ciainrcut h rncito ntaincmlxt induce Notch transcription the to with to signaling complex machinery. Notch response initiation coupling , transcription in target Notch the E(y)1/TAF9 recruits with activation NICD–Su(H)– the complex of association the Mastermind Taken that NICD. the suggest and data with Su(H) our together, signaling interacts Notch of physically discs. effectors E(y)1/TAF9 transcriptional imaginal that E(y)1/TAF9 S2 wing demonstrated in that studies cells Biochemical and indicated cleavage. tissues Notch cells of two reporter downstream functions follicle these activity in and the analyses Epistatic both target in in defects expression and phenotypes like Atosfrcrepnec riosn.b.cc;[email protected]) ([email protected]; correspondence for *Authors Beijing 15, Road Datun Sciences, of China. Academy 100101, Chinese the Biophysics, of 20-25 USA. 32304-4295, egin Xie Gengqiang signaling Notch of output in transcriptional the mediates E(y)1/TAF9 ARTICLE RESEARCH ß 3830 2014 June 23 Accepted 2014; April 4 Received stem embryonic mouse 1 and Pointud zebrafish both 2007; in al., and, et 2003), (Cheng al., differentiation et required germ-cell specifically is male and TAF7L for (Goodrich 2006). mouse example, such adipogenesis some For events, 2010). Chiang, and specific that Tjian, in spermatogenesis suggest vital and apoptosis, are studies species as Thomas different of cells in number found 2010; TAFs increasing eukaryotic an Tjian, all However, RNA- and for in required (Goodrich be transcription to the thought generally in polymerase-II-initiated were components role complex all this and essential complex, of 14 expression, an gene or of (TFIID) plays 13 regulation transcriptional and (TAFs), IID (TBP) factors factor TBP-associated box-binding TATA transcription the comprising eukaryotic The INTRODUCTION regulation Transcriptional pathway, complex, Notch TAF9, E(y)1, WORDS: KEY a of in in Knockdown involved cells. transition are follicle that mitotic-to-endocycle genes for Notch-dependent large-scale screen a (RNAi) in interference E(y)1/TAF9 the RNA identified the We of of signaling. output with component transcriptional Notch specifically the a facilitate interacts to E(y)1/TAF9, complex complex, NICD–Su(H)–Mam that TFIID Mastermind factor report coactivator transcription we and Here, mammals] of Suppressor in (Mam). protein RPBJ DNA-binding [Su(H), (NICD), Hairless receptor domain the Notch intracellular the the requires involves of that targets complex ternary signaling a of Notch formation of activation Transcriptional ABSTRACT eateto ilgclSine lrd tt nvriy alhse,FL Tallahassee, University, State Florida Science, Biological of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,33–89doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Drosophila 2 tt e aoaoyo ri n ontv cec,Institute Science, Cognitive and Brain of Laboratory Key State 1 hnsegYu Zhongsheng , e(y)1/TAF9 2 displayed oguJia Dongyu , Drosophila Notch TFIID , Drosophila 1 -mutant- ejeJiao Renjie , nvivo in lhuharcn eothssonta A4itrcswith interacts of signaling, TAF4 transcription (Wg) that Wingless induce 2010). of shown to al., activator family has transcriptional Integrin et a report Pygopus, AP-1 (Kalogeropoulou of recent progression a other expression Although cancer the and of regulate context c-Jun is to TAF4b with that members demonstrated associated have al. specifically et Kalogeropoulou 2009). pce htpasapvtlrl nmn ifrn developmental different many in role pivotal a plays that species understood. poorly Notch, is as TAFs such specific pathways, by signaling regulated other are how 2009), Tjian, and for essential is TAF3 and TRF3 (Ba between hematopoiesis interaction cells, atr(LF iecn opee omdaeepigenetic mediate to target Notch complexes RPD3-LID-associated the silencing at and silencing (RLAF) (LAF) associated factor differentially flies, factor In are 2013). LID-associated Nap1 al., et and and with Zeng Asf1 2013; Domanitskaya al., chaperones et 2005; histone Yu al., 2011; al., et et Mulligan epigenetically- (Bray genes expression Schu chromatin-associated target proper Notch for important of that very are mechanisms their regulatory suggested systems regulates different in complex findings have Recent unclear. ternary largely is 1995). Su(H)–NICD–Mam expression stages, in developmental the Schweisguth, identified different been during how organs and have or genes tissues Lecourtois target different to Notch of 1995; numerous (Bailey (Mam) by Although complex Suppressor Mastermind Posakony, ternary genes coactivator Su(H)–NICD–Mam protein and target functional the a DNA-binding its and the form to [Su(H)] the of translocated Hairless transcription with then (NICD) activates is interacting domain which and intracellular 1998), an Notch nucleus Adachi, the Such that of and 1996). processes release (Struhl al., proteolytic the in et consecutive result (Diaz- two Doherty cell induces 1995; neighboring the interaction Cohen, on ligand Notch signaling and its the and Notch between Benjumea cell interaction Artavanis- one 2014). direct in a and al., receptor by mediated (Louvi et is cancer Ntziachristos activation including 2012; a in diseases, Tsakonas, cell implicated is of Notch al., of of cells Dysregulation number et 2009). stem al., control Guruharsha et of Tien 2010; 2012; maintenance determination, Muskavitch, the and and fate (Artavanis-Tsakonas apoptosis, cell and proliferation including events, ai usin fhwteS()NC–a ope activates complex CAF-1 Su(H)–NICD–Mam target the how chaperone Notch of questions histone regulates basic in positively the expression and gene that epigenetically demonstrated complex and has mammalian study both complex in co-repressor expression SIRT1–LSD1 Drosophila gene target A Notch 2009). regulates al., et (Moshkin oc inln sa vltoal osre aha across pathway conserved evolutionally an is signaling Notch 1, pah 02 noe l,21;Kge n ae,2007; Nagel, and Kugler 2011; al., et Endo 2012; ¨pbach, n uMnDeng Wu-Min and * utrdcls(ulgne l,21) u eyrecent very Our 2011). al., et (Mulligan cells cultured ´ ta ta. 04 ate l,20;Hr tal., et Hart 2007; al., et Hart 2004; al., et rtfai Drosophila ae cuticle naked 1, * nacro Split of Enhancer Y ta. 03.Bttemost the But 2013). al., et (Yu in Drosophila [ E(spl) a nthe in 6 cluster ] (Wright

