of Identification 4. Fig. ARTICLE RESEARCH 1530 and Polyak that 2013; ascertained Berx, further as such and 2012), al., Pdfgc et Craene Tiwari 2009; (De Weinberg, malignant and/or progression target EMT in Tead2 implicated tumor candidate previously been other had that identified genes we Fig. material Furthermore, supplementary S4A,B). 4E,F; (Fig. investigated actin systems model TGF upon the increased also expression of including regulators ChIP-PCR, by for and encode junctions which 4D). cell (Fig. genes cytoskeleton controls of revealed analysis 2009) predominantly components al., ontology et Tead2 (Ikeda Gene GOstats that by S2). components cellular Table TGF for during material expression showing as (supplementary and their decreased regions to promoter identified or their defined kb in analysis increased 10 Tead2 overlay by than were bound The less genes (TSS). 132 distance genes sites a start at supplementary Tead2-bound transcriptional peak 4C; ChIP-Seq S1). (Fig. a showing analysis Table (adjusted ChIP-Seq material 1.5-fold by least at determined of cutoff Py2T a P in passing and level, genome-wide cells TGF expression Py2T performed with epithelial treated we in cells EMT, profiling during expression during gene expression Tead2 their by in targeted are motifs motifs GTIIC sequence and main TGF MCAT These the the known 4B). to the (Fig. among and that motifs MCAT binding suggest the Tead findings to core corresponds GTIIC that similar highly motif a revealed also as presented are expression TGF mRNA means with in treated RT- changes were by Fold respectively. cells determined NMuMG as EMT and Py2T during means qPCR. genes was as Tead2-bound region represented of intergenic are Expression An Data (F) IgG. control. control negative to as (IP normalized used background were above and enrichment fold input) qPCR as by over to presented determined subjected are regions data was Tead2-binding The A the ChIP-Seq. in spanning described pairs as primer PCR. treated using quantitative cells by Tead2 the by from bound Chromatin directly genes cellular of their Validation five and (E) top group groups. per the genes table for associated The analysis the GOstats. clustering compartments, using annotation was C functional analysis in the ontology described shows Gene genes (D) overlapping TGF genes. on of overlapping performed days were of 5 that number after genes and the before of and cells number Py2T the in analysis, differently regulated ChIP-Seq Tead2 the from genes , oietf iettre ee fTa2ta hwdachange a showed that Tead2 of genes target direct identify To mn hs 3 ee,w aiae nw edtre genes target Tead known validated we genes, 132 these Among .5,wr eetdfra vra ihTa2budgnsas genes Tead2-bound with overlay an for selected were 0.05), n b A N-idn oista r vrersne nTead2-binding in overrepresented are that motifs DNA-binding (A) , 5 6 idcdEMT. -induced Serpine1 ) h eunigdt eesbetdt Hypergeometric to subjected were data sequencing The 2). ...( s.e.m. n 5 n tes(upeetr aeilTbeS) We S2). Table material (supplementary others and ) * 3). D P cdcls niaigta ed snot is Tead2 that indicating cells, Ecad Elmo3 , .5 ** 0.05, b Ctgf Pard6b o as ee hthdadifferent a had that Genes days. 5 for eeipiae npaoyoi and phagocytosis in implicated gene a , and P enovo De , b eeecdn o polarity a for encoding gene a , .1 **** 0.01, Cyr61 o aswr ujce oChIP to subjected were days 5 for b siuaini h he EMT three the in -stimulation b eeaino eunemotifs sequence of generation idcdETi yTand Py2T in EMT -induced Zage l,21) whose 2011), al., et (Zhang P , 0.0001. Amotl2 b o r4days, 4 or 5 for b P 6 idcdEMT -induced vle ihnthe within -values ...( s.e.m. , Esrp2 b treatment n 5 , Mal 2). , cp nlsscnimdicesdlvl fzxnlocalized zyxin TGF during of formed are levels that in fibers increased stress found along confirmed micro- also analysis Immunofluorescence scopy S4E). was Fig. expression material (supplementary zyxin MT Higher Py2T mesenchymal in 5C). EMT (Fig. during increased cells substantially were zyxin of levels euae M-eeatgnsb ciga transcriptional motifs. sequences a GTIIC promoter or to as MCAT binding Tead-specific by acting containing mainly repressor, by or genes activator EMT-relevant regulates of promoters promoter the same including the S4C). to Tead2, Taz by Cyr61 and/or to Yap targeted induced of regions cells similar binding revealed Py2T increased analysis in RT-PCR Tead2- or Taz subsequent and and above-mentioned EMT Yap undergo the ChIP Tead2, complex. for Tead2–Yap–Taz S1I), of a experiments Fig. by some bound material were genes supplementary whether regulated 1D; assessed (Fig. Taz we and Yap with Bcel,19;Hrt ta. 08 mt ta. 00.I line In 2010). al., focal et Smith force to of 2008; traction cues al., sites of et mechanical Hirata generation to to 1997; and response (Beckerle, localizes polymerization in actin that fibers facilitate stress protein and regulatory is adhesions Zyxin the 5A). ( actin