idA aor* lkad iknv,Dn nraoasrs,SmaGoa,Re Gaouar, Samia Andriamoratsiresy, Dina fibroblasts Piskunov*, Aleksandr embryonic Tanoury*, mouse Al of Ziad in repertoire genes new target a receptor signaling: acid and retinoic adhesion cell in involved Genes ARTICLE RESEARCH ß eevd2 ac 03 cetd6Nvme 2013 November 6 Accepted 2013; March 27 Received { Ge de (Institut IGBMC tissue target (RARs; receptors, receptors nuclear of of in acid classes retinoic expression two the through and effects the its exerts differentiation, in It genes. modifications embryonic and in through growth roles homeostasis key cell plays regulates and development, A, processes Vitamin of biological metabolite multiple active the (RA), acid Retinoic INTRODUCTION Transcription, receptors, Adhesion Nuclear acid, Retinoic WORDS: KEY the expressing lines cell rescue RARsRAR RA of ligand, in advantage the and signaling Taking of in metabolism. involved presence genes the other of in compared expression contrast, filaments, the control In actin cells. of WT network the disrupted with a substrates display on of spread they to expression and and adhere the to unable are control MEFs knockout RARs the Consequently RA, adhesion. cell with of associated transcripts absence We gene several RARs. the three in the that lacking MEFs found (MEFs) to fibroblasts MEFs embryonic wild-type mouse comparing in genes by RARs the by of regulated analysis study are genome-wide the that a In conducted activity. we transcriptional here, their reported in phosphorylation importance their the showed of previously a we and of They phosphoproteins proliferation. also and expression are differentiation cell the in involved regulate genes of that battery factors (RAR transcription receptors dependent (RA) acid retinoic Nuclear ABSTRACT Ce Jost, Bernard Teeatoscnrbtdeulyt hswork this to equally contributed authors France. *These Cedex, Illkirch 67404 Universite UMR7104, CNRS, U964, eetieo ee htaergltdb Asadhglgtthe highlight gene. the and on by regulated depending RARs programs RARs, transcriptional by the regulated the of diversity increase are and results complexity that Our genes RA. by of regulation repertoire their for receptors phosphorylation a the the require by of that genes selected depending either also isotypes, We controlled RAR gene. of the combination be on a by can or genes RAR single RA-target specific of that found expression we background, the null the in sites) phosphorylation terminal utp,teeaea es w nw sfrs hc are which isoforms, known two each least For at 2006c). al., are et Germain there 2006b; subtype, al., et al., Germain et 2006a; (Germain regulators transcription RAR/RXR heterodimeric (RXRs; receptors uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,5153doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. a rRAR or c utps(ihrwl-yeo uae tteN- the at mutated or wild-type (either subtypes ´ a ne , ´ ln em n Ce and Keime ´line iu td ilgeMole Biologie de et tique b and c ´ ,wihfnto sligand-dependent as function which ), eSrsor,1reLuetFis P10142, BP Fries, Laurent rue 1 Strasbourg, de a , b and ´ uar tCluar) INSERM, Cellulaire), et culaire a cl Rochette-Egly ´cile , c b n h eiodX retinoid the and ) and c r ligand- are ) { As nteasneo iad N-on Asrepress RARs DNA-bound ligand, of absence by the regulation gene of in model (Moutier canonical promoters the RARs, to the According in 2012). al., located et (RAREs) polymorphic elements and the specific response control binding RA through RXRs genes with Rochette- target highly heterodimerized of and are expression a that and (Bastien RARs and 2004). (LBD) (DBD) well-structured Egly, N- domain domain two variable DNA-binding ligand-binding central C-terminal a and a of domains, (NTD), conserved consisting domain structure, terminal and organization domain 1996). (Chambon, regions N-terminal their splicing, alternative in differ and and usage promoter differential by generated ta. 03 ohteEl,20) ned eietfe two RAR in identified (S369 RAR LBD we in the Indeed, in S371 one 2003). Tanoury sites, Rochette-Egly, phosphorylation (Al main phosphoproteins 2013; are al., RARs et that Al discovery see the with reviews 2009). Germain, (for and the Rochette-Egly machinery for 2013; way al., transcription the et pave Tanoury the and of genes target structure recruitment chromatin of the promoter alter the recruitment which surrounding the complexes, and coregulator upon co-repressors other contrast, of of dissociation In the LBD, state. their induces in complexes, which changes repressed conformational undergo a corepressor RARs binding, in of ligand chromatin recruitment maintain the which through transcription 95 n h te n nteND(7 nRAR in (S77 NTD the in one other the and 1995) RAR pcfctre ee