rncitoa ciiysget htclaylclzto fGi is Gli2 of localization activation. The its ciliary and for localization localization. that required ciliary ciliary suggests both and activity for essential transcriptional nuclear domain its a distinct of regulate that identification reveals Gli2 ESCs of in analysis regions target systematic activate to localize our unable to Thus, was domain inability genes. central an this lacking to Gli2 addition cilia, In to a localization. was and ciliary domains, nuclear for characterized localization, contrast, required previously and nuclear By from distinct Gli2 localization. sumoylation region, restricted central ciliary Gli2 domains abrogate other for not several did required also are Sufu trafficking a that Mutating localization. and ciliary residues for cluster dispensable each were phosphorylation activation motif binding transcriptional domain, ciliary fingers, the repressor zinc for Gli2 domain, essential The are Gli2. residues of and localization domains which of testing ioeSno n eeyF Reiter* F. primary Jeremy and the Santos Nicole to localization activity its transcriptional regulates and Gli2 cilium of region central A ARTICLE RESEARCH ß 1500 2013 December 28 Accepted 2013; July 26 Received ([email protected]) USA. correspondence 94158-2324, for CA *Author Francisco, San Francisco, Institute, San Research California, Cardiovascular of Biophysics, University and Biochemistry of Department is converted Gli3R are tissues, Gli3 many and In Gli2 (GliA). length activators full transcriptional and into relieved, is Smo of the is as Tempe 2006; are (GliR), another Gli3, forms state, and repressor of Drosophila Gli2 off into signaling, this processed activity Hh proteolytically of In the effectors (Smo). transcriptional inhibits the Smoothened protein, Hh (Ptch1) the transmembrane ligand, Hh Patched1 of cell absence the receptor basal In di 2003). defects, Pasca Hebrok, including 2003; and al., congenital Magliano cancers, et (McMahon cause medulloblastoma and 2011). and can carcinoma holoprosencephaly, al., signaling as et embryonic (Ingham Hh such in homeostasis of roles tissue Misregulation important adult and plays patterning signaling (Hh) Hedgehog INTRODUCTION signaling, cilia Hedgehog Primary factors, Floxin transcription cell, Gli engineering, stem genetic Embryonic localization, Ciliary WORDS: KEY knock- create to system Floxin the used in activation. we Gli2 hypothesis, for this raising important test transcriptional is tip, To localization ciliary the ciliary the that signals, at possibility accumulates Hh the Gli2, to pathway, the response of In on activator depends cilium. proteins primary (Hh) the Hedgehog vertebrate through Signaling ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,10–50doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Gli2 lee nebyncse el EC)t lo methodical allow to (ESCs) cells stem embryonic in alleles l rhlg i(z-ln ta. 97 a tal., et Pan 1997; al., et (Aza-Blanc Ci ortholog, Gli ´ ta. 06.I h rsneo h th inhibition Ptch1 Hh, of presence the In 2006). al., et h aa oy(az ta. 00 uo ta. 01 and 2011) al., et Tuson 2010; al., to et localizes PKA (Barzi body. basal body the basal foundation, its the or cilium 1999). the al., to et that (Dai not pathway target the is transcriptional on domain, feedback Hh positive repressor a a a as In is functions 2009). lacks and al., Gli1 et processed Gli3, Pan proteolytically and 2007; Wang, Gli2 and to Pan contrast 2006; al., et Pan protein 2006; F-box the containing complex box unf ta. 03 i ta. 05.Udrcrn h link of the 2005; components Underscoring several Anderson, 2005). signaling, and al., Hh and (Huangfu et cilia Liu GliA primary 2003; between and al., GliR et Huangfu both the lacking the of to Mice attributable defects from 2010). loss patterning Anderson, tissue projects and display cilia (Goetz that functional cells most organelle of microtubule-based surface a the the cilium, by Ultimately, determined 1998). Anderson, and is al., (Eggenschwiler GliA signaling 2007). and et GliR Hh of Motoyama activity of relative 1997; output 2011; the Angers, al., is transcriptional and Gli2A Hui et and 1998; al., genes Mo et target (Ding Hh activator of predominant repressor predominant the ehp eas l2i erddi epnet PKA- to response in degraded is SCF by processed, Gli2 ubiquitylation is promoted Gli2 of because fraction smaller al., perhaps a et Gli3, Tempe (Pan to 2007; processing Compared Gli3 2006). Wang, and a and Gli2 of Pan of and region 2006; processing proteolytic a phosphorylation by the enhanced (PDD), controls (PKA) process domain a of A through determinant domain Gli3R kinase activator create repressor The to protein N-terminal cleaved 1999). is an al., their Gli3 to et possess contribute (Sasaki that Gli3 domain bi-functionality activator and C-terminal a Gli2 and (Kinzler domain 1988). DNA not target al., bind is that et fingers zinc it this C2H2-type for conserved processing, essential is Gli3 cilium is the cilium and to the localization regulation. that activity Gli clear al., whether is GliA et clear it Wen Although for 2009; 2010). al., Hh required al., et to et Kim response Zeng 2005; in 2010; there al., are accumulate et proteins but (Haycraft Gli tip, stimulation ligand, signal ciliary Hh the of at Hh absence present the in coordinates manner: be dependent to that proposed al., is site et cilium sub-cellular (Aanstad the transduction. Thus, cilium specialized 2005). strongly the al., pathway, a at et the functions Corbit fail activate that 2005; 2009; Smo cannot Smo al., cilium that of et Smo the forms suggesting (Corbit to mutant and Hh localize Moreover, of exiting 2007). to presence al., Ptch1 the et in with Rohatgi localize cilium manner, Smo the and ligand-dependent entering Ptch1 Both a cilium. the in to localize pathway the rtista euaeGitasrpinlatvt lolocalize also activity transcriptional Gli regulate that Proteins nrgigy etbaeH inln eursteprimary the requires signaling Hh vertebrate Intriguingly, nvrerts h he l rtis l1 l2adGi,share Gli3, and Gli2 Gli1, proteins, Gli three the vertebrates, In Hh- partially a in cilium the to localize also proteins Gli The b TRCP/Slimb ´ ta. 06 agadLi, and Wang 2006; al., et b RP Bai tal., et (Bhatia TRCP) , aSkp1 (a 0 eiusthat residues 200 2 Cullin 2 F-