Journal of Cell Science oc inln nasailyrsrce anrb affecting by manner restricted spatially for promotes a Corepressor (CoREST) in signaling cofactor factor Notch transcriptional transcription the element-1-silencing al., et whereas (Heck epithelium 2012), follicular a in manner in restricted activity temporally Notch inhibits signaling SMRTER Notch co-repressor of transcriptional control Schu precise and transcriptional in Interestingly, (Domanitskaya involved 2011). in also al., by are Notch cofactors et signaling (Poulton of Notch cells of inhibition follicle pattern Delta-mediated temporal microRNA repressing the the that regulates demonstrated finding pathway recent signaling Our 2011). Notch al., is et of mechanisms Su(H) multiple Schu timing through and signaling precise (Domanitskaya regulated Notch The is mid-oogenesis switch. of during the effector for nuclear is needed the which addition, cleavage, cell In protein Notch-dependent Notch by this requires achieved processes, also other transition and in cycle Sun As the 2007; Deng, 2005). by and Deng, (Sun mediated signaling as known Notch also is through (Hnt; Hindsight Pebbled) which of activation Lo and endoreplication, Cut of 2001; suppression into the from al., cycle cells follicle et the mitotic induces activation (Deng This germline-expressed 2001). developmental (Dl) Johnston, the by Delta its epithelia ligand, is follicular signaling entire and Notch in 7–10A), Deng, activated signaling (stages and mid-oogenesis Klusza During 2008; Notch Johnston, 2011). St and of (Bastock consequences regulation the ta. 09 ie l,20)o hr ari Ns(ie al., et (Ni RNAs hairpin short al., or et 2008) Ni al., 2008; (Ni et hairpin al., Ni (ds) et 2009; double-strand Ni al., long 2009; either et al., contain and et which Project (Ni 2011), negative RNAi Deng, lines Transgenic and both RNAi thousand Sun two (TRiP) 2007; around with Deng, tested We and oogenesis, 2005). (Sun of identified targets 7–10A positive pattern activation stages Notch temporal during a shows epithelium cell follicle in genes target Notch signaling Notch the coupling machinery. transcription genes, that the target with Notch initiation reveal induce transcription the to data recruits complex activation Notch our E(y)1 to response with together, in complex NICD–Su(H)–Mastermind Taken signaling, the of Notch NICD. association of step. and effectors cleavage transcriptional Su(H) both Notch physically with the E(y)1 that interacts demonstrated of cells S2 downstream in suggested studies Biochemical wings functions adult E(y)1 and S2 cells that follicle cultured both genes in in target analyses and Epistatic Notch development of wing regulation during transcriptional the that for showed required studies Further cells. cycle follicle mitotic-to-endoreplicating of transition the mediating in signaling identified Notch we cells, signaling Notch follicle of in regulators for screen (RNAi) interference RNA in Schu acetylation and (Domanitskaya K16 cells H4 follicle histone the and tri-methylation K27 H3 histone h eea rncito ahnr r unanswered. to are it machinery bring cofactor(s) transcription which general and the genes target Notch of expression ARTICLE RESEARCH oietf ee htmdlt oc inln,w efre a performed we signaling, Notch large-scale modulate that genes identify To during endocycle to Drosophila cycle mitotic from transition e(y)1 RESULTS The srqie o oc-inln-eitdflil cell follicle Notch-signaling-mediated for required is Drosophila nvivo in oogenesis c sceaecmoet rsnlnadNicastrin. and Presenilin components -secretase Nisre o eet nteepeso of expression the in defects for screen RNAi g hme sa da ytmt study to system ideal an is chamber egg rspiamelanogaster Drosophila pah 02 eke l,21;Poulton 2012; al., et Heck 2012; ¨pbach, pah 02 eke l,21)–the – 2012) al., et Heck 2012; ¨pbach, e(y)1 sbigrqie o proper for required being as pah 02.I genetic a In 2012). ¨pbach, ´ e-ciradSt and pez-Schier olcecls The cells. follicle Drosophila e(y)1 Drosophila salso is cells. SnadDn,20) oneuaino n nmid-oogenesis in Hnt 7–10A of stages Downregulation from normally 2007). epithelium Deng, is follicular and a entire (Sun which Hnt, the target, protein in zinc-finger expressed Notch of follicle-cell-specific expression the positive Notch examined a we suggesting cells, 1A), (Fig. 7–8 2006), stages Chiang, in ( at defect 74% and clones in Thomas RNAi initiation expression cell Cut 1999; transcription prolonged TFIID al., in the resulted et of (Soldatov component complex core a gene the TAF9, of knockdown signaling. that Notch found [ in early we defect screen, a in this to suggests Failure From upon 7 2005). expressed stage 7 Deng, at and stage Cut normally (Sun downregulate at cells is follicle downregulated in then activation Cut Notch and negative 1–6) 2005). the (stages Deng, oogenesis of then factor and pattern transcription expression were homeodomain-containing (Sun a the clones Cut, and target in RNAi Notch (GFP defects follicle-cell for fluorescent The screened red respectively). and the green RFP, by of of marked were expression that control clones the RNAi the the follicle-cell to random generate crossed under to were RNAi) lines These Gal4 as flip-out (UAS). site to activating referred upstream (both 2011) aZi e C n fGPi e D,rsetvl,i akdydcesdin decreased markedly (blue). is respectively, DAPI (D), with red in GFP of e(y)1 and (C) red in lacZ (C–D 7. stage at chamber (B,B chamber. the egg in stage-7 suppressed a in of pathway RFP) signaling (expressing Notch the for cells. required follicle is E(y)1 1. Fig. signaling, nacro elw1 yellow of enhancer ocnimta () srqie o oc inln nfollicle in signaling Notch for required is E(y)1 that confirm To dpee olceclsi i-tg g hmes uliwr labeled were Nuclei chambers. egg mid-stage in cells follicle -depleted ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal E(spl)m e(y)1 (A,A rvr(t ta. 97 inn n iusy 1997) Zipursky, and Pignoni 1997; al., et (Ito driver dpee olcecells. follicle -depleted b e(y)1 9 u surgltdi the in upregulated is Cut ) -CD2 kokonflil el epesn F)o negg an of RFP) (expressing cells follicle -knockdown and 9 h xrsino w ietrpreso Notch of reporters direct two of expression The ) ( e(y)1 E(spl)m7-lacZ ] hc noe ooo of homolog a encodes which )], aee yteepeso of expression the by labeled , e(y)1 9 xrsino n is Hnt of Expression ) RA olcecells follicle -RNAi n 5 2 ffollicle of 72) Drosophila CG6474 3831

Journal of Cell Science Fg B.Bt u peuainadHtdwrglto were downregulation Hnt and independent upregulation an in detected Cut Both 1B). (Fig. eeae hog mrcs xiino -lmn hthad that P-element 5 a the into of inserted excision been imprecise through generated aeilFg 1;dt o shown). not data S1A; Fig. down knock material to used was that e(y)1 (NIG) Genetics of Institute National ( downregulation 68% Hnt in Indeed, in signaling 2007). detected Notch Deng, disrupted was and of (Sun indication cells an follicle is 7–10A) (stages ARTICLE RESEARCH 3832 E(spl)m7-lacZ Both reporters. transgenic CD2 using by manner direct more wild- 2B–C neighboring (Fig. cells. with knockdown of compared cells levels when downregulated 7–8 type and stages Cut during of Hnt levels upregulated showed by caused signaling n ol ersudt dlho yaduplication of a clones by mitotic cell adulthood target recognition follicle to Flippase-flippase both shown). (FLP-FRT)-induced not rescued was (data sequence stages allele be larval This early [ could during details). lethal and for homozygous and Methods hemizygous and Materials hntpsof phenotypes Dp(1;3)DC335 oc-nue rncito ciaincnb sesdi a in assessed be can activation transcription Notch-induced Notch on effects off-target potential exclude further To d ei ta. 98 urosadBa,20)and 2001) Bray, and Furriols 1998; al., et Celis (de xrsini i-tgdegcabr (supplementary chambers egg mid-staged in expression As-ui ta. 07 ie ta. 00,which 2010), al., et Pines 2007; al., et (Assa-Kunik e(y)1 or