Dlcoxe l,2010). al., cell et of (Delacroix regulation the genes transcriptional to the target correlated to specific been and RARs has of response 2011; binding RA al., DNA et The RA- (Mahony 2012). neurons al., the into et during cells Moutier ES and Su and 2011; 2008) 2010) al., Gudas, et al., and (Mendoza-Parra cells et F9 Ross-Innes of differentiation 2009; induced al., lines cell cancer et breast in (Hua sites binding throughput RAR and high the genes RAR-target of and repertoire the (ChIP-seq) expanded technologies (RNA-seq) sequencing sequencing al., deep et with (Bruck coupled genes target and RA RAREs phosphorylation DR5 canonical to final 2009). receptor (Chebaro of This the of NTD transcription 2011). binding the the the al., for can et in crucial is then Samarut step located which 2013; site al., TFIIH, second et of the subcomplex recruitment the phosphorylate cyclin-H–cdk7 allows the p38MAPK–MSK1 residue the in this the located of RA-activated of residue of phosphorylation serine 2012). and the activation LBD at Rochette-Egly, RARs the phosphorylates and to MSK1 (Piskunov lead effects (Bruck that pathway non-genomic RA and RA to extranuclear response to of subsequent in 2009) phosphorylated al., rapidly et are sites two iia oms ula eetr,RR aeawell-defined a have RARs receptors, nuclear most to Similar uigtels eae hsseai eaemr complicated more became scenario this decade, last the During eety eoewd hoai immunoprecipitation chromatin genome-wide Recently, c )(ate ta. 00 ohteEl ta. 97.These 1997). al., et Rochette-Egly 2000; al., et (Bastien 1) c )(alade l,20;Rcet-gye al., et Rochette-Egly 2006; al., et (Gaillard 1) gsLtig a Ye, Tao Lutzing, ´gis a n 7 in S79 and a 521 and

Journal of Cell Science EERHARTICLE RESEARCH 522 adhesion. cell in involved genes of expression the control RARs RAR( RA, of in absence downregulated the In 1. Fig. and MEFs WT comparing by profiled absence the was Rarb in RARs by RA regulated are of that in genes of involved repertoire genes The of expression the adhesion to cell contribute RARs MEFs, In RESULTS utps(ihrW rmttda h N-terminal the their for selected at receptors we the mutated of background, RA. phosphorylation taking null by or regulation require the Finally, that in WT genes MEFs. RAR sites) the in (either phosphorylation expressing lines signaling the are cell adhesion subtypes that prevented rescue and cell and RA of also by advantage RARs regulated morphology with are of that the lack genes associated the The of all of cells. reduced expression adhesion disruption the with cell of associated the the with properties is in associated with RARs transcripts that, gene of and observed several lack We the of the out. which levels RA, knocked in of MEFs been absence with had MEFs RARs (WT) (MEFs), three fibroblasts wild-type embryonic comparing mouse in by RARs, by regulated are splmnaymtra al 1.Ti nlssrvae a revealed analysis This S1). Table material experiments (RNA-seq) (supplementary sequencing throughput high in hereafter] oneuaino ee novdi elahso nRAR( in adhesion cell in involved genes of downregulation he eaaeeprmns C TqC xeiet hwn htteteto TMF ihtepnRRatgns M 9 (1 493 BMS antagonist RAR means pan the the are with Values MEFs adhesion. WT cell of in treatment involved that genes showing the experiments of RT-qPCR expression (C) experiments. separate three eew odce eoewd nlsso h ee that genes the of analysis genome-wide a conducted we Here 2 / 2 Rarg 2 / 2 Es[eerdt sRAR( as to [referred MEFs a , b , c ) 2 / 2 Es(E O oprdwt Tcls h ee novdaelse.()R-PReprmnscnimn the confirming experiments RT-qPCR (B) listed. are involved genes The cells. WT with compared KO) (MEF MEFs a , b , a c , b ) a 2 , c ) / 2 rRAR or 2 Rara / 2 MEFs Escmae ihW Es ausaetemeans the are Values MEFs. WT with compared MEFs 2 / 2 6 c ..o rpiae rmtresprt experiments. separate three from triplicates of s.d. eit usata ee ftasrpinlatvto nthe in activation in transcriptional involved compound of RA. genes of level this can absence substantial RARs the with that a indicate of data MEFs mediate these Altogether, expression WT 1C). (Fig. the of adhesion Treatment repressed 2010). strongly with panRAR Maire le interaction al., 2010; the the al., that et et with (Bourguet not so enhanced 12 strongly (data that MEFs is helix agonist corepressors pairs inverse of WT an positioning base as the the is 8 displaces BMS493 fact treated and In BMS493. al., 0 also antagonist et between We (Moutier of (IRs) shown). spacing repeats with inverted and 2012) located adhesion. (DRs) regions repeats the response to direct of RA analysis associated had The 6 genes promoters. genes their these in whether RT-qPCR of elements analyzed in we transcription Therefore, corroborated the sustain was MEFs downregulated vanin-1, 1B). are knockout (Fig. genes vitrin, 11), these experiments 1), That RAR alpha alpha 1A). 