Journal of Cell Science uo ta. 01 ege l,21) hs K niisGli2 inhibits 2010; PKA activity transcriptional Thus, al., its 2010). restrains al., and et cilium et the forskolin Zeng (Tukachinsky to translocation 2011; by localization al., PKA et ciliary of Tuson GliA activation Gli2 increased exhibiting the cilia reduces of whereas of number localization, indicative the increases Gli2 PKA tube, Gli2. of loss neural of signal The activity. Hh the degradation of activation in the excess and transduction exhibit activity Gli3 PKA lacking of Mice processing the promotes ARTICLE RESEARCH eotie lxncmail eeta S ie XG045 line, ESC trap gene Floxin-compatible ( a obtained We activity and localization knock-in A function. Gli2 RESULTS for activity, required transcriptional is Gli2 localization ciliary for that essential suggesting regulate for is required Gli2 domain localization a and of and ciliary sites, stability regions subcellular distinct protein distinct to translocation, localization Thus, ciliary activity. plays in that Gli2 transcriptional Gli2 role of of We region exclusion important central function. the a an identified for Gli2 and crucial nucleus, in are the from domains implicated several residues that found or a created domains We 2010). modify GFP-tagged al., of the et of (Singla panel tailor (ESCs) control cells genetically stem the the embryonic to control system how Floxin that and activity. Gli2 mechanisms regulates Gli2, turn, Gli the in of localization, for we subcellular define mind, required localization in further are questions subcellular these events primary to With the ciliary entry. sought at nuclear other and occur whether activation Sufu or from they the PKA cilium from dissociation whether how unclear move and remains cilium, they it inactivation Moreover, the how nucleus. the and into to stimulation cilium trafficked Hh are upon accumulate they PKA how to 2010). al., including response et in Tukachinsky 2010; cilium of al., dissociation the et the (Humke at that inactivation occurs lacking suggesting complexes either PKA, cells activated 2010; Sufu-Gli in with al., occur or the genes. not cilia et target enter does Hh can of (Humke dissociation GliA outcome Hh-mediated transcriptional release, Gli3 the upon control of and, and to dissociation nucleus 2010) Gli2 the al., al., et promotes full-length et Tukachinsky Bishop, Hh Kogerman from and 2004). 2003; nucleus al., Cheng al., Sufu et the et 2009; Dunaeva Merchant al., 1999; in 1999; et al., cytoplasm negatively et Chen activity the Ding 2005; Sufu 2002; to transcriptional al., them 2003). et Gli tethering (Barnfield al., by suppressing et proteins and Gli (Dunaeva all manner among the conserved proteins regulates is at Gli-dependent Gli1 motif, Gli to SYGH a bind the can three which, Sufu in of 2010). one al., sites, cilium et two Zeng primary 2005; al., et the (Haycraft to localizes sites these of 2010). independently al., et localization (Zeng PKA and ciliary that either cilium suggesting Gli2 forskolin, the bearing by regulates enter activation PKA to alleles PKA to ability sensitive conserved are their Gli2 at retain However, sites residues phosphorylation phospho-dead 2011). or al., phospho-mimetic et (Tuson eetdtegn-rpmtto ocreate to mutation gene-trap the to reverted Gli2 of codons the containing Gli2 uu eaiergltro l rncitoa activation, transcriptional Gli of regulator negative a Sufu, nti td,w sda fiin nc-napoc aldthe called approach knock-in efficient an used we study, this In remain, proteins Gli of activation the about questions Many gt/+ .Tegn rpi oae ihnteito fe h exon the after intron the within located is trap gene The ). Gli2–GFP Gli2 b tr oo,pouigafso ftefrt46 first the of fusion a producing codon, start go(upeetr aeilFg 1.We S1). Fig. material (supplementary -geo leercptltswl-yeGli2 wild-type recapitulates allele Gli2 lee htsseaial eeeor delete systematically that alleles Gli2 Gli2 Rev/+ ou nmouse in locus Ss We ESCs. ihSG m gns Ce ta. 02 e ta. 00.In 2010). al., et Wen 2002; SAG, al., stimulation et to without (Chen response and agonist with Smo GFP well a ciliary SAG, as with of cilia, intensity at pixel GFP the exhibiting as cells of percentage the measuring oimnpeiiae ohwl-yeGi n h higher- the and Gli2 from known Gli2–GFP reciprocally wild-type molecular-mass Sufu recapitulates Sufu. both and to Gli2 Gli2–GFP Gli2–GFP co-immunoprecipitated Sufu, of wild-type co-immunoprecipitated binding whether to the Gli2–GFP assessed comparable test we interactions, levels Gli2 To at 1A). expressed (Fig. was GFP ontemo h eeta ihaCtria F a ocreate to tag GFP C-terminal a with the trap Gli2 encoding gene cDNA the a of insert downstream to system Floxin the used aeilFg 2) hrsosvns oSG sascertained of as SAG, levels Gli2 cells to (supplementary transcript responsiveness activation differentiated Hh pathway by S2B). similarly, Hh Fig. to material and, high- responsive more cells of compared are cells undifferentiated ciliary percentage differentiated in SAG-mediated of higher to prevalent 1D,E). more prevalence (Fig. a was increased localization GFP and an ciliary localization exhibited pixel-intensity ciliary LIF of GFP withdrawal the u ean h -emnlrpesrdmi n N-idn zinc DNA-binding domain and domain activation repressor N-terminal transcriptional the C-terminal retains but the presumably genes, lacks target it Hh because of expression the repress can N- Gli2-78 an generate to cilium of processed fragment the proteolytically terminal to be can localize Gli2 not Gli3, do Like Gli2 of forms Processed l2in Gli2 shown). not resistance, ta. 00.Teeoe eacrandwehrGli2–GFP whether in ascertained accumulation Wen we similar 2009; Therefore, al., a 2010). et displayed (Kim al., (MEFs) et fibroblasts embryonic mouse (Wen approaches standard 2010). of al., limit detection et the below detected be that might suggesting not it Gli2, similarly endogenous have of immunofluorescence researchers nuclear Other 1C). (Fig. nucleus ii nmueEC splmnaymtra i.S2A). Fig. material (supplementary of ESCs primary majority to the mouse localize al., Similarly, transduction also et in signal Smo, Wen Hh and cilia other 2008; Sufu and al., including Gli2 et components, that Kiprilov observed 2005; We al., 2010). et (Haycraft types interaction and regulatory expression endogenous wild-type Sufu. under with recapitulated Gli2–GFP we expressing control, by Thus, tteclaytp sdfndb osann ihtebasal- 1B). the (Fig. with Sufu co-staining with by colocalization defined and as Ninein tip, marker ciliary body the at lhuhw i o eetncerlclzto fGli2–GFP of of fractionation localization nuclear-cytoplasmic nuclear Gli2 detect immunofluorescence, not by did we Although osseaial eemn o pcfcdmisadpost- S2D). and of Fig. dynamics domains material subcellular (supplementary specific the Gli2 system regulate how Floxin modifications determine the translational used systematically we to Therefore, localization. subcellular tailor can system Floxin the the that demonstrate data these Together, of haplosufficiency ecmae h xrsino l2GPt hto wild-type of that to Gli2–GFP of expression the compared We l2acmltsa h iir i pnH tmlto in stimulation Hh upon tip ciliary the at accumulates Gli2 l2lclzst h i ftepiayclu nmn cell many in cilium primary the of tip the to localizes Gli2 Gli2 Gli2 gt/+ Gli2 , ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal 2 2 Gli2 ou ocet eeial rcal S oeso Gli2 of models ESC tractable genetically create to locus Gli2 GFP/+ GFP/+ b Rev/+ glcoiaeatvt n C eoyig(data genotyping PCR and activity -galactosidase Rev/+ Ssrvae htGi–F oslclz othe to localize does Gli2–GFP that revealed ESCs Ss neto a ofre yneomycin by confirmed was Insertion ESCs. and Gli2 and , Gli2 Gli2 Gli2 8ka emdGi-8(a ta. 2006). al., et (Pan Gli2-78 termed kDa, 78 Gli2 2 Gli2 Gli1 splmnaymtra i.S2C). Fig. material (supplementary GFP/+ Gli2 Gli2 2 GFP/+ 2 Gli2 and GFP/+ el htwr ifrnitdby differentiated were that cells Gli2 2 Ssb muolt Gli2– immunoblot. by ESCs GFP/+ Gli2 Ptch1 el,cnitn ihthe with consistent cells, Gli2 2 GFP/+ SsehbtdGFP exhibited ESCs seuvln for equivalent is , Gli2 2 Ss(i.1A). (Fig. ESCs GFP/+ Gli2 Ssby ESCs exons 1501