Dp(1;3)DC336 e(y)1 uat.A leenamed allele An mutants. n 0 9 5 ,spotn u idnsin findings our supporting ), URo the of -UTR e(y)1 1 of 51) RA,w e u oaayethe analyze to out set we -RNAi, RA ie(o 44-)fo the from 6474R-1) (no. line -RNAi e(y)1 abrn the harboring ] kokonflil cells follicle -knockdown e(y)1 ou Fg A see 2A; (Fig. locus e(y)1 e(y)1 E(spl)m genomic 190 e(y)1 e(y)1 was 190 b - Jae l,21) a lodcesdin decreased also was 2014), al., reporter et (Jia Notch the follicle-cell-specific in enhancer-lacZ reduced Indeed, another substantially of was activation. reporters cells Notch two follicle e(y)1 these in upon of expressed expression are mid-oogenesis sites, binding during Su(H) the contain olceclsrmie ntemttccce emntrdtwo monitored we (CycB) B cycle, Cyclin and mitotic H3 (supplementary histone phosphorylated the – switch markers in mitotic M/E remained cells the S2A follicle in Fig. failure material follicle a wild-type neighboring suggesting the cells, of those than nuclei distributed chambers egg of against mosaic RNAi Knockdown expressed in that markers nuclei clones mitotic examined follicle-cell containing of we expression event, and Notch-dependent 2001; size this al., for et required (Deng is mitotic mid-oogenesis the during from Lo transition) switch a M/E (endocycles) of cycles (the endoreplication induction to oogenesis the early is of divisions 7 stage during cell cells. follicle in suggest genes results target Notch These S1B). Fig. that material (supplementary cells ylsu osae6adaeasn uigedrpiainstages endoreplication during mitotic absent are early and in 6 oscillate stage to CycB up and cycles H3 chambers, egg wild-type histone In chambers. phosphorylated egg mosaic the staining by – ´ eeomna osqec fNthatvto nafollicle a in activation Notch of consequence developmental A e-ciradS ontn 01.T eemn whether determine To 2001). Johnston, St and pez-Schier e(y)1 RA olcecls(i.1,) nadto,teexpression the addition, In 1C,D). (Fig. cells follicle -RNAi ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal srqie o rprtasrpinlrglto fthe of regulation transcriptional proper for required is ( brE-lacZ e(y)1 9 .T eemn whether determine To ). eutdi mle n oedensely more and smaller in resulted ,rcnl dniidb u laboratory our by identified recently ), (B–C 5 indicate of regions coding the site of start transcription the denotes arrow black igmri yadpiainln arigthe carrying line normal e(y)1 duplication a a to by restored margin fully wing be edge can wing which defective (D), a has stages larval (B arrows Yellow green. in (B were Cut chambers for Egg stained and (B,C). RFP white of in absence outlined the by labeled clones i.2 eeaino an allele, of Generation 2. Fig. oc eet nflil el n adult and cells wings. follicle in defects Notch e(y)1 uliwr tie ihDP (blue) DAPI (B with stained Cell were suppressed. nuclei is normally Hnt is when Cut expressed which in cells, polar neto ie(ht rage n h deletion the and in triangle) (white site insertion the with locus, e(y)1 0 e(y)1 e(y)1 ,C 0 190 190 0 tg- g hmesbearing chambers egg Stage-7 ) eoi ein(E). region genomic .(B ). e(y)1 A eoi raiaino the of organization Genomic (A) 190 n t ieto.Bakbr indicate bars Black direction. its and oacwn htwsidcdat induced was that wing mosaic follicle-cell mutant homozygous 9 0 n 3 and - ,C bakdul ros.Tesingle The arrows). double (black 190 0 egdiae.(,)An (D,E) images. Merged ) n hrceiaino its of characterization and , p{GT}-BG00948 9 9 UR.Saebr 0 bps. 200 bar: Scale -UTRs. n n (C Hnt and ) e(y)1 e(y)1 dpee follicle -depleted e(y)1 9 e(y)1 ht bars white ; ,B 0 ,C,C 9 broad-early- ,bt shown both ), -knockdown ulmutant null P-element 0 on to point ) e(y)1 e(y)1 e(y)1 .

Journal of Cell Science xrse nteclso h oslvnrlbudr ftewing the of boundary dorsal–ventral the of cells the in expressed fNthtre ee ypTNthsol ecompromised be of should pMT-Notch perturbation for by a genes required expression by induced target is the Notch E(y)1 expression, which of gene that target Su(H), transient assuming Notch this with regulating system, In genes. interacts activation target Notch physically Notch of transcription and the nucleus activates the into irpinof in Disruption switch M/E Notch-dependent cells. Fig. the follicle material for (supplementary 2004), required that indicate 6 al., data these together, stage et Taken S2B,C). after Shcherbata expression 2001; random al., et in whereas (Deng 7–10A) (stages ARTICLE RESEARCH inln ntewn mgnlds,w nce down knocked we disc, tissue-specific a imaginal of wing use through the in signaling whether determine To pathway Notch e(y)1 poor very S3B,B displayed Fig. analysis material mosaic (supplementary Doherty FLP-FRT viability Because 1995; by Cohen, 1996). generated the the and al., on Serrate) between (Diaz-Benjumea or et (Delta interaction ligand cell its an and neighboring cell by one on boundary receptor activated Notch dorsal–ventral is pathway the signaling well-documented Notch at the two development, disc of wing expression genes, the target Notch examined we cells S2 tissues, cultured and whether disc assess imaginal To wing the in genes nefrn sNsagainst dsRNAs interfering h Niln osuyterl of role the study to line RNAi the euto nepeso ftepiayedgnu oc target Notch endogenous primary the genes of expression in reduction ( ciainsse nclue 2clsb rnfcigaNotch- Notch a artificially transfecting (Krejcı transient by is pMT-Notch cells well-documented signaling plasmid, S2 cultured expressing a Notch in system when employed activation genes we target induced, Notch of the posterior in disc. GFP-expressing wing those that the demonstrate with results in These e(y)1 compared 3K,L). Cut (Fig. as compartment or anterior decreased Wg were of compartment levels discs, lethality larval early circumvent To of development. wing during htEy1i lorqie o riiilyidcdNthsignaling cells. Notch induced S2 artificially cultured for in required also is E(y)1 that E(spl)m8 a ecevdt rdc osiuieyatv omo NICD of form active Notch constitutively full-length N a overexpressed by produce the (denoted to assay, cleaved this be In can 2013). al., et GFP- the (compare region). 