11 (Fig. and in 2 type robo 1, 1 and alpha 1 (alpha alpha robo II and (type 2 integrins alpha collagens 1, as (alpha 4), laminins 9, such protocadherin cells. 17, adhesion, cadherin knockout cell the involved in proteins in encode downregulated predominantly are genes these that Remarkably, genes of number 0k rmtegn iisrvae h rsneo several of presence the revealed limits gene the from kb 10 uhrslssgetta nteasneo iad Aswould RARs ligand, of absence the in that suggest results Such ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal A elahso stemjrbooia rcs,wihis which process, biological major the is adhesion Cell (A) 6 ..o rpiae rmtoor two from triplicates of s.d. m )ihbt the inhibits M)

Journal of Cell Science tbt h RAadpoen ees hra RAR whereas RAR specifically RAR recognizing levels, main two antibodies the proteins monoclonal experiments with and Immunoprecipitation performed 2A,B). mRNA (Fig. detectable the hardly both at EERHARTICLE RESEARCH eut ugs htteRAR the that suggest results RAR the constitutively express MEFs subtype, RA. RAR specific a by regulated akrud h eceln xrsigRAR expressing line rescue cell The rescue RAR MEF background. of expressing advantage stably took we lines genes, these adhesion. of cell expression in the involved genes the of expression the control hnteqeto a hte h xrsino hs ee is genes these of expression the whether was question the Then nodrt eemn hc A,apao am1 otiue to contributes gamma1, or alpha RAR, which determine to order In c sepesdi EsadntRAR not and MEFs in expressed is 1 c sfrs(RAR isoforms a a rRAR or n/rRAR and/or c n RAR and 1 a c , ntetil A null RAR triple the in 1 b or a n RAR and c a c c nteasneof absence the in , utpsmight subtypes 1 Fg C.Such 2C). (Fig. 2 c a led been already has )idctdthat indicated 2) c subtypes b is ie n 0 nteRAR the in 10% and RAR line) the in rescue two in downregulated the 15% in were lines, restored that cell were 15% genes cells, knockout the RAR triple among or the that shows 2E upregulated Fig. The RAR the were cells RAR S1). wild-type the versus with crossed knockout the Table experiments of RNA-seq genes in downregulated material cells knockout RAR (supplementary triple and WT the eeotie n n a eetd(i.2,ln 4). lane 2D, receptor (Fig. endogenous selected the was to one similar and obtained levels were at receptor the expressing hrceie (rc ta. 09 n i.2,ln ]adwe and 3] RAR lane expressing 2D, Fig. lines and rescue 2009) stable al., established et [(Bruck characterized h RAR The c ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal ececls(essteW el) h endarmin diagram Venn The cells). WT the (versus cells rescue a n RAR and a c ececls(essteW el)adwith and cells) WT the (versus cells rescue ececl ie eete oprdwith compared then were lines cell rescue a c ie(n o nteRAR the in not (and line ececl ie(n o nteRAR the in not (and line cell rescue rbt RAR both RAR or expressing in lines restored rescue not the is adhesion genes cell the in of involved expression the that showing experiments RT-qPCR (F) cells. WT C EsepesteRAR the express MEFs (C) means (KO), knockout RAR the of transcripts the comparing diagram knockout Venn RAR (E) triple background. the in with levels compared WT combination, in or alone RAR of Expression (D) o h RAR the not RAR RAR of analysis Immunoblotting (B) analysis. RT-qPCR by MEFs, WT RAR( of Comparison 2. Fig. eaaeexperiments. two separate from triplicates means of the s.d. are in Values involved adhesion. genes cell the of expression the RAR a with combined RAR with MEFs WT of treatment that showing experiments RT-qPCR (G) experiments. separate Ab1 isoforms, the recognizing specifically antibodies with (IP) immunoprecipitation RAR RA. of absence xrsigRAR expressing c a c b a A)adAb10 and (A1) n RAR and n RAR and n RAR and , b 6 , c ..o rpiae rmthree from triplicates of s.d. ) 2 c a / 2 sfr sasse by assessed as isoform 2 n RAR and c b c a EsadMEFs and MEFs A oprsno RAR of Comparison (A) a N eesi TMEFs WT in levels RNA ecelnsvru the versus lines rescue eesi TMEFs. WT in levels rRAR or n RAR and c b c A) respectively. (A2), a .Treclones Three 1. naoitinhibits antagonist c ausaethe are Values . n RAR and c c c antagonists sfr and isoform 1 c ie.From line). nthe in , a RAR , c 1, a , 6 523 a c a ,