revealed (Fig. region motif zyxin an MCAT this species-conserved for of a inspection of encoding intronic presence Close an S4D). gene to Fig. Tead2 the material of binding of direct EMT region a for revealed critical also gene ChIP-Seq target Tead2–Taz a is Zyxin n ohvrin fTz u o yYp(i.5,) These 5I,J). (Fig. Yap by not co-activation. but mediates Taz Taz, Tead2 Tead2-VP16 that Hippo- of Tead2-WT, demonstrate results of versions of expression (Pan, both expression expression forced Taz and zyxin by the that and of induced revealed by was Yap expression analysis of expression and Immunoblotting the 2010). versions the Taz by mutant and by Tead2-VP16, signaling-insensitive in and Yap function expression Tead2 wild-type Tead2 zyxin wild-type evaluated modulating of thus to We response Tead2. by controlled 5G,H). 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(Fig. gene , lo n Pdgfc and Elmo3 D cdclsa oprdt Tlcdcells MTflEcad to compared as cells Ecad splmnaymtra Fig. material (supplementary Zyx Zyx eeepeso via expression gene b idcdETin EMT -induced supplementary ; b o days 5 for Ctgf

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Sox11 regulation Sox4 Sox4, direct be include a which demonstrate to members, family remains SoxC EMT 2013). al., during et (Tiwari the EMT of that Whether regulator epigenetic indicate crucial studies a Sox4, our Moreover, Tead2 expression. Tead2 on TGF during Smad4 of ed srqie o h rpreeuino TGF a of of execution activity results proper transcriptional the These for and members. required presence is family the Teads Tead that of demonstrate ( depletion Tead2 mediated of TGF control version increased of dominant-negative to EnR a attenuation comparison display when an in observed cells metastasis Conversely, and cells. Tead2-overexpressing invasion migration, that previous 2008b). al., et report with Zhao 2008; consistent Taz Tead al., via et and EMT (Lei also induces Yap activity cells of transcriptional epithelial are breast expression normal MCF10A ectopic results in that transcriptional Our demonstrating cancer intact Tead reports various lines. an of and potential In on Tead, metastatic cell the depends in 2012). with Yap correlates effect al., activity for this et domain results, cancer (Lamar interaction our breast pro-metastatic with Hippo-signaling- renders cells accordance a study (YapS127A) melanoma another Yap of Recently, and of overexpression 2008). version breast al., that insensitive cultured et demonstrated ductal of (Chan has invasive is lines invasiveness co-activator cell and represent Tead cancer migration which this for of of expression responsible and most (IDCs), in cancers, carcinomas expressed breast highly a is evoke of Taz can Taz Indeed, or phenotype. 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EMT sepce rmclsta aeudroea M,we EMT, an undergone have that cells from expected As Tead elevated that suggested have studies recent Several edmntaeta ed xrsini nrae during increased is expression Tead2 that demonstrate We edtasrpinlatvt smil otoldb h direct the by controlled mainly is activity transcriptional Tead sepesd hsefc sas bevduo siRNA- upon observed also is effect This expressed. is ) eei ontemtre ftetasrpinfactor transcription the of target downstream a is gene ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal Tead2 eeepeso sdrcl ciae ySox4 by activated directly is expression gene b idcdEThdn usata impact substantial no had EMT -induced b inln,bcueteablation the because signaling, Tead2 b b inln n Hippo and signaling idcdETis EMT -induced eepooe by promoter gene b -induced Tead2- , 20%

Journal of Cell Science rms AJ AS nimtn and as angiomotins such proteins, PALS, junction reporting apical studies PATJ, with of clusters series Crumbs, Taz a and with Yap shown). consistent that not Taz (data is and junctions observation Yap tight This of of reminiscent pattern cells staining epithelial membranous in a observe also We ARTICLE RESEARCH uiid oylnlrbi niTa2atbd a eeae by of generated N-terminus the was Cruz to corresponding Affinity antibody (Santa peptide USA). a anti-Tead2 Taz with NJ, rabbits rabbit Princeton, and immunizing (Santa polyclonal (Covance, Yap zyxin HA USA), purified, CA), CA, Biotechnology), Diego, Francisco, San Cruz (IMGENEX, San Taz Biotechnology), (Zymed, (Sigma-Aldrich), vimentin (Sigma-Aldrich), N- ZO-1 GAPDH USA), St USA), (Sigma-Aldrich, CA, MO, fibronectin Japan), Jose, Louis, Shiga, San Otsu, Bio, (BD, (Takara Cruz E-cadherin cadherin (Santa actin TX), proteins: Dallas, following the Biotechnology, against as were used Antibodies reagents and Antibodies METHODS AND MATERIALS the for TGF required during is cytoskeleton be zyxin actin the can Notably, of therefore stress 1997). rearrangement on and and (Beckerle, adhesions focal cytoskeleton Zyxin in fibers membrane, manner. apical actin the Tead-dependent at the localized a with in gene associates EMT target