Journal of Cell Science eoe oetpxlitniy hra ++dntshgetpxlitniy A ramn fdfeetae el nrae ohteproportio the both increased cells differentiated of treatment SAG intensity. pixel * highest (left, denotes Gli2–GFP ++++ of localization whereas intensity, pixel lowest denotes + EERHARTICLE RESEARCH 06.Ulk ullnt l2GP u iia oprocessed to approximate al., et 1502 similar the (Pan but tag to Gli2–GFP, GFP trap C-terminal full-length a gene Unlike with whether the frame 2006). inserted in we investigated of site system, we downstream processing Floxin to 2010), the fails exons Using al., Gli3 the cilia. et to processed (Wen localizes Because Gli2-78 cilia 1999). to al., localize et (Sasaki fingers dynamics. ciliary and localization Gli2 wild-type recapitulates Gli2–GFP 1. Fig. F n uuwr eetdi ohtenceradctpamcfatos hra aii a nydtce ntencerfato.()Following (D) fraction. nuclear the in detected only was B Laminin whereas fractions, cytoplasmic and nuclear (1 SAG the or both DMSO with in treatment detected were Sufu and GFP ien n xiisc-oaiainwt h iir i rti uu nes h niiulclu niae ytewiearwed cl a:5 bar: Scale arrowhead. white the by indicated cilium individual the Insets, Sufu. protein tip ciliary the with co-localization exhibits and Ninein, n ohudfeetae n ifrnitdcls n nutetdadSGtetdcls(nes h niiulclu niae ytewiearrow white the by indicated cilium individual the (insets, cells SAG-treated and untreated in and 5 cells, differentiated and undifferentiated both Gli2 iir i of tip ciliary nat-l2atbd.Gi–F a xrse teuvln eest idtp l2 h yaewsas muorcpttdwt ihrat-F o anti-GFP either with immunoprecipitated in also Gli2 was co-immunoprecipitates lysate Sufu The Sufu. Gli2. and wild-type Gli2 to for levels immunoblotted equivalent was at and expressed Sufu, was Gli2–GFP antibody. anti-Gli2 an Gli2 5 m 0 e odto)adclayGPpxlitniy(e aeil n ehd,adWne l,21;treidpnetexperiments, independent three 2010; al., et Wen and Methods, and Materials (see intensity pixel GFP ciliary and condition) per 100 .()Udfeetae n differentiated and Undifferentiated (E) m. Gli2–GFP/+ Gli2–GFP/+ Gli2 S yae eesprtdit ula n yolsi rcin n rbdfrGP uuadLmnnB opnn ftencerenvelope. nuclear the of component a B, Laminin and Sufu GFP, for probed and fractions cytoplasmic and nuclear into separated were lysates ESC S yae niGPatbd muorcpttsbt l2GPadSf nyfrom only Sufu and Gli2–GFP both immunoprecipitates antibody Anti-GFP lysate. ESC Gli2–GFP/+ Ss F oaie oteopst n fcla(sietfe yat-ctltdtbln cu)cmae otebslbd marker body basal the to compared AcTub) tubulin, anti-acetylated by identified (as cilia of end opposite the to localizes GFP ESCs. P , m .5 Student’s 0.05, o hour), 1 for M Gli2 Gli2 Gli2–GFP/+ t ts)adteitniyo iir l2GP(ih,* (right, Gli2–GFP ciliary of intensity the and -test) Rev/+ and Sswr uniidfrtenme fclaehbtn F oaiain(he needn experiments, independent (three localization GFP exhibiting cilia of number the for quantified were ESCs Gli2 Gli2–GFP/+ Sswr tie o F n cu.Claylclzto fGi–F a eetdin detected was Gli2–GFP of localization Ciliary AcTub. and GFP for stained were ESCs omo l2i agtdt h ulu,adta h l2C- Gli2 the for the that required that indicated is and analysis and nucleus, Immunoblotting localization the localization. nuclear to ciliary repressor inhibits the targeted the both that is to terminus suggests Gli2 localization observation of 2A). This (Fig. form increased tip 2B). (Fig. ciliary showed the nucleus to Gli2-78–GFP localize not Instead, did Gli2-78–GFP Gli3, A hl-ellstsfrom lysates Whole-cell (A) Gli2 Rev/+ ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal S yaeadc-muorcpttsbt l2GPadGi in Gli2 and Gli2–GFP both co-immunoprecipitates and lysate ESC P , .5 h-qae et.Errbr ersn ..throughout. s.d. represent bars Error test). Chi-squared 0.05, Gli2 Gli2–GFP/+ Gli2 Rev/+ S yae B l2GPlclzst the to localizes Gli2–GFP (B) lysate. ESC and Gli2 Gli2–GFP/+ n Sswr rbdwith probed were ESCs 5 m 0prcondition). per 40 .(C) m. ed.Saebar: Scale head). fclawith cilia of n Gli2 anti- r Rev/+ and

Journal of Cell Science sls tbeta ullnt l2o l27 Fg 2C; (Fig. Gli2-78 SCF or bind including to ligases, carboxy-terminus Gli2 E3 Gli2 Gli2-110– the multiple of of to ability full-length instability the Gli2-110 reflect The might than S3B). GFP that Fig. stable material suggesting supplementary Gli2-110– levels, less 2A). mRNA (Fig. Gli2–GFP, is wild-type comparable of cilia those Gli2-78–GFP, than to despite lower Like were localize levels system. protein not GFP Floxin the did using the the Gli2-110–GFP at GFP, of inserted with downstream were frame from site processing To exons approximate S3A). peptide the Fig. (Gli2-110–GFP), sized material Gli2-110 does (supplementary similarly a Gli2-110 Sufu (supplementary create with that no by-product Gli2-110 interact suggesting However, C-terminal not Sufu, as with the S3A). to immunoprecipitated with referred Fig. processing, consistent material Gli2 is of that peptide EERHARTICLE RESEARCH ee fGi-8GPi oprbet hto ullnt Gli2– from length full of that 2C). to (Fig. comparable GFP is Gli2-78–GFP of level central or the domain for activator C-terminal dispensable domain, are repressor domains N-terminal finger 5 the zinc bar: lacking and Scale Gli2 repressor arrowhead. GFP-tagged activator, whereas the arrowheads), but (Gli2 (red cilia domain cilia to to localize localize not not do do Gli2 Gli2. of of forms localization ciliary processed Proteolytically 2. Fig. ta. 06 hn ta. 2009). al., Marcotullio et Di Zhang 2009; 2006; al., et al., Chen et 2006; al., et (Bhatia Numb–Itch from ihat-F n lte o F n uu hr sls l210poenta idtp rGi-8poen ete l27 o l210co-immuopr Gli2-110 nor Gli2-78 Neither protein. Gli2-78 or wild-type than protein Gli2-110 less is There Sufu. Gli2 and (D) GFP Sufu. for blotted and anti-GFP with ie nest,wees++ eoe ihs ie nest.* intensity. pixel highest denotes ++++ whereas intensity, pixel nadto ofl-eghGi–F,GPimmunoprecipitation GFP Gli2–GFP, full-length to addition In Gli2 Gli2 Rev/+ Gli2 Gli2 D D (Rev), e,Gli2 Rep, e–F,Gli2 Rep–GFP, 2 leeta rdcsaGPtge qiaetof equivalent GFP-tagged a produces that allele GFP/+ Gli2 D m Gli2–GFP/+ c rGli2 or Act S yaervae faster-migrating a revealed lysate ESC .