3J) GFP in anterior (Fig. GFP-negative by expression the However, with marked Cut region in posterior disc or changes positive detectable 3I) wing show (Fig. not a did Wg UAS- discs controlled of control express The compartment to expression. temporally 2003) posterior al., the the et used (McGuire system we shown), dsGFP odtriewehrEy1i eesr o h transcription the for necessary is E(y)1 whether determine To engrailed-Gal4 sivle nteepeso fNthtre ee nthe in genes target Notch of expression the in involved is eeial neat ihcmoet fthe of components with interacts genetically E(spl)m7 ,it h oc-ciaigS el e oasignificant a to led cells S2 Notch-activating the into ), - HLH e(y)1 ICD e(y)1 epciey Fg DE.Teerslssuggest results These 5D,E). (Fig. respectively) , en-Gal4 and kokonflil el,bt akr showed markers both cells, follicle -knockdown pntetetwt DA N EDTA. with treatment upon ) e(y)1 wg E(spl)m8 fet h xrsino oc target Notch of expression the affects ( en-Gal4 e(y)1 and , e(y)1 lomdltsNthsgaigi other in signaling Notch modulates also e(y)1 UAS-GFP cut sepce,teitouto of introduction the expected, As . e(y)1 )-induced as nw as known (also sfntoal novdi Notch in involved functionally is ntewn mgnlds.During disc. imaginal wing the in , uatclsi h igdisc wing the in cells mutant Gal4 [ dse(y)1 ; driver, e(y)1 tub-Gal80ts ´ e(y)1 ,btntaantGFP against not but ], n ry 07 Yu 2007; Bray, and C96-Gal4 nNthsignaling Notch in E(spl)m7 RA dt not (data -RNAi e(y)1 9 ,w employed we ), ICD e(y)1- . Gal4 spotentially is e(y)1- translocates Drosophila - hc is which , HLH Niin RNAi - Gal80ts RNAi e(y)1 and agnls,and loss, margin of Knockdown (B) between development. interaction Genetic 3. Fig. D.An (D). ehd o eal.Tashtrzgtsof Trans-heterozygotes details. for Methods igmri between margin wing eet of defects G oprdwt 9oto 0 of heterozygous 101 when of notching out mild 49 showing with wings compared 81 (G, of out 12 with agnnthn ntedsa ein() obnto f[ of Combination (C). region distal RNAi the in notching margin hwdn iil igdfcs(aantson,weesitouigone introducing whereas shown), not (data of defects copy wing visible no showed 6 u f12i ess4 u f11i C). in 101 of out 49 versus E in 132 of out (64 to phenotype similar a oprmn) h ee fCtwsas rmtclydcesdi the gene. in that decreased against dramatically anterior also the was in Cut posterior level of control level internal The with compartment). compared reduced (K, obviously (green) was level GFP Wg from and (K,L) I) discs larvae. the (red, third-instar Wing the in Wg (I,J) in of right. J) patterns (red, the expression Cut to of normal posterior showed white and flies by left control separated the wild-type are to discs is wing anterior of lines; compartments Knockdown posterior (I–L) (H). and 82 Anterior of out 50 to of and increased enhanced, remarkably visibly was was phenotype penetrance margin wing notched the incorporated, e(y)1 +N e(y)1 e(y)1 e(y)1 nwn ic opoie h xrsino oc agtgenes. target Notch of expression the compromised discs wing in ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal 264-39/+ e(y)1 N dpee otro oprmn akdb h xrsinof expression the by marked compartment posterior -depleted 264-39/+ 190 uatall [ allele mutant RA ra() uesrp iatragn eoe RNAi denotes gene a after Ri Superscript (L). area -RNAi A idtp dl igwt omlwn margin. wing normal a with wing adult wild-type A (A) ,hwvr eutdi erycmlt igmri defects margin wing complete nearly in resulted however, ], into Notch e(y)1 F.(,)Snritcehneeto ocigo the of notching of enhancement Synergistic (G,H) (F). N N 264-39/+ Notch 1/+ eeoyosfis( flies heterozygous ne the under and e(y)1 eeoyoe C,wt h aepenetrance same the with (C), heterozygotes le usatal nacdtewn margin wing the enhanced substantially flies e(y)1 190 C96-Gal4 a eeae;seMtrasand Materials see generated; was ] N 190/+ 264/39 e(y)1 . N N .We oyof copy a When ). N 1 264-39/+ and rvrcue ata wing partial caused driver 264/39 sahypomorphic a is e(y)1 Notch /e(y)1 hwdvr idwing mild very showed ) 190 eeoyosflies heterozygous nwing in BG00948 C96-Gal4 e(y)1 Notch E showed (E) . 190 e(y)1 3833 allele, was -

Journal of Cell Science oce igmri hntp a loosre nmosaic in observed also was which in phenotype flies ( adult margin 96.9% wing in the loss notched to margin similar wing 3B), partial (Fig. to led also ocigi h itlrgo n4.%( of 48.5% Combination in region distal the in notching tgsb etsokidcdFp( ( Flp animals heat-shock-induced heterozygous by stages oprdwt i.3)adpntac icesn rm48.5% from [increasing penetrance and ( 3F 3E) (Fig. Fig. expressivity both with in compared phenotype notching margin wing the h oslvnrlbudr ed ols fwn agn (de margins wing of of and Depletion loss 1996). in (Gustafson al., to signaling et margin leads Notch Celis Defective wing boundary 2010). dorsal–ventral adult al., the et the Saj of 1996; Boulianne, precursor the disc, ARTICLE RESEARCH 3834 when development vein wing and bristle specification lateral in observed we defects Indeed, 2010). cell (Artavanis-Tsakonas Muskavitch, in inhibition role and lateral important an through plays determination in also fate formation signaling boundary Notch disc, dorsal–ventral wing to the addition In 3G,H). (Fig. although both (100%, expressivity of and terms penetrance in phenotype wing notched enhanced synergistically ipae yegsi neato ihanother with interaction synergistic a displayed of copy a of incorporation shown), not the (data defects wing visible eltd(upeetr aeilFg 3–) hs genetic during pathway These Notch that S3C–H). suggest Fig. experiments material (supplementary depleted n 5 e(y)1 3;Fg E o9%( 90% to 3E) Fig. 132; 190 e(y)1 uaininto mutation eeoyosfis[ flies heterozygous C96-Gal4 e(y)1 Drosophila N 190 Notch . 264-39/+ n Notch 5 e(y)1 e(y)1 admcoe eeidcda larval at induced were clones random 0 i.3).Similarly, 3F)]. Fig. 60; oso-ucinpeoye This phenotype. loss-of-function e(y)1 hwdaml igmargin wing mild a showed ) RA and -RNAi ucinlyitrcswt the with interacts functionally eeoyoe ral enhanced greatly heterozygotes igdevelopment. wing n e(y)1 5 n ne the under hsFLP 2 i.3) Furthermore, 3D). Fig. 32; 5 190/+ 0)o ig Fg 3C). (Fig. wings of 101) N Fg D.Notch 2D). (Fig. ) i o hwany show not did ] 264-39/+ C96-Gal4 n Notch 5 4 fwings of 64) e(y)1 eutdi a in resulted e(y)1 allele, 190 driver also was N 1 ciefr fNth N Notch, of form active odce psai nlssi olceclsb expressing of steps by different cells in we acting follicle components possibilities, in encode two that analyses these NICD–Su(H)–Mam transgenes core epistatic between the distinguish conducted by Notch mediated To of second, are control complex. pathway; transcriptional that Notch the genes the in target of engaged directly regulator is a of E(y)1 expression or transcriptional component the signaling for core Notch required a in is role E(y)1 its first, for – possibilities exist two Thus, 1991). (Dynlacht al., which expression et complex, gene polymerase-II-mediated initiation for transcription required is TFIID the of 2012). ternary component al., coactivator a et the of al., (Guruharsha and Su(H) formation Mam et protein through DNA-binding (Guruharsha the genes with NICD target complex activates the nucleus of the of to release NICD transcription cleaved the the and of in Translocation S2 2012). results receptor, al., Notch et which the Zeng S3, of 2013; two al., cleavages induces et proteolytic receptor post-translational Yu and consecutive ligand 2009; Notch al., Fortini between Interaction et 2006; receptor 2013). al., Tien et 2009; (Fischer Bilder, expression and and gene epigenetic target and of trafficking, ligand regulation vesicle and multiple endocytosis at including modification, regulated delicately is levels, pathway signaling Su(H) Notch and The signaling NICD Notch with of association output through transcriptional the mediates E(y)1 h oc aha.Teetasei osrcsicuea include constructs transgenic N Notch, These of form pathway. active membrane-tethered Notch the rti uH–P6cmrsn h N-idn oanof domain transcription-factor viral of DNA-binding domain activation strong the the and Su(H) comprising Su(H)–VP16 protein ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal aha nedccigflil el n adult and Notch cells wings. the follicle in endocycling E(y)1 in of pathway analyses Epistatic 4. Fig. oso u peuaincue by caused (D,D upregulation Cut stop to ht ie.Mdsaeegcabr eesandfor (A–D stained green were in chambers Cut by egg outlined Mid-stage and lines. A,B,C,D) white (red; the RFP by of (blue; marked expression DAPI were with clones stained Flip-out were A,B,C,D). nuclei Cell cells. mid-stage follicle in cleavage proteolytic Notch of downstream rvne u xrsini mid-stage in expression Cut prevented a eobndi the in recombined was uesrp iatragn eoe Niagainst RNAi denotes gene. gene that a after Ri Superscript by caused defect C96-Gal4 margin wing Su(H)–VP16 notched the of suppressed expression Ectopic (E–H) cells. follicle ihntegr el.