Journal of Cell Science lhuhteW el pedrpdy RAR( rapidly, spread cells phalloidin. WT 488 green the fluorescent performed with Although cells experiments the fluorescence of incubation in after spreading and shape 3A). cell (Fig. IV and collagen fibronectin and attach not I laminin, did collagen cells to to knockout triple well efficiently the However, as 3A). (Fig. attached fibrinogen types with cell fibronectin) Both and fibrinogen laminin, (ECM matrix extracellular different lsi.Rmral,tetil A ulMF irtdless migrated MEFs null RAR triple the Remarkably, (collage plastic. substrates ECM different h deingnswr o etrduo eepeso of re-expression upon RAR both require restored would genes not these were genes RAR adhesion the RAR receptor, single specific restored these a of by expression regulated the is that genes suggest can one results, these ARTICLE RESEARCH 524 purified our cells with co-labeling WT that Note the RAR 4B). were (Fig. with antibodies, vinculin disrupted with compared also of labeled adhesions, network spreading disrupted, Focal the 4A). cells substantial was (Fig. knockout filaments triple without the actin in round Moreover, 4A). and (Fig. compact remained we hypothesis, this RAR( and corroborate WT of To the ability control MEFs. the monitored would of RARs that properties suggest adhesion results above the MEFs RA Collectively, of of migration absence and the adhesion in morphology, the to contribute RARs eeafce rnti Radius a in not or properties counterpart migration affected their whether WT were addressed we the Therefore, 1). than Movie proteins motile ECM 1). more different Movie with material supplementary coated 5; Fig. microplates by 3B; each corroborated (Fig. on splitting was line after cells microscopy cell round video compact RAR( time-lapse more That phase-contrast 4A). are (Fig. cells cells MEF knockout the in receptors role a suggesting not RAR 2F), were RAR, (Fig. third cells, line the rescue knockout for double triple either the in in restored downregulated were that RAR of control the under itself factor, ta. 00 u ta. 09 aoye l,21) hrfr we Therefore 2011). al., et Mahony 2009; al., et Hua 2010; al., et ergltdb igeRRuo nciaino h others. the of can inactivation which upon genes, RAR single of a class by of additional regulated an expression be highlighting upon 2E), repressed (Fig. or induced RAR were cells knockout the of genes none the by alone, exemplified of as added expression adhesion, the in When involved affected combination. significantly in antagonists either subtype, or RAR each for WT alone antagonists hypothesis, with a such treated corroborate were to MEFs order In expression. of level RAR both RAR expressing line and rescue stable additional an established bec flgn,sm ee eurn RAR RAR requiring between genes cooperation a the some others the ligand, highlight in expression of results genes absence regulating these mechanisms the Collectively, of complexity 2G). (Fig. expression RAR Vnn1 ihete RAR either with hnW n RAR( and WT Then ncnrs,6%o h oneuae ee icuigms of most (including genes downregulated the of 60% contrast, In ti neetn ont httetil ncotclsas seemed also cells knockout triple the that note to interesting is It ial,gnsta eentafce ntetil RAR triple the in affected not were that genes Finally, a a a c Fg G,adnihrddtecmiaino h RAR the of combination the did neither and 2G), (Fig. naoit Fg G.Hwvr h obnto fRAR of combination the However, 2G). (Fig. antagonists rRAR or rRAR or rRAR or c Fg D ae5.Ufruaey h deingenes adhesion the Unfortunately, 5). lane 2D, (Fig. c c efrthpteie htteepeso of expression the that hypothesized first We . c c nioiscrooae h bec fthe of absence the corroborated antibodies ntetil A ncotbackground knockout RAR triple the in rRAR or a , b b ept t o n adydetectable hardly and low its despite , , c ) 2 / a 2 rtis(olgnI olgnIV, collagen I, (collagen proteins ) ,lmnn irnci)o on or fibronectin) laminin, I, n TM infcnl erae their decreased significantly Eswr locmae for compared also were MEFs a a a rRAR or elMgainAsywith Assay Migration Cell n RAR and , b splmnaymaterial (supplementary s , c nvitro in a ) Itga1 2 rRAR or / 2 c a c ihRAR with , Est tahto attach to MEFs a , n/rapioneer a and/or b deinassays. adhesion Cdh17 , rRAR or c c ) a 2 a (Delacroix rRAR or / , 2 , b Vit , b c MEFs . c ) a 2 and and and / c 2 a b . irto n opooytruhterglto fthe of regulation adhesion, the cell of through control proteins. adhesion the morphology of in expression RARs and for role migration new a highlight RAR that fact reflect could the fact in but RAR unexpected the were observations the that Such shown). than note fibers to plastic actin interesting WT of and is the laminin it of However, morphology on RAR the 3B). recover (Fig. migrate adhesion, fully either to not cells and the did I and They collagen the recover 3C). to 3A) Accordingly, (Fig. attach (Fig. to not cells. ability IV the WT do recover collagen not the did lines of lines rescue migration rescue and the upon morphology restored that not expect were MEFs, RAR of knockout expression the plastic in or downregulated laminin on 2). seeded Movie