()Gi-8 Gli2 Gli2-78, (B) m. A l2GPEC eesandfrATb(e)adGP(re) h rcse om fGPtge l2(l27 n Gli2-110) and (Gli2-78 Gli2 GFP-tagged of forms processed The (green). GFP and (red) AcTub for stained were ESCs Gli2–GFP (A) D c–F n Gli2 and Act–GFP (WT), D F epciey outylclz oteclu wiearwed) nes h niiulclu niae ytewhite the by indicated cilium individual the Insets, arrowheads). (white cilium the to localize robustly respectively) ZF, Gli2 b Gli2-110–GFP/+ TRCP/Slimb D e n Gli2 and Rep D FGPclaylclzto rvlneadpxlitniywr oprdt l2GP eoe lowest denotes + Gli2–GFP. to compared were intensity pixel and prevalence localization ciliary ZF–GFP PPCl and SPOP-Cul3 , -1)and (-110) Gli2 D c oaiemr outyt h ulu hnwl-yeGi–F.Saebr 5 bar: Scale Gli2–GFP. wild-type than nucleus the to robustly more localize Act P , ou,in locus, .5(h-qae et scmae oWT). to compared as test, (Chi-squared 0.05 Gli2 Gli2-78–GFP/+ nc nGPfsoswt l2ta akteN-terminal the lack that Gli2 with to (Gli2 fusions Floxin domain used repressor GFP transcriptional we these localization, domain in whether ciliary DNA-binding knock for examine required the To are S2D). and domains Fig. domain material previously repressor activator (supplementary required the transcriptional the the are examined Gli2: the domain, Gli2 initially of to domains of We functional localize defined domains localization. to which ciliary failed Gli2 for investigated of we forms cilium, processed the Because localization are ciliary Gli2 for of domains dispensable DNA-binding and repressor activator, The n r ossetwt h oe htSf osntprevent al., not et (Humke does 2010). nucleus al., Sufu the et that entering Tukachinsky from model 2010; Gli Gli3 the of for forms with obtained processed results consistent to similar are are data and full-length These to 2C). binds (Fig. preferentially Gli2 Sufu that suggesting Sufu, with icfnesta r eesr o N idn (Gli2 five binding DNA the for lacking necessary Gli2 are encodes We that that 1999). fingers allele al., zinc an et in (Gli2 (Sasaki knocked 1184–1544) domain similarly acids activation amino C-terminal lacking the GFP; or 47–271) acids -8 Sswr lte o F n uui,o eeimmunoprecipitated were or tubulin, and GFP for blotted were ESCs (-78) ete l27–F o l210GPrbsl interacted robustly Gli2-110–GFP nor Gli2-78–GFP Neither ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal D e–F;lcigamino lacking Rep–GFP; m .()Lysates (C) m. D ZF–GFP; icfinger zinc ecipitate D 1503 Act–

Journal of Cell Science l2GP(T.+dntslws ie nest,wees++ eoe ihs ie nest.* intensity. pixel highest denotes ++++ whereas intensity, pixel Gli2 lowest (C) denotes + (WT). Gli2–GFP rmtstedgaaino l2b SCF cluster by phosphorylation Gli2 the of degradation of the Phosphorylation promotes 2006). al., et Pan akn mn cd 1–6)(altc n ao 1993). Pabo, and (Pavletich 417–569) acids Gli2 amino lacking ARTICLE RESEARCH 1504 (Rev), (Rev), Gli2. of localization ciliary Gli2 the of for intensity required pixel not and are prevalence sumoylation localization and ciliary phosphorylation Sufu, with Gli2 interaction The 3. Fig. a and Sufu with by interacts phosphorylated a GSK3 residues Gli2 PKA, contains includes that which Gli2 cluster domains, through phosphorylation activator motif and SYGH repressor the localization ciliary Between Gli2 are for sumoylation required and not phosphorylation PKA interaction, Sufu ln ahedo h icfne oan(o ta. 2010). al., et (Cox domain sites finger sumoylation zinc putative the Two of 2006). al., end et Han each 2010; Pan flank al., 2012; et Cox al., 2006; Gli2 et al., et inhibits (Bhatia that activity modification transcriptional post-translational a sumoylation, muolte o F n uu l2K Gli2 Sufu. and GFP for immunoblotted ciao einnrtecnrlzn igr hmevswere themselves fingers zinc localization. central C-terminal ciliary the the the for essential region, nor to repressor region translocate N-terminal activator to the Gli2, failed neither of repressor forms cilium, Gli2-110–GFP, or processed the and the import although nuclear Gli2-78–GFP that Thus, inhibit 2B). indicating (Fig. independently retention domains Gli2-78–GFP, full-length activator to to and relative localization similar localization ciliary Gli2, Gli2 nuclear Gli2 increased Both promotes displayed domain 2D). repressor (Fig. suggesting Gli2–GFP, the wild-type than Gli2–GFP that intensity full-length GFP as ciliary Gli2 cilia lower However, of proportion 2A,D). same (Fig. the in present niSf,adbotdfrGPadSf.Gli2 Sufu. and GFP for blotted and anti-Sufu, D D YH Gli2 SYGH, Gli2 Gli2 e–F,Gli2 Rep–GFP, D YHGPehbtdicesdncerlclzto oprdt l2GP iia oGi-8GP Gli2 Gli2-78–GFP, to similar Gli2–GFP, to compared localization nuclear increased exhibited SYGH–GFP Gli2–GFP/+ Gli2–GFP/+ b n K Bai ta. 06 uav ta. 2003; al., et Dunaeva 2006; al., et (Bhatia CKI and D CadGi K Gli2 and PC W)and (WT) W)and (WT) D D c–F n Gli2 and Act–GFP e–F n Gli2 and Rep–GFP Gli2 Gli2 . D Gli2 Gli2K Sswr tie o ii ATb e)adGP(ne fidvda iim ht roha) cl a:5 bar: Scale arrowhead). white cilium, individual of (inset GFP and red) (AcTub, cilia for stained were ESCs R e–F ipae significantly displayed Rep–GFP D SYGH–GFP/+ . R–GFP/+ . –F rti eesaeeuvln oGi–F n ohcnitrc ihSufu. with interact can both and Gli2–GFP to equivalent are levels protein R–GFP D YHGPpoenlvl eels hntoeo l2GP Gli2 Gli2–GFP. of those than less were levels protein SYGH–GFP b (K TRCP/Slimb ( D . YH S yae eebotdfrGPadtbln h yaewsimnpeiiae ihat-F or anti-GFP with immunoprecipitated was lysate The tubulin. and GFP for blotted were lysates ESC SYGH) D )EClstswr rbdfrGPadtbln rwr muorcpttdwt niGPand anti-GFP with immunoprecipitated were or tubulin, and GFP for probed were lysates ESC R) FGPwr all were ZF–GFP D D YHGP( SYGH–GFP c–F also Act–GFP n enhances and D YH,Gli2 SYGH), uuadSf eirclyimnpeiiae Gli2 immunoprecipitated reciprocally Sufu Gli2 and Sufu: Sufu Gli2 with of interacting expression to S3D). Fig. contrast material al., marked (supplementary of et expression in Kogerman the 1999; targets, nucleus, al., the in et Gli2 enriched (Ding preventing being in Gli2 supplementary Despite Sufu of for 1999). 3C; entry role (Fig. a nuclear with nucleus the consistent al., S3C), the et Fig. to (Chen material Gli2 localization Gli2 stabilizes 2010). enriched with Sufu al., et ciliary that consistent the Tukachinsky and for 2009; are lower required Gli2 not is of data to Sufu localization that These attributable show that 3A,B,D). studies potentially (Fig. previous is with levels albeit which protein Gli2–GFP, full-length intensity, as cilia reduced of proportion similar or localization. sumoylatable a whether ciliary lacking Gli2 tested putatively cluster regulate We residues Gli2 four phosphorylation S2D). and domains encode Fig. the these the material that (supplementary for lacking alleles lysines arginines motif, in substituting knock we SYGH sumoylation, to the and Floxin phosphorylation PKA from used dissociation of involve inhibition to postulated Sufu, is activation Gli Because YHmtfde o eit l fteitrcino l2with Gli2 of interaction the the of all that mediate indicating not 1A), does SYGH- (Fig. motif the Sufu SYGH for with account immunoprecipitate not interaction do not independent interactions does Gli2 Gli2–GFP homotypic Gli2, Because 3D). (Fig. GFP D eeino h YHmtfddntpeetGi from Gli2 prevent not did motif SYGH the of Deletion Gli2 CGP( PC–GFP D YHGPddntmsciaeH transcriptional Hh misactivate not did SYGH–GFP D ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal YHGP(akn mn cd 6–7)lclzdto localized 268–271) acids amino (lacking SYGH–GFP D P C n l2K Gli2 and PC) , .5(h-qae et scmae oWT). to compared as test, (Chi-squared 0.05 D . YHGPitrcswt uu (E) Sufu. with interacts SYGH–GFP D –F (K R–GFP e–F n Gli2 and Rep–GFP D YHGPimmunoprecipitated SYGH–GFP . D )wr oprdt hs of those to compared were R) A F-agdGli2, GFP-tagged (A) YHGPas showed also SYGH–GFP D c–F.(D) Act–GFP. D m Rep–GFP D .()The (B) m. SYGH– Gli2 Gli2 Rev/+ Rev/+