(,,,)Mre itrsof pictures (A Merged channels; (A,B,C,D) three cells. germ detected the were within signals green punctate , upesdtewn agnls eutn from resulting loss Gal4 margin wing substantially the Su(H)–VP16 suppressed of by Co-expression caused (H) defects RNAi. margin wing suppress same not the did under NICD loss of typical expression A in (F) margin margins. wing wing of normal had flies adult i-tg g hme hwdCtupregulation. Cut (B–C showed chamber egg mid-stage (A,A staining. 9 . 9 nuto ftesrn uH–P6activator Su(H)–VP16 strong the of Induction ) xrsino N of Expression ) e(y)1-RNAi (A–D ICD -driven 9 Rbye l,19) n fusion a and 1993); al., et (Rebay 9 () euae oc signaling Notch regulates E(y)1 ) olcecl lnsof clones cell Follicle ) e(y)1 e(y)1 l ig eefo aeflies. male from were wings All . C96-Gal4 9 9 .Bcuea Because ). ,B 9 ,C Ni (E) RNAi. EXT hsFLP 9 uaieyecdsacore a encodes putatively ,D 9 (B,B . igecanlo Cut of channel Single ) e(y)1 cryn X -carrying EXT 9 )orN stau:GFP C96-Gal4 RA le.()Co- (G) flies. -RNAi constitutively a ; e(y)1 C96-Gal4 e(y)1 ICD e(y)1 (C,C depletion. RA na in -RNAi transgene control -RNAi 9 failed ) driver C96- e(y)1

Journal of Cell Science rdcdatrS laaeadN and cleavage S2 after produced elto.Hwvr fEy1i ietyivle nteexpression N the genes, in target involved Notch directly of is E(y)1 if However, depletion. N hwdCturglto Fg A.Smlrpretgso Cut of N percentages when Similar detected 4A). were (Fig. upregulation upregulation Cut ( 7–8 showed 76% stages During component oogenesis, analysis. or epistatic this of expression in gene readout a target as employed of level in the upregulation Cut at expression. is E(y)1 involvement the of certain of whether distinguish Expression of therefore E(y)1. can expression bypass constructs these might that Su(H)–VP16 speculate by complexes we targets Notch 2002), factor multiple Struhl, transcription with and interact general (Hall directly different can of addition, domain in components Because, activation 1998). VP16 al., target et the Notch (Kidd a Notch activate of can independently and Su(H) the than activator in transcription expression of gene target absence Notch activate might Su(H)–VP16 rti P6(ide l,19;Sy ta. 09.N 2009). al., et Shyu 1998; al., et (Kidd VP16 protein ARTICLE RESEARCH o h xrsino n ftencsaypoen,epeso of expression proteins, necessary the of required one N is E(y)1 of function if expression components cleavage, the S3 core for or cleavage and S2 either regulators of Notch upstream of majority the ID eepoe iia psai nlssfrtefnto of function the for analysis of epistatic release similar the employed of expression We upstream the components NICD. to core related or regulators not Notch probably of is (24%, signaling Notch Su(H)–VP16 in E(y)1 of co-expression from n by resulting reduced upregulation dramatically Cut contrast, By 5 ICD EXT 7 i.4) hs eut ugs htteivleetof involvement the that suggest results These 4D). Fig. 57; (70%, n/rN and/or n e(y)1 5 2 i.4)wsc-xrse with co-expressed was 4C) Fig. 82; ICD eas uH–P6i oepotent more a is Su(H)–VP16 because , ol ecetedfcscue by caused defects the rescue would n 5 EXT 6 of 66) n/rN and/or e(y)1 e(y)1 ICD EXT kokonflil el was cells follicle -knockdown ICD RA olcecl clones cell follicle -RNAi fe 3cevg.Because cleavage. S3 after (79%, ol aen fet but effect, no have would n 5 e(y)1 6 i.4)or 4B) Fig. 46; e(y)1 RA was -RNAi -RNAi. EXT e(y)1 is Niaut a oce igmris(i.4) lhuhco- Although 4F). 3B, (Fig. Fig. N margins in of wing seen expression notched As had wing. adults adult RNAi the in E(y)1 h IDS()Mmtraycmlxhsbe eotdto reported been has Flag-tagged complex expected, ternary As NICD–Su(H)–Mam 5A). cells. (Fig. The Su(H) -tagged S2 immunoprecipitate to in able was E(y)1 Actin5C control Flag-tagged the the under express both of respectively, to Su(H), Myc-tagged constructs and two E(y)1 co-transfected We the to again. turned we hypothesis, to expression. this gene test us target To Notch encouraged initiate to the analyses recruit machinery transcriptional to E(y)1 Genetic with interacts physically 2010; signaling. Su(H) that hypothesize transcription al., Notch core executive et of the (Kalogeropoulou is factor Su(H) genes 2009). Tjian, target and Wright their to transcriptional the machinery recruit to TAFs specific with interact directly can that transcriptional genes. suggest in target engaged wings Notch is of developing and activation NICD and of downstream cells acts follicle E(y)1 epistatic both these 4H). together, in (Fig. Taken results S4). Fig. defects during material (supplementary phenotypes margin function wing Gal4 alleviated greatly Gal4 inln yitrcigwt IDadS()biochemically. Su(H) and NICD with Notch interacting of output by transcriptional signaling assay the co-immunoprecipitation mediates E(y)1 a Thus, in 5B). detected (Fig. was NICD with and associated between E(y)1 association physically the Indeed, activation. also Notch promote we is to NICD and E(y)1 organisms, whether many asked in activation therefore gene target Notch regulate rvossuishv hw htseii rncito factors transcription specific that shown have studies Previous . . Su(H)–VP16 e(y)1 ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal RA nml xrsigS()V1 showed Su(H)–VP16 expressing animals -RNAi ICD epciey hnNthsgaigi nue by pAc5.1-Myc-N induced of is transfection (E), signaling fivefold Notch and when (D) respectively, 39-fold increased were levels lgtge () n y-agdS()() n Myc- and N (A), tagged Su(H) Myc-tagged and E(y)1 Flag-tagged N in Su(H) tagged cells. expression S2 gene and in target NICD Notch with for required associated and physically Su(H) is E(y)1 5. Fig. y-agdN Myc-tagged genes, target and Notch (D) two of with Expression treatment (D,E) by unchanged were levels iteefc nterexpression. their on effect the and little in chart, each shown of vector, side empty left pAc5.1 the of transfection of by suppressed ciainidcdb h xrsino N of expression the by induced activation of Knockdown r ral eue ytetetwith treatment dsGFP by levels reduced were protein greatly that E(y)1 are extracts (C) cell immunoprecipitation. S2 for the used Input of left. 5% the represent blotting to lanes western indicated by are detected immunoprecipitation proteins after the at and indicated are top, A,B the in (IP) Antibodies immunoprecipitation antibodies. for indicated used the or (control) assay IgG co-immunoprecipitation with the extracts for cellular Total prepared cells. were S2 cultured into transfected *** ICD E(spl)m7 P i o leit hsdfc Fg 4G), (Fig. defect this alleviate not did , ln ipae yia oc gain-of- Notch typical displayed alone sascae ihFa-agdEy1i 2cells. S2 in E(y)1 Flag-tagged with associated is 0.001. nS el,weesN whereas cells, S2 in E(spl)m8 ICD and A lgtge () neat ihMyc- with interacts E(y)1 Flag-tagged (A) n lgtge () B,wr co- were (B), E(y)1 Flag-tagged and ICD e(y)1 e(y)1 Drosophila E(spl)m8 Drosophila nS el,wssignificantly was cells, S2 in E,srnl nue ytransfected by induced strongly (E), infcnl upesdthis suppressed significantly ncdw.Tebslexpression basal The knockdown. Drosophila e(y)1 svr o,idctdby indicated low, very is 2cls B Myc-tagged (B) cells. S2 ICD ICD E(spl)m7 RA aiuainhad manipulation -RNAi nnra 2cells. S2 normal in igdevelopment wing n uui protein Tubulin and C96-Gal4 dse(y)1 2cl system cell S2 and ICD dse(y)1 . E(spl)m7 E(spl)m8 u not but . e(y)1 . 3835 C96- C96- -