during Tead2 upregulated the upon repressor. Taz of transcriptional by a as bound function be also same could to The complex found EMT. also during TGF were reduced regions significantly promoter are expression rca euaoyrl ncnrligYpadTzsubcellular a Taz play and processes. also these Yap during but controlling activity Taz and metastasis, in and localization role and Yap and of regulatory EMT EMT executors crucial promoting mere of only in process not the functions are during Teads that factors suggest transcription phenotype Tead cellular for invasive and migratory controls a EMT. activity during of transcriptional induction of Tead one (Mori which the least at by migration be mechanisms therefore cell could the enabling upregulation Zyxin thereby 2009). al., cells, et NMuMG of EMT like genes target the Tead2 new of identified expression the regulating Cyr61 in of example co-activators for transcriptional as Teads, have act Taz exclusively and to Yap reported far, been function Thus co-factors. their transcriptional Tead domain, on their Because transactivation depends repressor. a and a lack as factors well genes acts transcription as Tead2 activator Tead2-bound that al., transcriptional indicates a EMT of as et during expression inspection gene (Anbanandam in closer changes regions motifs A to Tead-binding binds TGF 2006). in predominantly that MCAT that during Tead2 indicate harboring genes EMT, results Tead2 Our undergoing identified S1). by cells Table has Tead2 material bound (supplementary analysis activated directly by ChIP-Seq are regulated EMT. transcriptionally during are that and the genes in activity. complex complexes, transcriptional Tead Tead2–Yap–Taz promote a junctional finally published to of of nucleus levels of basis nuclear by the increased released disassembly are On that, Taz speculate and to 2011). EMT-induced Yap tempting membrane-bound Wang al., is cells, Silvis 2010b; it epithelial et results, al., in 2011; own et Zhao our Varelas al., and 2011; 2010a; observations et al., al., et (Chan Varelas et 2011; entry al., nuclear et their counteracting efrhrrpr htTzbtntYpat saco-activator a as acts Yap not but Taz that report further We nsmay u eut sals rca euaoyrole regulatory crucial a establish results our summary, In on report first the is this knowledge, our of best the To b tetet hc ol mlct htteTead2–Taz the that implicate would which -treatment, ee Zage l,21;Za ta. 08) u analysis Our 2008b). al., et Zhao 2011; al., et (Zhang genes Zyx yi rti xrsinis expression protein Zyxin . Pard6b a and ctnn thereby -catenin, b idcdEMT -induced Elmo3 b Ctgf -induced whose , and etodLmnmtrL90 iel uieaevle were values luciferase Firefly LB960. internal Luminometer to normalized and Berthold (Promega) system assay a reporter Dual-Luciferase the using 1 analyzed using lysed were nirgn(albd A.Raet sdwr:rcmiathuman (Clontech/Takara). recombinant from Doxycycline (BD); were: reduced was used phalloidin–Alexa-Fluor-568 Reagents TGF CA). 16–32); (Carlsbad, acids Invitrogen (amino Tead2 98 t n aai 08.TeMA eotrwsdrvdfo this from derived 8 was CCTGACAC of reporter MCAT replacement al., The by et 2008). construct (Davidson Sasaki, USA) Tead and WI, Ota Biology, Madison, GTIIC 1988; Developmental (Promega, pGL4 for the into Center Japan) RIKEN subcloning Kobe, Sasaki, p Hiroshi by from by promoter generated provided basal the was including elements reporter response GTIIC The Plasmids VA). Manassas, from obtained (ATCC, originally previously Collection was MT as Culture and and was Type 2005) (NMuMG/E9) MTflEcad al., American cells et the NMuMG (Maeda of described subclone lines The cell and culture Cell el eetasetdwt 0 grpre n 0n fplasmids of ng 10 and reporter plating, after ng day 800 1 with At plates. transfected encoding 24-well in were triplicate cells in plated were Cells assay reporter the Luciferase using calculated S3. Table was material supplementary induction in listed fold were ( and method assays Ct comparative triplicate, PCR normalization. in for used performed were (Invitrogen). primers MasterMix L19 reverse PCR SYBR-green Riboprotein using (Sigma-Aldrich), PCR transcripts by TriReagent and quantified (Promega), were transcriptase using reverse M-MLV prepared with transcribed was RNA Total RT-PCR and isolation RNA was Transfection the to experiments. according (Invitrogen) instructions. knockdown manufacturer’s RNAiMax Zyxin transient Lipofectamine CO, and with Taz for Lafayette, performed Yap, Tead4, (Dharmacon, used Tead3, siRNAs Tead2, were Tead1, pool murine against smart USA) siGENOME nM 10 interference RNA yXaln ag(ahlg n oeua eiie Queen’s Medicine, Molecular and (Pathology Yang provided kindly and Xiaolong 2011) TeadMut Larkin al., Cyr61prom et by to and (Lai WT described according Cyr61prom previously The were were 1996). reporters al., sequences et flanking (Larkin al. and et underlined, is motif ervrlH-agdTa2Y4Hwsgnrtdb PCR-mediated USA). by