material when supplementary MEFs 5; WT (Fig. the than efficiently epnet Awspoie ycmaigW Esand MEFs in WT RARs comparing by by regulated profiled are was that RA genes to of response repertoire but signaling MEFs in the of involved adhesion genes Then the of affect expression not the does enhances RA of addition The RAR( euae yRAR by 2012). regulated that al., indicating et shown), (Moutier not RARs (data for pairs targets base are 8 they triple- and p 0 the between the in spacing revealed expression promoters analysis Moreover their differential S2). of Table significant material (supplementary any cells knockout have not did cells. differentiated line already C.K., in are B.J., fibroblasts data), that Urban, unpublished fact C.R.E., Sylvia the and Dierich, with Su T.Y., 2012; Andree A.P., al., Davidson, S.G., et Irwin Moutier (Z.A.T., (Kashyap 2011; 2008) lines al., genes et cell Gudas, homeobox Mahony other canonical 2010; in the Gudas, activated include and and not development did in list involved the that Note 1). Rhpn2 receptor), A2b Cxcr7 Tnfrsf1b as such signaling in Pfkfb3 as involved proteins encoding such genes several included metabolism RA and in involved migration. the genes Cyp26b1 In included 3). and genes RA-target Movie upregulated canonical material adhesion of (supplementary list the the RA cell in same fact, of the presence in were and MEFs absence of involved spreading and did genes adhesion list Accordingly, this include Remarkably, S2). not Table only. material hours (supplementary 2 MEFs two for the RA genes, with target treated primary were the lines select MEF to order In experiments. eeurgltdi TMF,2%(xmlfe by (exemplified 25% MEFs, WT in upregulated were RT- 1). in Table corroborated and were 7 results (Fig. The experiments 6). qPCR Fig. S2; (supplementary RAR Table experiments material RNA-seq expressing in i.e. background, triple-null lines, rescue different olcieysc eut orbrt h N-e eut and results RNA-seq the corroborate results such Collectively were that adhesion cell in involved genes the of most As hni re oivsiaewihgnsaespecifically are genes which investigate to order in Then cells WT the in RA-regulated were that genes the all Remarkably WT with generated was genes downregulated and up- of list A h endarm(i.6 hw htaogtegnsthat genes the among that shows 6) (Fig. diagram Venn The Dhrs3 c a ceoie(-- oi)rcpo 7], receptor motif) (C-X-C [chemokine rohln h Taebnigpoen2 and 2) protein binding GTPase Rho (rhophilin, n RAR( and , b 6poporco2kns/rcoe2 -ihshts 3), 6-biphosphatase (6-phosphofructo-2-kinase/fructose-2, tmrncoi atrrcpo ue aiy ebr1b), member family, super receptor factor necrosis (tumor , ctcrm 40 aiy2,sbaiyb oyetd 1) polypeptide b, subfamily 26, family P450, (cytochrome ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal c dhdoeaerdcae(D aiy ebr3.I also It 3]. member family) (SDR [dehydrogenase/reductase ) 2 / 2 Rab20 a Es Atetdvru nrae,i RNA-seq in untreated, versus RA-treated MEFs, c , a c a etrsmr ee hnRAR than genes more restores ecelnsrcvrdamr es network dense more a recovered lines rescue ) ,RAR rRAR or RB0mme A noeefamily), oncogene RAS member (RAB20 c rbt RAR both or c a TMF eecmae othe to compared were MEFs WT , eceln Fg Baddt not data and 4B (Fig. line rescue eec fsvrlD oiswith motifs DR several of resence a n RAR and a Adora2b Rar rRAR or b a 2 Fg 2E). (Fig. Stab1 eeadthe and gene c n could one , (adenosine Cyp26b1 c (stabilin nthe in ,

Journal of Cell Science Fg C.Tu h Arglto fteegnsapasto S1A,B). appears genes RAR( RAR double these specific the of (RAR in RAR regulation corroborated single RA a the involve Thus 7C). (Fig. n 5 eepiidby (exemplified 15% by and (exemplified 5% contrast, Rhpn2 ( In S1A). RAR Fig. double material further the not was in genes these regulated of expression the Consequently, other. by mediated be could genes RAR these either of expression the Thus 7A). (Fig. EERHARTICLE RESEARCH Dhrs3 , eersoe nyi h RAR the in only restored were ) Cxcr7 a rRAR or a and rRAR or Tnfrsf1b c ahRRbigal osbttt o the for substitute to able being RAR each , c gnss(upeetr aeilFig. material (supplementary agonists eersoe ntetorsu lines rescue two the in restored were ) Stab1 a rRAR or a , a c nteRAR the in ) eceln (supplementary line rescue ) , c eceln n yusing by and line rescue ) c c ececl ie(i.7B) (Fig. line cell rescue .Ti ocuinwas conclusion This ). a eceline rescue Rab20 and otitrsigy h xrsino hs ee erae othe to decreased genes these of expression the interestingly, Most and 7B); 1); polypeptide atrrcpo;TF ue aiy ebr1)o ntetwo the in or 16) member [ family, lines super rescue TNFR RAR receptor; the factor in 3), RAR homeobox the less in activated were RAR MEFs, for WT role a but RA 1996) RAR al., to excluded. et both be response