Journal of Cell Science oc ciiydrn igdvlpet(i.6,) mligthat implying 6C,F), unlike (Fig. development wing during activity Notch of expression of that as signaling Notch 90,btol () sblee ob opnn fteTFIID the of component a be to whether believed investigate To is complex. E(y)1 only but 1990), EERHARTICLE RESEARCH ait ftsusadseis agn from 3836 ranging and a Fortini in species, 2010; Muskavitch, documented and and well (Artavanis-Tsakonas tissues been humans has of pathway variety signaling Notch The DISCUSSION the of phenotype the enhance mutations y respective the as identified of ablation However, 6D). of (Fig. margins TAF1 wings wing normal adult had animals the 6A), consistently, (Fig. Wg and of expression the by revealed as activity, Notch affect the Notch from obtained of examined RNAi Knockdown through TRiP. complex we TFIID the activation, of components gene target are during complex activity Notch TFIID the for Notch of for components required complex all TFIID whether the of and/or factor signaling specific determine TFIID To a the 1993). is al., of E(y)1 et components whether Goodrich core 1991; al., the et of (Dynlacht one complex as identified initially was the the E(y)1, for signaling. of required Notch is program E(y)1 that transcriptional indicate NICD–Su(H)–Mam-complex-mediated far thus signaling described Notch results in The TAFs other of Requirement eurdfrNthsgaig tlatduring all least that are at TAF12, suggest signaling, The except tested, development. Notch results we for complex These TFIID required shown). the different of with not components defects margin (data the wing notched at expressivity showed available them of TAF6) all and and TAF5 and (TAF2, complex Vienna components 6B), TFIID specific (Fig. to Furthermore, the corresponding 6E). of lines disc (Fig. RNAi flies several adult tested wing in we margins the wing notched caused of of overexpression compartment expectedly, posterior the 2 uain(erivadGrsmv,18;Goge tal., et Georgiev 1989; Gerasimova, and (Georgiev mutation ne the under e(y)2 Drosophila , e(y)1 e(y)2 Drosophila en-Gal4 sseiial eurdfrNthsignaling. Notch for required specifically is (),e(y)2 e(y)1, RA n on hti a o eurdfor required not was it that found and -RNAi NiCne ne h same the under Center RNAi TAF12 rvrvsbydcesdtelvlo gin Wg of level the decreased visibly driver e(y)1 igdvlpetatrdpeigother depleting after development wing Drosophila ythe by ecompromised we , TAF1 e(y)2 and en-Gal4 RA nue by induced -RNAi e(y)3 or 9-Gal4 C96- ooo fhmnTAF9, human of homolog e(y)3 ee eegenetically were genes rvrddntobviously not did driver ly iia oein role similar a plays Drosophila C96-Gal4 e(y)2 . Drosophila TAF12 ucinby function C96-Gal4 -RNAi driver, wing to nacn h hntp fthe of phenotype the enhancing cusa ifrn eesadi seilywl-hrceie for pathway well-characterized this especially of is regulation and The levels 2009). different al., at occurs et Tien 2009; Bilder, h rncitoa ciaino oc inln in signaling Notch mediate of to activation complex Su(H)– TAFIID transcriptional TFIID with the The the the recruits interacts of and genes. complex directly component NICD–Mam target complex, a Notch initiation E(y)1/TAF9, transcription of that expression suggest factor results initiate transcription general to the with studies of complexes interacts Our complex pathway 2013). transcriptional Notch al., Su(H)–NICD–Mam the et the Zeng in chaperone 2013; how complex al., chromatin address remodeling et chromatin (Yu CAF1 SWI/SNF tissues different the the by or be by can complex signaling from epigenetically Notch findings that Recent shown regulated have 2009). others 2011; al., and al., et laboratory et Tien our (Andersson 2012; NICD al., of et entry Guruharsha nuclear before stages the ta. 90.Afloigsuydmntae that demonstrated study following A 1990). al., et ait fseishv eeldacnieal iest nthe in diversity considerable a revealed have species of variety effector. transcriptional for a required and signaling through TAF is mediated the given complex is between a interaction which TFIID direct pathway, for the this that, of in output likely TAF transcriptional very specific is a It pathway, output transcriptional signaling. TAF4 the Notch mediates that TAF9 of that shown suggests study has Our report of transcription signaling, recent induce Wg of to activator A transcriptional a 1997). Pygopus, with interacts al., transcription is that of et protein activation crucial VP16-dependent (Uesugi a and as - identified for been and homologrequired also human has VP16 The TAF9 1995). of al., E(y)1 binding et of domains Thut 1993; direct al., activation of et (Goodrich the revealed p53 intensity and has the E(y)1/TAF9 assay between that A dependent. interaction suggesting context probably protein–protein disc, is signaling wing Notch in involvement the flies in studies female previous that our of of this, effect the with sterility that Consistent indicate and 1999). al., ovaries et (Soldatov the decreased of underdevelopment that and during oocytes and cells follicle in expressed rspiae(y)1 Drosophila eoewd rncitm tde ndfeetcl ye rma from types cell different in studies transcriptome Genome-wide ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal gis htgene. that against the by flies induced Gal4 from constructs wings indicated adult the of expressing Pictures (D–F) which in expressed. discs is marks wing (green) of GFP compartment (red). posterior Wg the for stained constructs. were indicated discs the Wing plus expression with discs GFP wing driving third-instar wandering of signaling. images Notch on genes encoding i.6 fet fdepleting of Effects 6. Fig. a ntal hntpclycaatrzdby characterized phenotypically initially was rvr uesrp iatragn eoe RNAi denotes gene a after Ri Superscript driver. e(y)1 e(y)1 ae cuticle naked osi olceclsi togrthan stronger is cells follicle in loss y rncito assdramatic causes transcription 2 leewe uae (Georgiev mutated when allele Wih n ja,2009). Tjian, and (Wright e(y)2 Drosophila rohrTAF- other or AC Confocal (A–C) e(y)1 Drosophila oogenesis shighly is en-Gal4 en-Gal4 e(y)1 C96- .