generated WI, was Madison, mutagenesis. Y440H subcloning site-specific (Clontech, Tead2 by pLVX-tight-puro HA-tagged generated Retroviral into were pRFTO constructs La from HA– HA–Tead2-EnR UCSD, doxycycline-inducible and lentiviral, Center, The Tead2 Cancer TazS89A). Sasaki (Taz, Moores USA) H. and Jolla, from Pharmacology Guan Kun-Liang gifts of and YapS127A) kind (Department (Yap, Kohler were FMI, R. cDNAs Tead2-VP16), Kohler, (Tead2, Reto Original pRFTO by provided vector Switzerland). (kindly retroviral Basel, tag HA pBabe-derived HA-tagged N-terminal the an inserting Retroviral into containing by created cDNAs 2008). were respective Yap Sasaki, the and Taz and Tead2-EnR, Research, Tead2-VP16, (Ota Tead2, as Cancer were of previously constructs were Tead2-VP16 Institute described and (The pcDNA3.1 Tead2-WT Retroviral Carvajal in UK). Jaime Tead1-4 London, by Murine provided Promega. kindly from was The (pRL-CMV) Canada). Ontario, University, eeclue nDlec’ oiidEgesmdu (DMEM) FBS TGF medium ng/ml 10% 2 Eagle’s and with treated cells streptomycin were modified All penicillin, Cells 2012). (Sigma-Aldrich). Dulbecco’s glutamine, al., with et in supplemented Waldmeier cultured 2008; al., were et (Lehembre previously b RDSses inaoi,M,UA;Mtie,got factor growth Matrigel, USA); MN, Minneapolis, Systems, (R&D 1 ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal Renilla CTCTAC (8 ACATTCCTCAGCT uieaeuigLpfcaie20 Ivtoe) Cells (Invitrogen). 2000 Lipofectamine using luciferase 6 D cdclsadP2 el r sdescribed as are cells Py2T and cells Ecad asv yi ufr(rmg)adlstswere lysates and (Promega) buffer lysis passive Renilla DD t.Piesue o uniaieR-C are RT-PCR quantitative for used Primers Ct). 6 uieaecnrlvalues. control luciferase TI ihegtcpe ftesequence the of copies eight with GTIIC Renilla 6 CT,weeteMA core MCAT the where MCAT), uieaeepesn vector expressing luciferase d b 1LcI(kindly 51-LucII . 1533

Journal of Cell Science elcdb omlgot eimadatr1dy niitcselection antibiotic day, 1 after and performed. medium was growth normal by replaced muoltig muoloecnesann,retroviral staining, immunofluorescence SDS-PAGE. Immunoblotting, by to analyzed according were extracts Samples cell instructions. Protein-G- cytoplasmic manufacturer’s and co-immunoprecipitation instructions. the nuclear for Waltham, total, manufacturer’s used on Scientific, were the experiments (Invitrogen) to (Thermo Dynabeads according coupled reagents nuclear USA) NE-PER extraction with MA, prepared cytoplasmic were extracts and cytoplasmic and Nuclear co-immunoprecipitation and fractionation Cell ARTICLE RESEARCH luia(a ig,C,UA n eesqecduigIllumina using sequenced were 1534 and from Kit USA) Prep CA, sample Diego, ChIP-Seq the (San using Illumina prepared were libraries ChIP are ChIP-Seq ChIP-qPCR for was S4. used and Table Primers samples material Input supplementary control. in over of negative listed IP enrichment IgG by Fold to calculated reaction. normalized was per genes PCR an target quantitative specific for achieve used to were 150 DNA sonicated with Starting was bp. 500 5 chromatin of size crosslinked fragment al., average brief, et (Weber In described previously 2007). as performed were experiments ChIP immunoprecipitation Chromatin staining H&E and processing 0.5 tissue injection, vein Tail structures microscope. growth, DMIL of Leica days a 5 After using days. photographed 3 were after tissue replenished a was in medium solidification 50 gel of incubator, 10 minutes culture 20 in After protein cells a Germany). mg/ml of Martinsried, 2500 well 4 one medium. to to transferred diluted growth was serum-free (BD) with Matrigel factor-reduced Growth culture Matrigel 3D culture tissue a in hours 24 After 37 FBS. at 20% 700 incubator contained with chambers the supplemented of Bottoms medium (BD). growth TGF 500 in ng/ml in 2 resuspended cells and were FBS 0.2% cells containing washed and assay Trypsinized invasion and migration Transwell to (8 added polybrene were envelope- of supernatants presence the viral the in vectors infection, cells target helper For and a pVSV. the vector produced pRC-CMV-RaII in using encoding were instructions pHDM-Tat1b, Tead2-EnR particles manufacturer’s pHDM-HGPM2, Tet- Lentiviral or the Lenti-X to (Clontech). Tead2-WT the according system using expressing Advanced generated On cells were fashion of doxycycline-inducible pools Stable 2012). infection al., Lentiviral et (Waldmeier previously described as performed profiling were expression These gene Affymetrix and infection %CO 5% B o 0mnts el hthdntcosdtemmrn were membrane the crossed not had that Cells minutes. 