Journal of Cell Science htteSG idn iei motn o ula xlso and Gli2. exclusion of nuclear stabilization for important C- the is for and a site domains, binding multiple through SYGH through the that Sufu Sufu that reveal with findings interacts with Our Gli1, 2004). like interacts Gli2, al., et Gli1 (Merchant Apart motif, domain S3C). terminal SYGH Fig. its material (supplementary from fractions nuclear and Gli2 Sufu. ARTICLE RESEARCH el xiie nices nteitniyo iir GFP ciliary of Notably, 4A,B). intensity (Fig. the cells Gli2–GFP in Gli2 displaying full-length increase altered, to cells not an compared were of exhibited GFP percentages cells of localization the ciliary fused Although NESs 1037–1051). predicted (Gli2 several Gli2 GFP or (supplementary NLS to cilium described we the previously Similarly, from cilium. exit S2D). signals the Fig. Gli2 export material into nuclear affected leucine-rich entry (NESs) putative Gli2 whether were in examined signals involved localization nuclear nuclear importins, also putative Because with whether interaction RP2 2011). examined through and we al., Kif17 localization et within ciliary sequences promote Hurd import (NLS)-like nuclear 2010; signal of that localization al., with et similarities shares (Dishinger RP2 import exit ciliary and of ciliary Kif17 mechanism Gli2 the of in that revealed role but have a studies entry, play Recent ciliary might Gli2 signals mediate export not nuclear post-translational do signals import repressive Nuclear a activity. although transcriptional be Gli2 in included), limits that might cilia not modification to sumoylation (data localizes Thus, Pias Ubc-9 cilium, elegans enzyme ligase 5 primary Caenorhabditis Sumo-conjugating Sumo 3, E2 the Senp2, E3 the near proteases the desumoylation or or the at several Sumo1–3, detect components including not did pathway We in accumulation. Sumo seen nuclear activity or increased Fig. localization the material that suggest (supplementary Gli2K data 2012) our al., Thus, S3E). et this increased (Han mutations multiple hyperactivity and nucleus Gli2 wild-type the than to in activity enriched ability K Gli2-containing highly findings, individual its previous not with was Consistent retained 3A,B,E). and (Fig. Gli2–GFP, Sufu with full-length interact to Gli2K localization unsumoylatable create Gli2K with K716R–GFP). K630R, lysines to sumoylatable of arginines putatively each substituted four we S3E), the ciliary Fig. material Gli2 (supplementary promote 2011) other might GSK3, by reach and to phosphorylation of CKI loss Gli2 localization. that as does, of but such 2010) ability cluster al., kinases, the et phosphorylation Zeng affect 2011; the not al., the et to does (Tuson cilium sites Gli2 the PKA of the substitution localization phospho-mimetic of observed or efficient phospho-dead that the we Given cilium. promotes Gli2 3A,B; However, (Fig. region ciliary this S3D). Gli2 of amounts wild-type Fig. diminished to material Hh similarly supplementary activates the and in targets Gli2–GFP length present full transcription as was cilia cluster) of proportion phosphorylation same the contains which sGi a esumoylated be can Gli2 As Gli2 eue lxnt nc nallsta noeGi akn a lacking Gli2 encode that alleles in knock to Floxin used We D E–F,lcigaioais2921 8–9 and 887–895 229–241, acids amino lacking NES–GFP, . D –F sidpneto lee uuitrcin ciliary interaction, Sufu altered of independent is R–GFP CGP(akn mn cd 6–5,teregion the 767–852, acids amino (lacking PC–GFP D YHGPitrce ihSf nbt cytoplasmic both in Sufu with interacted SYGH–GFP . usiuin ipae rae transcriptional greater displayed substitutions R D L–F,lcigaioais715–725; acids amino lacking NLS–GFP, hnfsdt F L ta. 2012). al., et (Li GFP to fused when nvitro in . . –F osse iia ciliary similar possessed R–GFP –F GiK7R K398R, (Gli2K375R, R–GFP D Cxe l,21;Lee al., et Lee 2010; al., et (Cox CGP ugsigthat suggesting PC–GFP, D NES–GFP hte ula xotdrcl eitsGi xtfrom might exit exit B ciliary the Gli2 affect leptomycin Gli2. that interactors of and mediates Gli2 of distribution Exportin1 elucidated the directly affect Alternatively, be cilium. export to interaction an the remains nuclear detect it to Exportin1, whether unable and were export Gli2–GFP we ciliary between Gli2 as in However, role 4E). a (Fig. play construct, might siRNA Exportin1 the on that increased depending suggesting degree Gli2–GFP Exportin1 varying nuclear a of in to Gli2 increase inhibition ciliary subtle siRNA-mediated a as 4C,D). well (Fig. as Gli2 endogenous tip, and ciliary Gli2–GFP the treatment of at intensity B pixel leptomycin increased stimulation, to Hh led to similar Exportin1. Gli2–GFP that, of inhibitor found differentiated an We B, treated leptomycin with we fibroblasts and export, ESCs nuclear to Gli2 similarly for required not are NESs export. predicted nuclear these that suggesting Gli2 euaeGi rncitoa ciiy eit neato with interaction mediate that activity, is regions transcriptional Gli2 the Gli2 of particularly majority localization, regulate the ciliary that for demonstrated dispensable analyses our efficient Because for necessary is localization Gli2 ciliary of region central A Fg AB.Smlry mle ecnaeo Gli2 of percentage smaller Gli2–GFP a displayed Similarly, that 5A,B). percentage Gli2 (Fig. displayed the cilia to of compared percentage 1183–GFP small very a 1999). contrast, al., By et (Sasaki previously domain activation region secondary a that a 852–1183), the as allele and acids described an cluster (amino created phosphorylation domain also the activation We between 2007). region Wang, the and spans removed and domain which (Pan finger 562–707), PDD zinc acids the a the (amino created between cluster localization we region phosphorylation the ciliary protein, the to the lacks for Specifically, that of domains. allele portions required ciliary finger remaining zinc Gli2 for the or screen of activation required the region of is outside a that sought we Gli2 Gli2 of that Gli2 we region Given modifications, a post-translational localization. known for of sites searched are or Sufu oaiainrgo aioais8218)nrC ue othe ciliary to in fused Gli2 expressed Ci the nor when Neither 852–1183) 2010). cilium acids (amino al., the region et to localization (Zeng localize cells ciliary not mammalian to fused the direct does or the Ci, to Ci. GFP proteins, of to whether sufficient half fused Gli2- N-terminal domain assess is the this to expressed region To we Similar localization localization, S4B). deleted ciliary Gli3. Fig. we Gli2 human proteins, material Gli of (supplementary Gli3 other region of CLD–GFP, homologous localization ciliary the the in defective defective’. localization exhibit ‘ciliary for Gli2 not Gli2-CLD–GFP, renamed as did we Therefore, sub-deletions localization. ciliary these However, Gli2 S4A). and Gli2 alleles GFP Floxin localization, created ciliary mediates we domain Gli2 smaller a ciliary whether localizationdetermine of ciliary steady- levels in the that increase state indicating Gli2– significant, fold not to was The compared treatment 5B). SAG as upon Fig. SAG, 1E; to (Fig. response in GFP cilia to localized GFP ofrhrts hte h iir xto l2cudfunction could Gli2 of exit ciliary the whether test further To Gli2 oass hte h iir oaiainrgo participates region localization ciliary the whether assess To D D E–F i o xii nihdncerlocalization, nuclear enriched exhibit not did NES–GFP FGPrahteclu,btta l27–F osnot, does Gli2-78–GFP that but cilium, the reach ZF–GFP D ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal 6-0–F oaie oteclu outy(i.5A). (Fig. robustly cilium the to localized 562-707–GFP D 0518–F splmnaymtra Fig. material (supplementary 1045-1183–GFP D 1-23GPfie orahtecilium the reach to failed 912-1223–GFP D 5-13GPaedcesd To decreased. are 852-1183–GFP D D e–F,Gli2 Rep–GFP, 5-4–F,Gli2 852-940–GFP, Drosophila ooo fteGli the of homolog D D 852-1183–GFP c–F and Act–GFP D D 941-1044– 852-1183– D 1505 852- Gli2