Journal of Cell Science e(y)1 3 CATAAGCTCACCGATTTC-3 mrcs upotsrtg.Ecsosof Excisions strategy. jump-out imprecise soitdwt t agtehnesadta trepresses al., it et (Andersson that activated and not enhancers statically is is target Notch Su(H) when its that transcription is with view associated conventional the partially only The explain is diversity understood. might transcriptome contexts. this cellular which different for basis many 2006), the so However, al., in signaling et Notch of Weerkamp function 2010; Krejcı al., al., 2009; et et al., (Aoyagi-Ikeda et genes Chadwick target 2011; Notch-induced of expression ARTICLE RESEARCH o lnlaaye,temtto of mutation the analyses, clonal For genetics and stocks Fly The site. insertion element P the Fly of characterization and Generation initiation METHODS AND transcription MATERIALS genes. TFIID target then Notch the of is expression activate complex to to Su(H)–NICD–Mam complex E(y)1/TAF9 this by and recruited Mam with complex and activator transcriptional NICD a form that, to Su(H)– the complex suggests from Hairless released study is Su(H) activation, Our signaling Notch manner. upon a in temporal-specific genes target and Notch of expression spatial- regulating remains the in tissues of involved intensity are and all output TAFs the certain in that expression plausible is gene It is unclear. target E(y)1 Notch Whether for dependent. context required probably is of signaling intensity Notch the that suggesting cells, follicle Notch certain the addition, of of In effect 2010). activation for Tjian, and for (Goodrich required tissues specific studies dispensable not in genes are with TAFs consistent be some formation, that showing margin to wing during seemed signaling TAF12 development, in Notch after that and before different revealed largely (Krejcı activation is has loci target at genes 11 occupancy at binding target Su(H) of analysis Notch Su(H)– systemic a of the but activation transcriptional genes, to target to then leads co-activators which Mam, brings complex, with NICD–Mam and together co-repressors NICD, activation, displaces Notch Upon 2011). seFg 2A). Fig. (see 5 the of part hsFLP ptemo h tr oo fthe of codon start the of upstream e(y)1- eiyoslta eesqecdb h s fapi fpies(5 primers of pair a of use the by sequenced were lethal hemizygous eerqie o oc inln during signaling a Notch in for our controlled tested we In required that is complex TFIID 2009). were the signaling of Oswald, TAFs many Notch and although studies, (Borggrefe by manner activation spatiotemporal their and genes. target Notch Su(H) of activation transcriptional of to leading change complex, ternary Su(H)–NICD–Mam association the the the and through E(y)1/TAF9 enhancers between mediates gene target complex Notch on occupancy TFIID the Notch-activation-induced after both that, cleavage, propose with We interact Su(H). can and E(y)1/TAF9 NICD that shows study Our 2010). biochemical Tjian, and (Goodrich activator absence transcription specific the a in of even promoters core to binds complex TFIID general htaebt ooyosadhmzgu ibeadfriewr used were fertile and the viable of hemizygous generation and for homozygous both are that 9 htfakdtegnmcrgo fthe of region genomic the flanked that ) ayNthtre ee aebe on ndfeettissues, different in found been have genes target Notch Many TRiP#HMS00336 Nilnswr sdt nc down knock to used were lines RNAi 122 e(y)1 and 9 UR xn1 nrn1adpr feo fthe of 2 exon of part and 1 intron 1, exon -UTR, w FRT19A osi h igds per ob ekrta htin that than weaker be to appears disc wing the in loss 1118 ´ rmteBloomington the from e(y)1 n ry 07.Pealn oesipyta the that imply models Prevailing 2007). Bray, and P{GT1}e(y)1 e(y)1 nteXcrmsm.ToidpnetUAS independent Two chromosome. X the on 190 190 uaini eeino 3 p including bps 339 of deletion a is mutation leeb tnadP-element-mediated standard a by allele 9 BG00948 n 5 and e(y)1 ee sgnzdPeeetflies P-element Isogenized gene. ´ e(y)1 9 a netd7 aepis(bps) pairs base 78 inserted was -CCTCCATCTTGAGATCTC- ta. 09 Meier-Stiegen 2009; al., et P{GT1}e(y)1 Drosophila e(y)1 e(y)1 190 e(y)1 a eobndwith recombined was 190 eesi hsppr– paper this in levels Drosophila P{GT1}e(y)1 Drosophila allele tc etrand Center Stock BG00948 novmn in involvement e(y)1 htwere that ,Su(H) BG00948 E(spl) wing locus 9 - - lo eimi ahwl eetetdwt 15 with CuSO treated with induction were or well transfection each in plasmid cells in before plate, medium 6-well of a ml For sequences). 2 promoter T7 indicate bold in (bases TCACTATAGGGGAGA AGCAAGGGCGAGGAGCTG-3 GFP CTCACTATAGGGGAGA TCATGTCCATCCTGAAGGAG-3 eu-reisc elcluemda(oh) rnfcino 2cells S2 of Transfection (Roche). media culture cell insect serum-free assays RNAi and Drosophila transfection culture, cell at S2 room Facility confocal Adobe in Resource LSM-510 arranged at and Imaging Zeiss processed minutes were Science Illustrator. 15 Figures a University. Biological for State on the Florida against captured Invitrogen) in were were microscope (1:1000, used Images DAPI temperature. antibodies with 1:50; Other (mouse, stained CD2 (1:15). Upstate), and 1:400; Serotec) Hnt (rabbit, H3 histone and (1:10), Bank CycB phosphorylated (1:20), (1:20) Hybridoma Cut against were Studies IA) Wg al., City, Developmental et Iowa Iowa, the (Deng of (University described from previously Antibodies as 2001). performed was staining Antibody Immunocytochemistry aucit hqagSu(lrd tt nvriy alhse,F)frassisting for the FL) Tallahassee, on University, comments State helpful (Florida Shu and State Zhiqiang reading (Florida manuscript; critical Calvin for Gabriel FL) technical and Tallahassee, for University, Palmer the University William from State Kennedy, Fellers Florida Jen J. of support; Thomas Resource thank Imaging Studies also Science Developmental We Biological the antibodies. and stocks; for fly Bank for Hybridoma School Medical Harvard at TRiP the Bloomington the Vienna NY), the York, Center, New University, (Columbia Mann Schu Trudi thank We Acknowledgements 4 at specific co- (Abmart) with beads For agarose incubated (Calbiochem)]. A/G were protein cocktail EDTA and mM extracts inhibitor antibodies 1 protease assays, NaCl, inhibitors mM immunoprecipitation protease PMSF; 150 of presence the 7.4, in mM SDS) pH [1 0.1% X-100, Tris-HCl Triton 1% 7.4, mM pH (50 immunoprecipitation in buffer prepared were lysis cells S2 from extracts protein Total assay Co-immunoprecipitation 500 using normally