Taneja cannot in 2008; that Gudas, expression and their suggest (Su control can would combination One 6). (Fig. ial,svrlohrgns hc eentR-euae in RA-regulated not were which genes, other several Finally, restored not were genes upregulated the of 55% Remarkably, Nrip1 ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal Cyp26a1 ncerrcpo neatn rti ) Fg 7D). (Fig. 1)] protein interacting receptor (nuclear Wnt7b ctcrm 40 aiy2,sbaiya, subfamily 26, family P450, (cytochrome wnls-eae MVitgainsite integration MMTV (wingless-related c A deino TMEFs, RAR( WT of Adhesion (A) migration. and adhesion, MEF morphology control RARs 3. Fig. lsi 1 or fe eoa fthe of spot). removal hydrogel after hours (12 plastic RAR( WT, MEFs (B) experiments. separate three from duplicates means the are Values RAR and RAR expressing lines rescue Radius RAR expressing RAR both or TMF,RAR( of MEFs, Invasion WT (C) plates. on micro seeding plastic after hours 6 microscopy, phase-contrast by their morphology for compared were background, ftersu ie xrsigRAR expressing RAR lines rescue the of eceln ( line rescue c a a rbt RAR both or , , TM b b a , , c c c elMgainAsyon Assay Migration Cell eceln ( line rescue ) ) 2 2 ndfeetsubstrates. different on a / / 2 2 n RAR and Esado the of and MEFs EsadMEFs and MEFs Ngfr a a a , rRAR or b a , c n RAR and n RAR and ev growth nerve ; ) 2 6 c Dlx3 / rbt RAR both or 2 ..of s.d. c Esand MEFs ntenull the in a distal- ; RAR , c na in c 525 a b in c a , 2

Journal of Cell Science EERHARTICLE RESEARCH 526 ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal hlodnsoigtedsuto fteactin RAR( the in of network disruption the showing 488 phalloidin green with performed experiments network. and filament morphology actin MEF the control RARs 4. Fig. efre ihgen48phalloidin. 488 green with performed experiments fluorescence in compared MEFs, WT (C) RARs. RAR( three the for out MEFs knocked in disturbed is network microfilaments that the showing antibodies vinculin with performed cells. experiments KO Immunofluorescence the (B) in RARs these of RAR RAR raised against antibodies polyclonal purified with a a , rRAR or b , c ) 2 a / 2 c rRAR or ntenl akrud were background, null the in Es n Esexpressing MEFs and MEFs, a , b , c ) 2 c / ofre h absence the confirmed 2 Es Co-labeling MEFs. A Fluorescence (A)

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Journal of Cell Science oegns xmlfe by exemplified genes, some RAR requirements. phosphorylation distinct with gene same a of is RAR Thus 7C). This (Fig. was gene state RAR this that by Note effects. 6C). restored that (Fig. also receptor inhibitory WT the indicating by not but have S79A phospho-mutants, the the also by the in exemplified can only expressing restored phosphorylation were lines genes other cell Reciprocally, 7A). (Fig. RAR of EERHARTICLE RESEARCH ciae ntecl ie xrsigteRAR the expressing 528 lines cell the in activated the example, an of As 8E,F). phosphorylation (Fig. the RA by the on activation their RAR depend for by receptors thus restored and not respectively, were RT-qPCR 20% RAR and and WT upregulated by the restored 8E,F) among were that that Fig. show genes results The S2; 7). (Fig. Table experiments experiments. RNA-seq material the in selected (supplementary were that lines MEF different the in RA by regulated genes the 6 of analysis RT-qPCR 7. Fig. ..o rpiae rmtret iesprt experiments. separate five to three from triplicates of s.d. iial,aogtegnsta eeatvtdol nteWT the in only activated were that genes the among Similarly, Rab20 a n TRAR WT and c and Fg B n the and 7B) (Fig. Rhpn2 Stab1 c ee r otoldb h phosphorylation the by controlled are genes a ececl ie adnti h Tcells), WT the in not (and lines cell rescue u neednl fisphosphorylation its of independently but a ee hc a etrdb RAR by restored was which gene, n RAR and Cyp26a1 Tnfrsf1b a c eento esefficiently less or not were , rb TRAR WT by or a euaeteexpression the regulate can a eeb hto RAR of that by gene 7Ao RAR or S77A c c n RAR and ny13% only , c S79A, a a c oee,teoiiaiyo h rsn td eie nthe in 2008). or RARs resides Gudas, three all study and which in present Su MEFs with the 2010; MEFs Mendoza-Parra WT of al., 2011; of comparison originality et cell al., other the et Ross-Innes in Mahony as However, 2011; well 2009; al., as al., 2010) et Several et al., et RA. (Hua (Delacroix activated of types MEFs is genes presence in certain RA embryonic and of by expression mouse absence that reported in the have studies in RARs both by that genes fibroblasts, regulated the of transcriptionally analysis genome-wide are a described have we Here DISCUSSION hsh-uat,rsetvl Fg D.Rcpoal,other Reciprocally, as 6D). such (Fig. genes respectively phospho-mutants, fet nteratvto yR,icesn h opeiyo the of complexity the process. increasing negative RA, transcriptional or by positive activation their either on have effects can RARs of phosphorylation the phosphorylation. RAR WT the in Dlx3 not and line cell rescue loehrteerslsidct ht eedn ntegene, the on depending that, indicate results these Altogether a loatvtdb RAR by activated also was ora fCl cec 21)17 2–3 doi:10.1242/jcs.131946 521–533 127, (2014) Science Cell of Journal Dlx3 eeatvtdol nteRAR the in only activated were a u neednl fits of independently but c n Fg D.Nt that Note 6D). (Fig. one ausaetemeans the are Values c S79A