Journal of Cell Science EERHARTICLE RESEARCH 1506 type Gli2-CLD–GFP assessed we expression, diminished to Gli2 due not 5C). was cilia (Fig. to targeting localize ciliary that for indicating sufficient cilia, not is to region localized this region localization ciliary Gli2 exit. ciliary Gli2 in role a play might signals export nuclear but Gli2 of localization ciliary mediate not do signals (A) import Nuclear 4. Fig. el eesandfrcla(cu) F rGi t akGi–F nEC n l2i Es n ien(omr h aa oy.()Qatfcto fpa of Quantification (D) body). basal the mark (to differentiated Ninein for and MEFs) assessed in are Gli2 Gli2 and and ESCs GFP in Gli2–GFP for mark intensities (to pixel Gli2 the or GFP where (AcTub), C cilia for stained were Cells on htdlto fteclaylclzto eini Gli3 in region localization ciliary S4C). the Fig. material (supplementary turnover of its abrogated similarly deletion we possibility, Wang this that with 2006; Consistent 2009). found al., al., et et Zhang Marcotullio 2010; al., ubiquitin (Di might et turnover region E3 protein this the suggesting Gli for respectively, promote region motifs SPOP, localization binding and ciliary Itch contain ligases Gli2 to the shown and Gli1 been to of have homologous regions are The 5D,E). that (Fig. Gli3 Gli2 wild-type of those than l2GP(T.+dntslws ie nest,wees++ eoe ihs ie nest.* intensity. pixel highest denotes ++++ whereas intensity, pixel lowest (C) denotes + (WT). Gli2–GFP 5 bar: Scale -eodepsr;mdl ae,3-eodepsr) iir l2pxlitniywsasse nclssoigdmnse p1immunofluoresce Xpo1 diminished (Ac showing cilia cells for in stained assessed were was ( and intensity levels (sc), pixel Xpo1 control Gli2 scramble Ciliary normal a exposure). and or 30-second (si1,2,3) panel, Exportin1 middle 10 against exposure; bar: 5-second siRNA Scale with hours (red). 72 Xpo1 for and transfected were fibroblasts NIH3T3 (E) oesr httefiueo l2CDGPt efficiently to Gli2-CLD–GFP of failure the that ensure To Gli2 Gli2 and Gli2 Gli2 Gli2–GFP/+ GFP D n h rti eeso l2CDGPwr greater were Gli2-CLD–GFP of levels protein the and , NLS–GFP/+ m .()Teclaylclzto rvlneadpxlitniyo Gli2 of intensity pixel and prevalence localization ciliary The (B) m. xrsin swl spoenlvl yimmunoblot. by levels protein as well as expression, SsadMF eetetdwt MO A (1 SAG DMSO, with treated were MEFs and ESCs RAlvl eecmaal otoeo wild- of those to comparable were levels mRNA and 2 Gli2 D treidpnetexperiments, independent (three KD) Gli2 m .Tekokonefcec a loasse ya p1adtblnimnbo ftettlcl yae(o panel, (top lysate cell total the of immunoblot tubulin and Xpo1 an by assessed also was efficiency knockdown The m. D NES–GFP/+ Sswr tie o ii ATb e)adGP(nesso h niiulclu niae ywiearrowheads). white by indicated cilium individual the show (insets GFP and red) (AcTub, cilia for stained were ESCs n 5 m 0prcniin.* condition). per 50 ,1hu)adLpoyi LB 0n,9 iue) nihbtro xotn (Xpo1). Exportin1 of inhibitor an minutes), 90 nM, 20 (LMB, B Leptomycin and hour) 1 M, Gli2 D oes ndfeetae Ss l2CDGPpartially Gli2-CLD–GFP ESCs, the differentiated tested we In cell Therefore, different two models. expression. using Gli2-CLD–GFP will of gene activity Gli2-CLD–GFP transcriptional target that activate predicts not activation transcriptional its role localization. its ciliary of independently promoting turnover in Kif3a localization protein promotes ciliary without Gli2 the of and of Thus, region with Levels S4D). cells Fig. S4D). in material Fig. (supplementary equivalent material also Gli2-CLD–GFP were (supplementary similar Kif3a, Gli2–GFP cells Gli2–GFP, of wild-type ciliated absence ciliogenesis to to for compared the stabilized required In was is 2008). in protein that al., Gli2-CLD–GFP protein et and a (Corbit Gli2–GFP Kif3a, expressed lacking we MEFs cilia, reach to L–F ( NLS–GFP Gli2–GFP/+ h yohssta l2lclzto oclai rca for crucial is cilia to localization Gli2 that hypothesis The failure its by stabilized was Gli2-CLD–GFP whether assess To P , ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal .5(h-qae test). (Chi-squared 0.05 SsadMF,rsetvl.* respectively. MEFs, and ESCs D L)adGli2 and NLS) P , .5(h-qae et scmae oWT). to compared as test, (Chi-squared 0.05 D E–F ( NES–GFP D P E)wr oprdt hs of those to compared were NES) , .5(h-qae test). (Chi-squared 0.05 u,green) Tub, c (+KD) nce nel

Journal of Cell Science EERHARTICLE RESEARCH anr norsuy eue h lxnsse oexpress to system Hh-responsive Floxin a the in used we genes study, target our activates In or manner. represses induce Gli2 to is factors DISCUSSION signals. Gli2 transcription Hh localization Gli for to response of ciliary crucial in finding ability genes also that target and the The is for suggests Gli2 5F). domain essential (Fig. of activity localization repressor domain ciliary transcriptional constitutive the activator a that C-terminal as Gli2 transcriptional the of functions activity lacks Gli2 dominant-negative the Gli2 which inhibits to wild-type In similar Gli2-CLD–GFP 5F). with activity, (Fig. that Gli2-CLD–GFP negative dominant of confirmed a as co-expression act MEFs, might genes it target that Hh indicating the of expression the suppressed activity. transcriptional and stabilization localization, ciliary for necessary is Gli2 of region central The 5. Fig. xrsinwsnraie ota of that to normalized was Expression ii ATb e)adete F rMc(re) nesdpc l2GPo ooosa h niiulclaidctdb rohas cl a:5 bar: Scale arrowheads. by indicated cilia individual the s at and homologs cells NIH3T3 or into Gli2–GFP transfected depict transiently were Insets Gli2) (green). of Myc half C-terminal or the GFP and Ci either (D) of and half red) N-terminal the (AcTub, of cilia fusion (a 5 Ci–Gli2 bar: and Scale Ci–Myc CLD–GFP, localization. ciliary of lack depict arrowheads red whereas rti ees (E) levels. protein lte o F n uu l2CDGPi tblzdcmae owl-yeGi–F.Gi-L–F neat ihSf.()Lf n ide differe middle: and Left (F) Sufu. with interacts Gli2-CLD–GFP Gli2–GFP. wild-type to compared stabilized is Gli2-CLD–GFP Sufu. Gli2 and GFP for blotted fSGo eil oto htdslyclaylclzto ffl-eghGi–F W)o l2CDGP(L) * (CLD). Gli2-CLD–GFP or (WT) Gli2–GFP full-length of localization ciliary display that control vehicle or SAG of of xrsino l2CDGPi ihrEC rMF nefrswt l-eedn rncito.