by were induced for Full-length cells experiments. was further used S2 expression for The transfection Notch constructs after request. hours (Roche) The upon 48 reagents harvested available instructions. kit are transfection manufacturer’s transfections HD the FuGENE following using performed was n abtat-y 120 im)atbde eeue o co- and for Sigma) (1:2000, blots. western used for anti-Myc used were antibodies rabbit were Sigma) Sigma), (1:2000, anti-Tubulin Sigma), mouse (1:1000, antibodies (1:1000, anti-E(y)1 anti-Flag Sigma) rabbit mouse (1:200, experiments; anti-Myc Sigma) immunoprecipitation 2 (1:100, anti-Flag rabbit in Rabbit beads. boiled the and from were elution Immunoprecipitates for buffer elution. loading and washes before e(y)1 vrxrsincntutudrtecnrlo h A rmtror promoter at UAS twice heat-shocked the were progeny of adult 37 control Their interest. the of combination under construct overexpression admcoe nflil el,the cells, follicle in act clones random Poea speiul ecie Hage l,21) rmr used Primers 2011). al., et was – (Huang dsRNA kit described were system-T7 1990). previously production al., RNA as large-scale et (Promega) RiboMAX (Fehon the with pMT-Notch prepared with transfection after o – asbfr dissection. before days 2–3 for rmRcadS an(sel n an 00.Ohrfysok and stocks material fly supplementary Other in listed 2010). S1. are listed Table Mann, figures and the to (Estella related genotypes Mann S. Richard from ˚ . o 0mntsadte utrdi e-es-atdva t29 at vial wet-yeast-pasted a in cultured then and minutes 30 for C D o y (or CD2 6474R-1 owr 5 forward ora fCl cec 21)17 8033 doi:10.1242/jcs.154583 3830–3839 127, (2014) Science Cell of Journal e(y)1 rmteNtoa nttt fGntc,Jpn oinduce To Japan. Genetics, of Institute National the from 2clswr utrda omtmeauei Hyclone in temperature room at cultured were cells S2 b Glcoiae(abt :00 im) uliwr co- were Nuclei Sigma). 1:2000; (rabbit, -Galactosidase owr 5 forward + 9 ) Drosophila - . TTAATACGACTCACTATAGGGGAGA pah(rneo nvriy rneo,N) ihr S. Richard NJ), Princeton, University, (Princeton ¨pbach Gal4 a rse ofiscryn nRA ran or RNAi an carrying flies to crossed was ] 9 CTTGTACAGCTCGTCCATGCCGAGAG-3 - NiCne,teNtoa nttt fGntc and Genetics of Institute National the Center, RNAi TTAATACGACTCACTATAGGGGAGA- CGCTAGTTGGTCACAAACTC-3 b-F M(1) ubi-GFP w 9 and 9 and GFP e(y)1 lpotGal4 flip-out ees 5 reverse ees 5 reverse m CuSO M osp 4 m . sN o days 4 for dsRNA g 9 FRT19A - TTAATACGAC- 9 Drosophila - TTAATACGA- rvr[ driver 4 ˚ o 4hours 24 for Covernight ATGGTG- a gift a was 9 ;control hsFLP; Stock 6 3837 SDS ˚ C 9

Journal of Cell Science oet,D,Fgr . one-hped . a,L .adJn .N. Y. Jan, and Y. L. Jan, S., Younger-Shepherd, G., Feger, D., Doherty, M. S. Cohen, and J. F. Diaz-Benjumea, A. M. Muskavitch, and S. Artavanis-Tsakonas, .. .MD n ..peae n rt h ae ihiptfo ..adD.J. and Z.Y. from input with data. paper the the analyzed wrote and W.-M.D. prepared and and R.J. Z.Y. R.J. and Z.Y., G.X., W.-M.D. G.X., G.X., experiments. experiments. the the designed performed and D.J. conceived R.J. and G.X. W.-M.D., contributions Author interests. competing no declare authors The interests Competing suggestions. and feedback for for screen ARTICLE RESEARCH 3838 H. J. Ruohola-Baker, and S. C. Bray, Althauser, and M., J. W. Deng, Celis, de M., D. Tyler, F., J. Celis, de J. S. Bray, and A. Garcia-Bellido, F., J. Celis, de L., G. Gerton, C., D. Page, M., Goodheart, M., Kouadio, G., M. Buffone, Y., Cheng, and S. Hoyle, F., Warrander, C., Fennessy, V., Portillo, L., Zeef, N., Chadwick, M. Bienz, and H. Musisi, S., Bray, F. Oswald, and T. Borggrefe, D. Johnston, St and R. Bastock, Ba W. J. Posakony, and M. A. Z. Bailey, B. Shilo, and S. D. Johnston, D., E. Schejter, L., I. Torres, E., Assa-Kunik, F., Aoki, Y., Aoki, K., Hara, M., Ueno, U. H., Matsui, Lendahl, T., Maeno, and K., R. Aoyagi-Ikeda, Sandberg, R., E. Andersson, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154583/-/DC1 online available material Supplementary material Supplementary in Deposited months. GM072562]. 12 R01 after number release [grant for and Health PMC IOS-1052333]; of is number Institutes W.-M.D. [grant National Foundation R.J.]. the grant Science to a National 2012CB825504 and the number W.-M.D.]; by to [grant supported 31228015 program and 973 R.J. Foundation the to Science from 31271573 Natural number National the [grant from China grants of by supported was work This Funding ylct .D,He,T n ja,R. Tjian, and T. Hoey, D., B. Dynlacht, Schu and E. Domanitskaya, sel,C n an .S. R. Mann, and C. Ito, M., Estella, Siebert, E., Kinameri, A., Krejci, H., Taniguchi, R., M. Karim, K., Endo, rfi . adf . itn . ahan . aziv . oa . Orba L., Tora, Y., Hadzhiev, Y., Rathmann, T., Hilton, C., Balduf, R., ´rtfai, sals igesdpnetognzra h oslvnrlcompartment dorsal/ventral wing. the Drosophila at the of organizer boundary Wingless-dependent a establish nue rniinfo ioi elccet noyl nDoohl follicle Drosophila in endocycle to cycle cell mitotic from cells. transition a induces disc. imaginal wing the of boundary dorsal-ventral 122 the at Notch of J. gene. P. Wang, and I. Davidson, leukaemia. M. A. Buckle, modification. histone and genes. target Notch 1646. at regulation transcriptional R1082-R1087. zebrafish. of development embryonic in required is Mu and L. Notch to response in genes complex pathways. Notch split activity. 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Journal of Cell Science a,A,Azmn . tmfe . a el,W,Sue,U,Hr,T., Horn, U., Sauder, W., Belle, van D., Stempfle, Z., Arziman, A., Saj, J., S. Schleede, Artavanis-Tsakonas, and N., G. R. Leake, Fehon, I., J., Rebay, Sun, L., Smith, C., Y. Huang, S., J. Poulton, Sassone- M., Parvinen, L., Monaco, S., Brancorsini, G., Mengus, C., J. Pointud, Ro and J. S. Bray, F., Bernard, E., B. Housden, K., M. Pines, I. L. Aifantis, S. Zipursky, and and F. J. Pignoni, Sage, S., J. Lim, P., Shim, Ntziachristos, D., Yang-Zhou, L., Holderbaum, P., L. Liu, B., Czech, R., Zhou, Q., J. Ni, EERHARTICLE RESEARCH i .Q,Lu .P,Bnr,R,Hry . hm .S,Cvlao . okr M., Booker, A., Cavallaro, S., H. Shim, R., Hardy, R., Binari, P., L. Liu, Q., J. Ni, M., Booker, C., Villalta, P., L. Liu, B., Pfeiffer, R., Binari, M., Markstein, L., Q., J. J. Ni, Nishikawa, J., Ouyang, Y., J. Ji, L., Stefano, Di F., Yang, P., Mulligan, xvv n nvv Nisre o oc euaosi rspiarvasan reveals Drosophila in regulators notch for screen network. 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