10 for PBS eesandwt AIadqatfe sn ec M 4000 DMI Leica a using quantified membrane and the of DAPI bottom microscope. the with on stained cells and were swab, cotton a with removed fie(FO n h euain fteCnoa eeiayOfc of Office 1908). Veterinary 1907, Cantonal Veterinary 1878, the Federal (licences of All Stadt Swiss regulations Basel the 2012). the by al., and approved (SFVO) et Office were staining (Waldmeier mice post H&E described days involving and previously 33 studies processing as at Tissue killed performed isolated. were were were Mice lungs mice. and nude injection Balb/c female old months ora 30 at hour m 6 fatbd,14 fteCI apeada110dlto finput of dilution 1:100 a and sample ChIP the of 1/40 antibody, of g 10 6 2 yTclswr netdotooial noteti eno three of vein tail the into orthotopically injected were cells Py2T natsu utr nuao o or.Vrlspraatwas supernatant Viral hours. 2 for incubator culture tissue a in ˚ t1000 at C m eimwr eddit elclueisr chambers insert culture cell into seeded were medium l ˚ ne %CO 5% under C m g fnra rwhmdu a de.Growth added. was medium growth normal of l n eesbeunl nuae t37 at incubated subsequently were and m sieagoeei irsoysie(ibidi, slide microscopy angiogenesis -slide 2 el eefxdwt %PAin PFA 4% with fixed were cells , b hr prpit.2.5 appropriate. where , m /l.Clswr pnfr1 for spun were Cells g/ml). m lofgrowthmedium m fcrmtnand chromatin of g m arglwere Matrigel l ˚ under C 6 10 4 h nihetoe h nu hoai n nypaswt tlat4- least at with on peaks based only used. and were filtered chromatin enrichment further input fold the were over enrichment candidates the peak Resulting estimate)/2. the 2011). data against al., mapped et ChIP-Seq Stadler were 2009; protocol. reads al., et manufacturer’s Briefly, (Langmead described the as to processed were according 2000 HiSeq oi ciiyrsos analysis response activity Motif 2005). al., PhyloGibbs, Motif For et of 2010). Optimization al., novo et Hypergeometric (Heinz de Tead2-binding the v4.1) of (HOMER; using tools vicinity out EnRichment the carried in was detection sites motif analysis, HOMER For analysis Motif cou,H,Aas .S,Fswc,K,BonrFae,M n it,M A. M. Nieto, and M. Bronner-Fraser, K., Fishwick, S., M. Adams, H., Acloque, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139865/-/DC1 online available material Supplementary material Supplementary League. release. Cancer immediate Swiss for the PMC framework and in EU-FP7 2008-201662]; Deposited the TuMIC Cellplasticity), number (RTD [grant the Biology program Foundation; Systems Science National for Swiss Initiative the Swiss by supported been has research This Funding manuscript. the experiments, wrote the and designed G.C. data analysis analysis. the MARA profiling analyzed expression the gene performed initial E.v.N. the and and and P.A. and P.B., ChIP-Seq data of profiling. analysis the expression bioinformatical analyzed gene the performed experiments, R.I. the manuscript. performed the and wrote designed L.W. and M.D. contributions Author interests. competing no declare authors The interests Competing Switzerland). Basel, (Department Schmidt, of Jost Petra Roland University to and Biomedicine, Schmieder grateful of Ursula are Galm, We and Isabel reagents. Carvajal Antoniadis, important Jaime Helena Yang, sharing Xiaolong for Guan, Kohler Kun-Liang Reto Sasaki, Hiroshi thank We Acknowledgements accession (GEO Omnibus Expression TGF Gene GSE55711). during at number: ChIP deposited TGF Tead2 are days cells of five Py2T data versus ChIP-Seq untreated and Py2T cells of data expression Gene numbers Accesssion performed legends. were figure in analyses indicated Prism Statistical GraphPad as Inc.). the Software using (GraphPad generated were software graphs and analysis Statistical analysis Statistical http://ismara.unibas. to uploaded analysis. were for files) ch/fcgi/mara (.CEL data raw Microarray sn Padiptsmlswt olwn aaees nomodel, parameters: following with samples 2008) al., input length et and fragment gsize (Zhang (v.1.3.7.1) IP estimated software the using MACS of employing ChIP-Seq 3 half identified from Alignments their by length. shifted towards fragment were the estimate experiments and to (http://sourceforge.net/projects/chip-seq) weighted read used on software was based per the were by quantifications Chipcor positions All weighted hits. was alignments. alignment of alignment number the Each best of mismatches. inverse hundred two -m most to -a at 2 allowing up -v parameters tracking with 0.9.9.1) 100, (version software bowtie using mm9) 20) pteilmsnhmltastos h motneo hnigcell changing of disease. importance and development the in state transitions: Epithelial-mesenchymal (2009). 5 70000 tsize 2700000000, ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal oi eeainwsue sdsrbdpeiul (Siddharthan previously described as used was generation motif 9 n.Cutr fCI-e edainet were alignments read ChIP-Seq of Clusters end. 5 0 pvalue 50, .Ci.Invest. Clin. J. 5 u musculus Mus e5adshiftsize and 1e-5 119 1438-1449. , b idcdETin EMT -induced eoe(UCSC, genome 5 b (chipcor -treated