* transcription. Gli-dependent with interferes MEFs or ESCs either in Gli2-CLD–GFP of expression Gli2 Gli2 Gli2 Rev/+ Gli2-CLD–GFP/+ Gli2–GFP/+ Rev/+ (Rev), (Rev), Gli2 and lcigaioais8218)EC eesandfrcla(cu,rd n F gen;wiearwed eitclaylocalization, ciliary depict arrowheads white (green); GFP and red) (AcTub, cilia for stained were ESCs 852–1183) acids amino (lacking Gli2 Gli2–GFP/+ Gli2 Gli2 Gli2–GFP/+ Gli2–GFP/+ Gli2-CLD–GFP/+ n Gli2 and and W)and (WT) Gli2 b Gli2-CLD–GFP/+ atn ih:teefc fGi n l2mtn rti xrsinin expression protein Gli2-mutant and Gli2 of effect the Right: -actin. Sswspoe o F n uui.Teasneo h iir oaiainrgo osntrdc l2mN or mRNA Gli2 reduce not does region localization ciliary the of absence The tubulin. and GFP for probed was ESCs Gli2-CLD–GFP/+ Gli2 Gli2-CLD–GFP/+ Sswr sae for assayed were ESCs S yae eebotdfrGPadtbln rwr muorcpttdwt niGPo niSf and anti-Sufu or anti-GFP with immunoprecipitated were or tubulin, and GFP for blotted were lysates ESC Gli1 CD Sswr sesdfor assessed were ESCs (CLD) and Ptch1 D Act, m .()Tepretg fEC rdfeetae el df)i h presence the in (diff) cells differentiated or ESCs of percentage The (B) m. , Gli1 and 00.Hwvr etse h oaiaino Gli2 of localization the tested we However, al., et 2010). (Zeng is cilia a to that localize identified to 570–967) GFP–Gli2 overexpressed al. acids for required (amino et region Zeng central localization. overlapping ciliary partially for sufficient, not but an is cilium primary activation. the Gli2 ciliary at in Gli2 for step of required important regulation a the is support which findings that in These model Gli2 activity. transcriptional within and domain approach, localization a this Using precisely identified S2D). to we Fig. combination, material of subcellular of (supplementary in control function tailoring Gli2 and the genetic alone studying investigate systematic residues, of the and domains allowed means Floxin tractable dynamics. genetically the from a Gli2 of forms GFP-tagged eta eino l2(mn cd 5–13 snecessary, is 852–1183) acids (amino Gli2 of region central A Ptch1 ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal xrsinlvl olwn 8huso A treatment. SAG of hours 18 following levels expression Gli2 and P , GFP .5(Student’s 0.05 Smo xrsin omlzdto normalized expression, 2 / 2 P , Eso l–uieaerpre ciiy The activity. reporter Gli–luciferase on MEFs .5(Student’s 0.05 (A) Gli2 t ts scmae oWT). to compared as -test Gli2 ne noeosregulatory endogenous under Gli2–GFP/+ Gli2 ou nEC,creating ESCs, in locus t ts) C l2GP Gli2- Gli2–GFP, (C) -test). , b Gli2 atn hl ellysate cell Whole -actin. Gli2 D D 562-707–GFP, 562-707–GFP/+ m andfor tained m. ntiated 1507 and

Journal of Cell Science lentvl,bcueGi-L–F ol edtce tavery a at detected GFP–Gli2. overexpressed be examined could 2010) Gli2-CLD–GFP because al., whereasAlternatively, expressed et elements, (Zeng Gli2–GFP regulatory al. endogenous examined et its Zeng studies of control our the as differences under or levels, tag GFP protein the of locations in different the observations to our due in be Differences could deletions. individual three all localization ciliary with intact observed and system Floxin the using GFP Gli2 ARTICLE RESEARCH PPmdae erdto ih con o h decreased the 1508 for 2010), account al., et might and (Wang SPOP stability degradation Because Gli2 2010). on SPOP-mediated al., effects et protein opposing Gli2 Wang have consistent decreased 2009; Sufu to al., destabilized, leads et Sufu (Chen was of levels loss and the that Gli2 findings with full-length to nuclear N-terminal important compared an inhibit Gli2 lacked the each site, that For protein site Sufu-binding Gli2 domain, behavior. Sufu-binding the Notably, a its activator accumulation. and of domain other C-terminal aspects protein, repressor the the important of other activity example, and control localization ciliary regions the in role other or domain repressor its to cilium of mechanisms. the inhibition be induce will through through signals It Gli2 Hh absolute. activate requirement whether not repressor determine is to ciliary N-terminal requirement interesting this the that the of indicates idea deletion domain circumvent the through with or to However, overexpression consistent activation. inhibits ability Gli2 is Wong for Gli2-CLD–GFP genes 2002; required the is that al., target localization finding et Hh ciliary that our Sheng of the 2009; in expression Thus, even al., the 2009). targets et an al., Hh (Han Similarly, et activate cilia cells. can of non-ciliated Gli2 absence and in of the ciliated wild- version in genes both overexpressing oncogenic cilia by in target for circumvented Gli2 be requirement Gli2-CLD–GFP Gli type can the Gli2 Smo, of that of Unlike activation activation MEFs. found and the we ESCs inhibited activity, dominantly 2006; this transcriptional al., et and for Pan 2009; Gli3 localization al., and ciliary et 2009). Chen al., central Gli2 2006; et the Zhang al., of et require (Bhatia C-termini also region the might of interaction interacts portions ligases, ubiquitin Cul3-based protein with to the proteins nuclear-speckle-associated Gli recruits within a Itch-dependent that SPOP, are as function The Similarly, Gli2 might 2006). degrons. and in al., region, et found localization Marcotullio ciliary motifs (Di proline-rich ubiquitin turnover within its homologous E3 motifs promote the proline-rich to recognizes in with Itch Gli1 SPOP. role interaction and through its Itch ligases are of could domain Gli2 independently this which destabilize stability by mechanisms Two protein domain localization. central ciliary reduces the we cilia, Gli2 of cilia, absence of the reach by unaffected to Gli2-CLD–GFP were and levels failure protein Gli2–GFP a Because its Kif3a, ciliogenesis. for lacking to required MEFs in due the levels was Gli2-CLD–GFP whether assessed ascertain Gli2 To of Gli2–GFP. stabilization wild-type than stable more cilia. to retention entry or Gli2 promote to domain localization ciliary the with cooperate might the domains repressor N-terminal that the suggesting and cluster phosphorylation localization, ciliary diminished exhibited both Gli2 cilia. to Gli2 facilitate of might localization domains the additional tips, ciliary of percentage small nadto ofiigrahteclu,Gi-L–F was Gli2-CLD–GFP cilium, the reach failing to addition In lhuhteclaylclzto eino l2pasacrucial a plays Gli2 of region localization ciliary the Although required is localization ciliary whether investigating While D CGP(akn mn cd 6–5)adGli2 and 767–852) acids amino (lacking PC–GFP D YH a nihdi h ulu as nucleus the in enriched was SYGH, D CGPadGli2 and PC–GFP D D Rep–GFP 852-940– 8husfrtasrpinlasy r1hu o oaiainassays. minutes. 