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RAR null individual two or one Dlx3 Nrip1 Wnt7b Rhpn2 Rab20 Stab1 Cxcr7 Tnfrsf1b Signaling Cyp26a1 nte motn idn fti td sta,aogthe among that, is study this of finding important Another c c a uae tterNtria hshrlto iealwdthe allowed site phosphorylation N-terminal their at mutated oprt ihRAR with cooperate rRAR or c nteasneo iad ncnrs,6%are 60% contrast, In ligand. of absence the in ) TK RAR + + + + + KO 62 ++ ++ WT RA to response lines: MEF 226 22 22 b ept hslte A being RAR latter this despite , a 2 2 22 2 2 2 2 ,- b ,- c a ncotMEFs knockout n/rRAR and/or a n RAR and a ++ +++ ++ ++ ++ ++ ++ ++ ++++ ++ + ++ + ++ ++++ +++ + ++ ++ a and or c c c TRAR WT hi rmtr.Hwvr hc ie oRAR do sites which in sites However, RAR-binding promoters. potential several their contain ligand, of absence ta. 00 are ta. 02.RAR that 2012). al., complexes et (Delacroix of Laursen regions 2010; promoter recruitment al., specific et the of methylation through the the regulate in ligand activation of transcriptional RAR of absence that levels reported condensed substantial studies mediate a recent can in However, state. chromatin repressed maintain and that complexes corepressor ilrqiefrhrivsiain.Acrigt h classical the cells, to the According of spreading investigations. of and further adhesion expression require proper the the will maintain for RARs genes unliganded these how and occupy t ekaiiyt idcrpesr Frode l,2003; al., et (Farboud corepressors reflect bind would 2003). property al., to et this ability Hauksdottir that proposed weak been Gudas, its has and (Su it ligand and of absence 2008) the in transcription gene control DNA- and unliganded 2001), RAR Chambon, bound and (Dilworth model nooyaayi hwdta,i hs el,telretcasof class largest ( metabolism the in cells, involved are these genes in target Gene MEFs. that, in showed RA by analysis regulated ontology are that genes genes signaling the of profiled cell also expression in We the involved control proteins RARs essential ligand, encoding of presence the In nsga rndcin( transduction signal in on htteatvto falteR-euae ee was genes RA-regulated the all null of RAR activation we triple the Consequently, that the each redundancies. analyzing found of without in means two requirement, a out or provides receptor one strategy knocked or a RARs Such three were background. all which RAR in MEFs individual with MEFs WT of (Delacroix type 2010). 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C.K., S.G., unpublished B.J., (Z.A.T., C.R.E., development Urban, in Sylvia involved are RAR as of such which types form cell phosphorylated other other the contrast, in cells, that ES In indicate laboratory survival. our and/or from results proliferation cell for essential RAR of form EERHARTICLE RESEARCH A hshrlto a lohv eaieefcso gene RAR of forms on effects negative signaling. cell have in by involved exemplified also genes, These can expression. phosphorylation RAR Indeed, RAR RAR that of fact activation RA The for required is genes RARs specific of phosphorylation MEFs, In rgasb As eedn ntegn n ntefaueof feature the on and gene type. regulatory the cell the on the al., of depending diversity RARs, et the by highlight programs (Mendoza-Parra gene and of RARs site models by the regulation same complicate observations a these and Overall, dynamic at 2011). their and/or recruitment or sites the one different sequential of that at binding subtypes speculate the RAR requires can several genes RAR-target one the of sites, activation RAR-binding several contain ciae nyb h hshrltdfr fRAR of form phosphorylated ( the ( genes background, by two knockout only identify RAR activated to triple us the or allowed in RARs WT the phospho-mutants RA expressing MEFs the the The with forms. of MEFs phosphorylated WT repertoire their of by comparison the regulated question are that into genes brought target RA, to response Tnfrsf1b ncnlso,tecretsuyicesdterpror of repertoire the increased study current the conclusion, In codn oorrcn tde,popoyaincnrl the controls phosphorylation studies, recent our to According hsoecnseuaeta,i Es h phosphorylated the