Journal of Cell Science alr,R n eneg .A. R. Weinberg, and R. Kalluri, and T. Dolle Suzuki, A., J. A., Martial, J., Matsuki, J. Hwang, K., P., Jacquemin, Yajima, S., Kosugi, T., Kanda, Y., Ikeda, og .adGa,K L. K. Guan, and W. Hong, iaa . asm,H n oae M. Sokabe, and H. Tatsumi, H., Hirata, en,S,Bne,C,San . etln,E,Ln .C,Lso . Cheng, P., Laslo, C., Y. Lin, E., Bertolino, N., Spann, C., Benner, S., Heinz, iaaa M. Kitagawa, G. Berx, and B. Craene, P. De Chambon, and A. Staub, R., Rosales, H., J. H. Xiao, Song, I., and W. Davidson, Hong, J., C. Lim, M., Walsh, X., W. Zhang, Hong, W., and S. Chan, C. L., Huang, Chen, V., A. Pobbati, F., Y. Chong, and J., Q. C. Zeng, Lim, W., W., Hunziker, S. I., Chan, Lee, P., C. Ng, K., Guo, J., A. C. Lim, R. W., S. Weinberg, Chan, and L. C. Chaffer, htaa,P,Penzo-Me P., Bhattaram, and X. C. M. Gao, Beckerle, G., Halder, T., C. Nguyen, C., D. Albarado, A., Anbanandam, ARTICLE RESEARCH iesi .C,Ci .C,L,J,Bon . u .M n pti,J A. J. Epstein, and M. M. Lu, C., Brown, G. J., Li, K. C., N. I. Chi, Farrance, C., R. and Milewski, B. M. Yaffe, J.-H., Hong, Jr, M., W. Mahoney, P. C. Ordahl, and K. I. Farrance, B., S. Larkin, L. S. Salzberg, and M. Pop, C., O. Trapnell, R. B., Hynes, Langmead, and Z.-G. Jiang, W., J. Schindler, H., Liu, P., Stern, M., J. Lamar, X. Yang, and Y. Hao, C., K. Ho, D., Lai, L. M. DePamphilis, and J. K. Kaneko, ae,J . ikuoa .adMrio,S J. S. Morrison, and E. Piskounova, A., J. Magee, J. M. Wheelock, and R. K. Y. Johnson, Xu, and M., K.-L. Maeda, Guan, H., Yang, Z., Dong, F., Chen, P., Wang, B., Zhao, Z., Li, Y. Xiong, S., Zhao, X.-H., Pei, F., Bai, Z.-Y., Zha, B., Zhao, H., Zhang, Q.-Y., Lei, D., Ziegler, K., Strittmatter, T., Schomber, A., Wicki, M., Yilmaz, F., Lehembre, transition. factors transcription domain. mammalian binding DNA regulated 21785. TEA/ATTS developmentally the of containing family novel Surg. Gastrointest J. World K. Hatakeyama, e ontemefcoso h amla ip pathway. Hippo mammalian the Biol. of effectors downstream key 192-195. h ehntasuto tcl deiestructures. adhesive cell at mechanotransduction the o arpaeadBcl identities. cell K. B C. and required Glass, elements macrophage cis-regulatory and for prime H. factors Singh, transcription C., lineage-determining Murre, X., J. eacl rti E- id pcfclyadcoeaieyt w SV40 two to progression. and cooperatively initiation and cancer specifically sequence. binds unrelated of TEF-1 motifs pathway. protein enhancer hippo cell the in HeLa TEAD4 by recognition YAP Dev. of Genes basis Structural (2010). angiomotin. by YAP and Chem. TAZ Biol. of J. restriction pathway-independent Hippo (2011). cells. cancer breast W. Hong, metastasis. dniiaino iia nacreeet ufcetfrPx xrsinin expression Pax3 for sufficient elements enhancer minimal of Identification transcriptional with members. differentially family (TEF-1) interacts factor-1 TAZ enhancer co-activator transcriptional The elements. M-CAT of specificity cell genome. the human modulate the to sequences DNA Biol. short Genome of alignment memory-efficient TEAD- its its through metastasis domain. and promotes YAP, interaction target, TAZ pathway Hippo component The (2012). pathway hippo the CTGF. and by 2738. Cyr61 targets mediated transcriptional is downstream cells cancer TAZ. and YAP co-factors Commun. the Res. with Biophys. interaction its Biochem. affects Tead1 mouse in mutation transcription of family TEAD the and development factors. mammalian of beginning the V. Lefebvre, and and neural M. of survival the Wegner, for progenitors. factors L., mesenchymal transcription SoxC M. on relies Organogenesis Depamphilis, A., Vassilev, 949-957. domain. TEA the of structure solution USA the from activity S. Veeraraghavan, mat eeoeet,aduncertainty. and heterogeneity, impact, complex. TEAD and YAP the into 235-240. insights Structural (2010). pathway. hippo the by inhibited 28 is and transition K.-L. mesenchymal Guan, and al. E-cadherin. et of loss A. upon Berns, switch functional 27 W., a P. assembly: Derksen, adhesion P., focal Went, A., Kren, 3755. seta o eairlbtntmrhlgclcagsdrn nepithelium-to- an during changes morphological transition. not mesenchyme but behavioral for essential 2426-2436. , 2603-2615. , 23 103 785-793. , e.Genet. Dev. 17225-17230. , .Ci.Invest. Clin. J. 20) oefrTZi irto,ivso,adtmrgnssof tumorigenesis and invasion, migration, in TAZ for role A (2008). Science 20) viso’ hroeia toh-soitdmissense atrophy-associated chorioretinal Sveinsson’s A (2007). 24 19) yi:zn igr tstso eladhesion. cell of sites at fingers zinc Zyxin: (1997). 10 286 290-300. , R25. , 20) ati acrsreyfrptet ihlvrcirrhosis. liver with patients for surgery cancer Gastric (2009). 7018-7026. , rc al cd c.USA Sci. Acad. Natl. Proc. 20) nihsit rncito nacrfco (TEF-1) 1 factor enhancer transcription into Insights (2006). 20) A rmtscl rlfrto n epithelial- and proliferation cell promotes TAZ (2008). acrRes. Cancer 331 22 ne,A,Sc,E,Cleae,C,Knk,K J., K. Kaneko, C., Colmenares, E., Sock, A., ´ndez, 43-55. , .Cl Sci. Cell J. 1559-1564. , 119 21) h A n A rncito co-activators: transcription TAZ and YAP The (2012). a.Commun. Nat. 1 21) euaoyntok eiigETduring EMT defining networks Regulatory (2013). 