90 localization for for nM 20 hour at 1 used was or L2913) assays (Sigma B transcriptional Leptomycin for hours 18 ocntanGi ucini h bec fH pathway determine Hh to of interesting be absence will the It 2012). in al., function et (Li Gli2 non- activation constrain the cilia to to localize Because can Ubc-9 activity in enzyme localization. Sumo-conjugating transcriptional E2 the increased ciliary and displays for mutant a sumoylatable dispensable Because sumoylation cilia, localization. to localizes is ciliary still Gli2 affect of form not non-sumoylatable did sites sumoylation transcriptional Gli2 restrict 2004). might site motif a activity. al., SYGH through the et binding from Sufu distinct Merchant transcription, misactivating 2002; transcriptional without Bishop, their Gli2 and suppresses Because inhibits Gli2 (Cheng and Sufu in proteins, localization activity plays Gli Sufu nuclear stabilizing that to their roles addition multiple In the regulation. with correlate might or other directly but either interaction, Gli2 Sufu–Gli indirectly. the of to stabilization contribute Sufu- also for and sites important exclusion is motif and nuclear cytoplasm SYGH the mediated the in Therefore, both nucleus. Sufu the with in interact to ability its retained Gli2 of stability ihclaylclzto en rca n seta tpi the in step essential consistent Gli2. and are of crucial activation a findings Hh-induced being These essential localization also ciliary activity. is with and The transcriptional Gli3 regulation. and Gli2 Gli2 the for this for both necessary of whether for is localization identified essential clear ciliary have the we is been that cilia not region localization to has ciliary Gli functions it repressor of factors, and localization transcription activator the Gli or modulate of to directly signals needed Hh Exportin1 for is Gli2. of of work exit inhibition ciliary further the the regulates Thus, whether indirectly Exportin1. elucidate interaction Both and an Exportin1 to GFP. detect not Gli2 of ciliary did we knockdown between of but intensity phenotype, siRNA-mediated led this the signals and recapitulated in export B nuclear increase of leptomycin putative detectable localization three a ciliary of to the deletion control The trafficking Gli2. nuclear to similar of de-sumoylation inducing cilia. by at Gli2 Gli2 activate signals Hh whether eeclue n01 eai nkoku MM(Invitrogen) 100 DMEM pyruvate, mM knockout 1 glutamine, in mM gelatin 1 FBS, m 0.1% 10% with on supplemented cultured were etr nfaewt h -emnlGPtg ukhneI XL-site II QuikChange tag. GFP C-terminal the with frame in vector, Gli2 cloning and constructs cDNA culture Gli2 cell and lines Cell METHODS AND MATERIALS itfo .Tiae n I33clswr utrdi MMH21 DMEM in cultured were 50 cells and FBS NIH3T3 10% and with Taipale) supplemented J. from gift mrodbde nteasneo I o ih days. eight for LIF of absence the in bodies grown were embryoid lines cell Floxin 150 (LIF). factor in inhibitory leukemia U/ml 1000 o-seta mn cd,100 acids, amino non-essential M A Ez)wsaddt rs eima 1 at medium fresh to added was (Enzo) SAG oehtsrrsnl,mttn l2popoyainand phosphorylation Gli2 mutating surprisingly, Somewhat Sufu with Gli2 of interaction the mediating sites Multiple lhuhi a rvosybe vdn htclaaerequired are cilia that evident been previously has it Although mechanisms that possibility intriguing the explored also We .elegans C. gt/+ xn –4wr mlfe n lndit h Foi shuttle pFloxin the into cloned and amplified were 2–14 exons m X05 1 S ie eeotie rmByeois Cells BayGenomics. from obtained were lines ESC E14 (XG045) ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal /lG1.Dfeetae el eegona ooae ras or monolayer a as grown were cells Differentiated G418. g/ml ti osbeta uolto cusa h cilium the at occurs sumoylation that possible is it , D YHGPsosicesdncerlocalization nuclear increased shows SYGH–GFP D YH ept t etblzto,Gli2 destabilization, its Despite SYGH. m m M /lpenicillin/streptomycin. g/ml b mratehnl(M)and (BME) -mercaptoethanol m nDS for DMSO in M Smo 2 / 2 D Es(a MEFs SYGH

Journal of Cell Science E o 8huswt rwtotSG h el eeasydusing assayed Student’s was were unpaired significance Opti- cells Statistical the The in (Promega). using SAG. 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RNeasy Synthesis using synthesis cells cDNA differentiated or (Qiagen). ESCs from extracted was RNA PCR Quantitative hta . hygrjn . lhv,I,Sle,M,Duoz . uha,H. Mukhtar, A., Dlugosz, M., Saleem, I., Elcheva, S., Thiyagarajan, S. N., Pons, Bhatia, and R. Alvarez-Rodriguez, A., Menendez, J., Berenguer, M., Barzi, C. C. Hui, and M. Yoshida, V., Thanabalasingham, X., Zhang, C., P. Barnfield, Ramı P., Aza-Blanc, W., Salvenmoser, A., Trinh, J., P. Scherz, C., K. Corbit, N., Santos, P., Aanstad, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139253/-/DC1 online available material Supplementary months. 12 material after Supplementary release for PMC Family in Sandler Deposited the J.F.R. and to Foundation; Foundation the Packard Supporting the and the R01GM095941]; N.S., Fund; R01AR054396, to Wellcome numbers Foundation Burroughs [grant Science Health National of the Institutes by National supported was work This Funding the wrote J.R. and N.S. analysis. data and manuscript. experiments the performed N.S. contributions Author interests. competing no declare authors The interests Competing reagents. for and Francisco) Ingraham San Holly (UC (Genentech), Scales Kornberg Jussi Jonathan Tom Suzie thank Curie), Georgia), We (Institut of discussions. (Univ. Sillibourne helpful Eggenshwiler James for Helsinki), Lab of Reiter the (University Taipale of members the thank We Acknowledgements MAPad2 and ATP mM 2 ecin eete eovdb .%SSPG n eetdby detected and SDS-PAGE 7.5% by resolved immunoblot. then were Reactions otiig5 MTi-C H80 0 MNC,1 MMgCl mM 10 NaCl, mM 100 8.0, pH Tris-HCl mM 50 containing 9 erdto ybt-rPuiutnligase. ubiquitin S. beta-TrCP by V. degradation Spiegelman, and PKA base. of cilium localization the the to requires proliferation Sonic-hedgehog-mediated (2010). of Suppressor by function mechanisms. activator multiple Gli2 and through Gli1 fused of regulation Negative (2005). 1043-1053. Hh high-level for required is and localization signaling. ciliary Y. regulates D. Stainier, Smoothened and F. J. Reiter, J., Huisken, nerpu rti otencesadcnet tt repressor. a to it converts and nucleus the to protein interruptus B. T. n thR 5 Ptch1R, and 19) rtoyi hti niie yhdeo agt Cubitus targets hedgehog by inhibited is that Proteolysis (1997). ora fCl cec 21)17 5011 doi:10.1242/jcs.139253 1500–1510 127, (2014) Science Cell of Journal uolto assays sumoylation 9 uolto fimnpeiiae l2GPwsperformed was Gli2–GFP immunoprecipitated of sumoylation -GGACATGCACATCATTACGC-3 m t ts rANOVA. or -test ur Biol. Curr. 1 4 E1, M 9 l2,5 Gli2F, ; 9 ttsia infcnewsasse sn h unpaired the using assessed was significance Statistical . b rzWbr .A,Lgt .P,Shat,C n Kornberg, and C. Schwartz, P., M. Laget, A., F. ´rez-Weber, atn rmrsqecsare: sequences Primer -actin. 9 m .Cl Sci. Cell J. -TGCCGCAGTTCTTTTGAATG-3 m rs ihohetl(T)a 37 at (DTT) dithiothreitol fresh M 19 b9ad10 and Ubc9 M 9 1034-1039. , thF 5 Ptch1F, ; 20) l2i agtdfruiutnto and ubiquitination for targeted is Gli2 (2006). 9 -GCTGCACCAAGAGGTACACA-3 123 9 9 n l1,5 Gli1R, and 62-69. , and 9 Differentiation -CTCTGGAGCAGATTTCCAAGG- 9 n fR 5 gfpR, and m b uo nasmyainbuffer sumoylation a in Sumo1 M ciR 5 actinR, .Bo.Chem. Biol. J. 20) h xrclua oanof domain extracellular The (2009). 9 Xpo1 l1,5 Gli1F, ; 73 9 -GTGCCAATCCGGT- 9 9 -GAACTCCAGCAG- 397-405. , -CGTCATCCATGG- b iN (Invitrogen, siRNA ciF 5 actinF, 281 9 ˚ n fF 5 gfpF, and ; o . hours. 1.5 for C 9 19320-19326. , -GGTGCTGC- 9 -CACA- Cell 9 1509 and 89 9 2 - , ,

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