MEFs, in that, speculate can one Thus aigit con httemjrt fteR-agtgenes RA-target the of majority the that account into Taking a l hs ee noekypoen novdi elsignaling. cell in involved proteins key encode genes these All . ´ ta. 00 n hrfr h erimn fRR to RARs of recruitment the therefore and 2010) al., et e Rab20 eog otetmrncoi atrrcpo ue family super receptor factor necrosis tumor the to belongs ,wihi otoldb h hshrltdfr of form phosphorylated the by controlled is which ), a a rRAR or n RAR and noe Tae hc soitswt and with associates which GTPase, a encodes a c n RAR and n o yteW eetr,idctn that indicating receptors, WT the by not and c oto h ciaino ee htare that genes of activation the control c ihlk l h Tae fthe of GTPases the all like hich r ail hshrltdin phosphorylated rapidly are -Taebnigpoenand protein A-GTPase-binding Rab20 Stab1 c ´ ta. 00.Further 2010). al., et e euae te genes, other regulates and loecd proteins encode also , Rhpn2 c n one and , htare that ) Rhpn2 ciainb A ial tse ih ntedvriyand type. diversity cell the the of on feature on depending the RARs, light on by and shed programs gene the regulatory it the new their Finally of revealed for complexity RARs RA. also of It state by al., 2002). phosphorylation al., et activation the et on (Goetz depend Tari that 2013; signaling genes al., RA et aberrant Siletz 2011; by characterized cancers abtplcoa nioisaantRAR against antibodies polyclonal Rabbit reagents and Antibodies METHODS AND MATERIALS RAwsprfe rm2m oa N sn lg(T magnetic oligo(dT) using 94 RNA at cations total divalent mg using 2 fragmented and from Illumina beads Briefly, purified the modifications. some was following with protocol mRNA created v2’ for prep was suitable sample molecules RNA sequencing ‘Truseq template DNA of library throughput a high RNA, total of isolation (RNA-seq) After sequencing mRNA throughput High qPCR by transgene the of presence the with immunoblotting. for selected analyzed and and were days lines 10 rescue for stable G418 The (Roche). reagent RAR Transfection the to into coupled promoter introduced to early also CAG coupled a promoter with vector early pCA CAG a with Lipofectamine vector pCA a PGK/ into 2000) al., deleted, et RARs RAR RAR WT and expressing three RAR( MEFs a as in all well S77A as 2002) with al., et Chapellier (MEFs) fibroblasts RAR( embryo immunoprecipitation Mouse and immunoblotting lines, Cell ed AecutBocecsCroain ihaBoe 3000 Biomek a with Corporation) AMPure Biosciences using purification (Agencourt by removed were beads primers PCR surplus Then 0scnsa 60 at seconds 30 olwdb C mlfcto 3 eod t98 at seconds [30 amplification in PCR fragments by and followed blunted, dimers, were of adapter Inc.). 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C.R.E. paper. and results. T.Y. the the and wrote of experiments analysis the RNA-seq bioinformatic generated the the R.L. performed performed and B.J. analyzed experiments lines. and RT-qPCR cell work the rescue experimental performed all S.G. undertook data. and the devised D.A. and A.P. Z.A.T., contributions Author interests. competing no declare authors The interests Amandine Competing to analysis. thanks bioinformatics Special for help. Genomique) monoclonal for France mouse facilities by the culture (supported for cell Velt (IGBMC) the Abdelghani to Oulad and M. antibodies to grateful are We request. upon Acknowledgements available previously are described sequences as Primer performed 2005). al., were et RT-qPCR (Bour and isolation RNA RT-qPCR and isolation RNA hbr,Y,Aa,I,Rce,N,Rcet-gy . tt,R .and H. R. Stote, C., Rochette-Egly, N., Rochel, I., Amal, Y., Chebaro, and P. Chambon, M., LeMeur, A., Dierich, J., Bastien, M., Mark, B., Chapellier, P. Chambon, C. Rochette-Egly, and A. Piskunov, Z., Tanoury, Al B. B. Aggarwal, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.131946/-/DC1 online available material Supplementary material respectively. Z.A.T., Supplementary and A.P. supported Lady INCA the from from and Fellowships [grant trust Cancer C.R.E.]; Memorial du to Tata National PL07-96099 Institut and the INCa-PL09-194 and number C.R.E.]; to DEQ20090515423 number betv sdwsLiaHXP P 63 APO PL HCX Leica was used objective rc,N,Vtu,D,Fry . un,V,Bur . eThe de A., Bauer, V., Duong, C., Ferry, D., Vitoux, N., H. Bruck, Gronemeyer, and R. A. Lera, de W., Bourguet, Laleve A., Bauer, L., J. Plassat, G., Bour, Y. Hochberg, and Y. Benjamini, Rochette- and P. Chambon, M., J. Egly, T., Riedl, S., Adam-Stitah, J., Bastien, C. Rochette-Egly, and J. Bastien, W. Huber, and S. E., Anders, Wilhelm, D., Vitoux, A., Nebbioso, O., Hirsch, A., Rossin, L., Altucci, uhnn .Q,Rcet-gy .adAsnBte,M A. M. Asson-Batres, and C. Rochette-Egly, Q., F. 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