1420-1428. , 49-55. , 20) h aiso epithelial-mesenchymal of basics The (2009). 68 a.Rv Cancer Rev. Nat. 361 118 2592-2598. , o.Cell Mol. 19) euaino eeepeso at expression gene of Regulation (1998). 20) yi mre sakypae in player key a as emerges Zyxin (2008). 1022-1026. , 21) esetv ncne cell cancer on perspective A (2011). acrCell Cancer 873-887. , Cell 1 21) ao eitnei breast in resistance Taxol (2011). ,9. ice.J. Biochem. 54 ,P n aisn I. Davidson, and P. ´, 21) ipecmiain of combinations Simple (2010). 38 109 931-942. , 576-589. , 19) lnigsequences Flanking (1996). .Bo.Chem. Biol. J. 20) ahrnswitching: Cadherin (2005). E2441-E2450. , 21) acrse cells: stem Cancer (2012). 21 o.Cl.Biol. Cell. Mol. 13 omn ner Biol. Integr. Commun. 283-296. , 97-110. , acrRes. Cancer 20) NCAM-induced (2008). rc al cd Sci. Acad. Natl. Proc. 388 20) lrfs and Ultrafast (2009). 217-225. , ei.Cl Dev. Cell Semin. ee Dev. Genes o.Cl.Biol. Cell. Mol. Bioessays 271 18) The (1988). 16 71 19) A (1996). MOJ. EMBO 21775- , 3742- , 2728- , (2010). (2004). (2005). 19 24 1 , , , hey .P,Alqe . un,R .adNeo .A. M. Nieto, and Y. R. Huang, H., Acloque, P., J. Thiery, J., P. Balwierz, O., C. Daub, E., Nimwegen, van R., A. Forrest, H., Suzuki, tde,M . ur . ugr . vnk . inr,F,Scho F., Lienert, R., Ivanek, L., Burger, R., Murr, B., M. Stadler, mt,M . lnmn . adl .L,Letoan . aemn .M. C. Waterman, L., Luettjohann, L., M. Gardel, E., Blankman, A., M. Smith, ivs .R,Kee,B . in .H,Keoic,O,Rdkv,G M., G. Rudakova, O., Klezovitch, W.-H., Lien, T., B. Kreger, R., M. Silvis, cel .adWibr,R A. R. Weinberg, and C. Scheel, idata,R,Sgi,E .advnNmee,E. Nimwegen, van and D. E. Siggia, R., Siddharthan, aaa . ioai . kt,K,Nsik,N,Iua .adSsk,H. Sasaki, and Y. Imuta, N., Nishioka, K., Ukita, H., Kiyonari, A., Sawada, S., Yamamoto, R., Nakayama, Y., Yada, H., Sato, Y., Nishizaki, A., Sawada, Sa ee,M,Hlmn,I,Salr .B,Rms . Pa L., Ramos, B., M. Stadler, I., Hellmann, M., Weber, oyk .adWibr,R A. R. Weinberg, and K. Polyak, D. Pan, ag . un,J n hn J. Chen, and J. Huang, W., Wang, G. Christofori, and M. Diepenbruck, N., Meyer-Schaller, L., Waldmeier, L. M. DePamphilis, and Y. Zhao, H., Shu, J., K. Kaneko, A., Vassilev, A., F. Fellouse, A., Gregorieff, S., Song, R., Sopko, W., B. Miller, X., Varelas, G. Christofori, and M. Tatari, A., Gheldof, N., Tiwari, t,M n aai H. Sasaki, and M. Ota, aea,X,Smvrh-ern,P,Nrmtu . es,A,Ccbr,K., Cockburn, A., Weiss, M., Narimatsu, P., Samavarchi-Tehrani, X., Varelas, M., Pachkov, P., Arnold, J., P. Balwierz, L., Waldmeier, K., V. Tiwari, N., Tiwari, X. Luo, and D. Pan, R., D. Tomchick, J., Yu, W., Tian, oi . aaai . obci . ir,K,Tkm,Y,Krym,H., Koriyama, Y., Takami, K., Miura, N., Koibuchi, H., Nakagami, M., Mori, A. Cano, and F. Portillo, G., Moreno-Bueno, it,M A. M. Nieto, ´nchez-Tillo ewr htcnrl rwhars n ifrnito nahmnmyeloid disease. human and development a in in transitions mesenchymal differentiation and arrest al.; line. growth et cell J. leukemia controls M. Hoon, that de Y., network Hasegawa, Center T., Science Omics Ravasi, Riken T., Consortium; FANTOM Lassmann, M., K. Irvine, iwgn . iblur . aee,E . adti,D tal. regions. regulatory et distal D. at Gaidatzis, methylome J., mouse Nature the E. shape Oakeley, factors C., DNA-binding Wirbelauer, E., Nimwegen, aneac n repair. and maintenance C. M. Beckerle, and Yap1. the coactivator regulating transcriptional by the accumulation cell of ra33. controls activity that and suppressor localization tumor a is catenin aag,F . at,D . ekr,J .adVsoki,V. Vasioukhin, and T. J. Seykora, M., D. Lantz, D., F. Camargo, e67. eecya rniin ocpsadmlclrlinks. molecular 396-403. and concepts transition: mesenchymal 3189. ib apigmtffne hticroae phylogeny. incorporates that finder motif sampling Gibbs 20) eudn oe fTa1adTa2i oohr development notochord in survival. Tead2 and and proliferation cell Tead1 of of regulation the roles and Redundant (2008). factor. second a with cooperation in node H. 132 the Sasaki, in and enhancer H. A. Foxa2 Kondoh, Postigo, N., and Nishioka, A. invasiveness. tumor Castells, and Sci. EMT Life C., beyond Mol. cancer: D. Cell. in factors Dean, transcription activating S., D. Darling, fpooe N ehlto ntehmngenome. human the in methylation DNA promoter of Schu and eecya tts custo fmlgatadse eltraits. cell stem and malignant of Cancer acquisition states: mesenchymal Cell n eaieyrglt YAP1. regulate negatively TGF and of model versatile vivo. a in cells, and cancer vitro breast murine Py2T cytoplasm. the Src/ a in YAP65, 1241. of localized domain activation protein the Yes-associated utilize factors transcription TEAD/TEF al. et signaling. H. Wnt/beta-catenin McNeill, W., regulates Hunziker, pathway T., Pawson, R., Sakuma, 768-783. rlfrto n otc niiina rncitoa eitr fHippo of mediators transcriptional as inhibition contact signaling. and proliferation ope eldniysnigt ip-eedn oto fteTGF- the of control Hippo-dependent L. to J. sensing Wrana, pathway. and density J. cell Rossant, couples G., B. Larsen, by transition epithelial-mesenchymal reprogramming. of epigenetic regulator and master expression a Ezh2 controlling is Sox4 (2013). Schu N., Meyer-Schaller, cells. cancer of mechanism TEAD2. survival human of domain USA YAP-binding Sci. the Acad. of analysis functional aah,H,Sb,H,Mciui . oiht,R tal. et morphogenesis. R. endocardial transition Morishita, epithelial-mesenchymal to N., in contributes reorganization Mochizuki, fiber and H., actin mediates Sabe, Zyxin H., Hayashi, cancer. and EMT in polarity cell Pax3. of regulator a as Tead2 of 829-837. implication and crest neural elhaddisease. and health 3124. 4719-4729. , 19 21) h ip inln aha ndvlpetadcancer. and development in pathway signaling hippo The (2010). 491-505. , ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal 480 9 ¨beler,D. 265-273. , e.Cell Dev. Development 21) h n n uso h pteilt eecya rniinin transition mesenchymal to epithelial the of outs and ins The (2011). . i,Y,d aro,O,Sls . al,L,Carcss M., Cuatrecasas, L., Fanlo, L., Siles, O., Barrios, de Y., Liu, E., , ´ 490-495. , 107 LSONE PLoS a.Genet. Nat. 20) itiuin iecn oeta n vltoayimpact evolutionary and potential silencing Distribution, (2007). 69 19 7293-7298. , nu e.Cl e.Biol. Dev. Cell Rev. Annu. 21) yi-eitdmcaimfratnsrs fiber stress actin for mechanism zyxin-mediated A (2010). 3429-3456. , 831-844. , 135 e.Cell Dev. ee,D,vnNmee,E n hitfr,G. Christofori, and E. Nimwegen, van D., beler, ¨ 20) amla edpoen euaecell regulate proteins Tead Mammalian (2008). 4059-4069. , 7 e48651. , 41 .Bo.Chem. Biol. J. Oncogene 21) nimtnlk rtisascaewith associate proteins Angiomotin-like (2011). 553-562. , 20) rniin ewe pteiland epithelial between Transitions (2009). 19 21) acrse el n epithelial- and cells stem Cancer (2012). ei.Cne Biol. Cancer Semin. 365-376. , 27 20) rncitoa euainof regulation Transcriptional (2008). 20) edpoen ciaethe activate proteins Tead (2005). 6958-6969. , 286 e.Cell Dev. 27 21b.TeCub complex Crumbs The (2010b). 4364-4370. , 347-376. , o.Bo.Cell Biol. Mol. 21) M steultimate the as EMT (2012). a.Genet. Nat. o.Cl.Biol. Cell. Mol. 20) h transcriptional The (2009). ee Dev. Genes ei.Cne Biol. Cancer Semin. Cell ¨a LSCmu.Biol. Comput. PLOS 20) hlGbs a PhyloGibbs: (2005). 21) tutrland Structural (2010). 18 b,S,Rba,M. Rebhan, S., ¨bo, 22 21a.TeHippo The (2010a). 579-591. , 139 194-207. , Development b 20) Epithelial- (2009). idcdETin EMT -induced acrCell Cancer 871-890. , 39 c.Signal. Sci. 21) EMT- (2012). Development e,A,van A., ler, ¨ 457-466. , rc Natl. Proc. 15 20 (2011). 28 a.Rev. Nat. b 1229- , 3115- , (2009). 3177- , -SMAD (2001). (2012). (2011). 1535 Dev. 131 22 23 a 4 1 - , , , , ,

Journal of Cell Science hn,H,Lu .Y,Za .Y,Za,B,Yo . ho . in,Y,Li Q.-Y. Lei, Y., Xiong, S., Zhao, J., Yao, B., Zhao, Z.-Y., Zha, C.-Y., Liu, H., Zhang, E., B. Bernstein, S., D. Johnson, J., Eeckhoute, A., C. Meyer, T., D., Liu, Y., Zhang, Vullhorst, G. J., Christofori, K. and M. Kaneko, Yilmaz, I., Karavanova, J., M. Kohn, R., Yagi, ARTICLE RESEARCH 1536 ncl rwhadeihla-eecya transition. 13362. epithelial-mesenchymal and growth cell in al. et K.-L. Guan, W. and Li, M., Brown, (MACS). M., ChIP-Seq of R. analysis Myers, C., Nusbaum, invasion. development. mammalian of A. beginning Development the Buonanno, at lineage trophectoderm and the specifies L. M. DePamphilis, acrMtsai Rev. Metastasis Cancer 134 20) EDtasrpinfcosmdaetefnto fTAZ of function the mediate factors transcription TEAD (2009). 3827-3836. , 20) M,tectseeo,adcne cell cancer and cytoskeleton, the EMT, (2009). eoeBiol. Genome 28 15-33. , 20) rncito atrTEAD4 factor Transcription (2007). 9 R137. , .Bo.Chem. Biol. J. 20) Model-based (2008). 284 13355- , Zhao,B.,Lei,Q.-Y.andGuan,K.-L. E. Fuchs, and A. H. Pasolli, H., Zhang, ho . i . u . ag .H,Lu .Y,Li .adGa,K.-L. Guan, and Q. Lei, C.-Y., Liu, H., L. Wang, C.-Y., Q., Wang, Lu, D., L., Li, J. B., Lin, Zhao, K.-L. J., Guan, Yu, and Q. S., Lei, L., Li, Li, W., B., Zhao, Li, L., Li, J., Yu, X., Ye, B., Zhao, ewe euaino ra ieadcancer. and size organ of regulation in between differentiation and growth balancing in skin. functions coactivator transcriptional nimtni oe ip aha opnn htihbt A oncoprotein. YAP inhibits that component Dev. pathway Hippo Genes novel a is Angiomotin version. updated an tumorigenesis: and control size al. control. et growth and M. induction A. Chinnaiyan, rc al cd c.USA Sci. Acad. Natl. Proc. ora fCl cec 21)17 5313 doi:10.1242/jcs.139865 1523–1536 127, (2014) Science Cell of Journal 25 51-63. , 20b.TA eitsYPdpnetgene YAP-dependent mediates TEAD (2008b). ee Dev. Genes 20a.TeHpoYPptwy e connections new pathway: Hippo-YAP The (2008a). 108 2270-2275. , 21) h ip-A aha norgan in pathway Hippo-YAP The (2010). 21) e-soitdpoen(YAP) protein Yes-associated (2011). ur pn elBiol. Cell Opin. Curr. 22 1962-1971. , ee Dev. Genes 20 638-646. , 24 862-874. , (2011).

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