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1 oi:.38/nture997 Acetyltion-epenent regultion of enothelil Notch signlling y the ecetylse Virgini Gurni, Ginluc Deflorin *, Cluio A. Frnco 3 *, Mrcus Krüger, Li-Kun Phng 3 {, Ktie Bentley 3, Louise Toussint, Frnck Dequiet, Rul Mostoslvsky 6, Mirko H. H. Schmit 7, Brr Zimmermnn, Rlf P. Brnes 8, Mrin Mione, Christoph H. Westphl 9, Thoms Brun, Anres M. Zeiher, Holger Gerhrt 3,, Stefnie Dimmeler & Michel Potente, Notch signlling is key intercellulr communiction mechnism tht is essentil for cell specifiction n tissue ptterning, n which coorintes criticl steps of loo vessel growth 3. Although sutle ltertions in Notch ctivity suffice to elicit profoun ifferences in enothelil ehviour n loo vessel formtion,3, little is known out the regultion n pttion of enothelil Notch responses. Here we report tht the NAD -epenent ecetylse cts s n intrinsic negtive moultor of Notch signlling in enothelil cells. We show tht cetyltion of the Notch intrcellulr omin () on conserve lysines controls the mplitue n urtion of Notch responses y ltering protein turnover. ssocites with n functions s ecetylse, which opposes the cetyltion-inuce stiliztion. Consequently, enothelil cells lcking ctivity re sensitize to Notch signlling, resulting in impire growth, sprout elongtion n enhnce Notch trget gene expression in response to DLL stimultion, therey promoting non-sprouting, stlk-cell-like phenotype. In vivo, inctivtion of Sirt in zerfish n mice cuses reuce vsculr rnching n ensity s consequence of enhnce Notch signlling. Our finings ientify reversile cetyltion of the s moleculr mechnism to pt the ynmics of Notch signlling, n inicte tht cts s rheostt to fine-tune enothelil Notch responses. We investigte the role of in the regultion of enothelil Notch signlling. Notch ctivity ws nlyse in response to DLL stimultion in scrmle or short interfering (si)rna-trnsfecte humn umilicl vein enothelil cells y ssessing the ctivity of the Notch reporter genes TP-luciferse n 3CBF-luciferse s well s the expression of the enogenous Notch trget genes NRARP n HEY. Knockown of y i not lter sl Notch ctivity, ut significntly enhnce Notch reporter gene ctivity n trget gene expression fter DLL stimultion (Fig. c n Supplementry Fig., ). Comprle results were otine with non-relte pool of s (Supplementry Fig., c). Importntly, enhnce Notch responsiveness to DLL in -eficient enothelil cells ws rogte y the c-secretse inhiitor ienzzepine (DBZ) or y muttion of the CBF-ining sites in the Notch reporter gene (Fig., ). Conversely, ctivtion of signlling using the smll molecule SRT83 (ref. ) inhiite DLL-meite inuction of Notch reporter ctivity n trget gene expression, inicting tht negtively moultes DLL/Notch signlling in enothelil cells (Fig., e). Anlysis of the sl messenger RNA expression of criticl Notch pthwy components such s NOTCH, NOTCH, MAML n NCOR in control n -eficient enothelil cells revele no significnt ifferences (Supplementry Fig. ). However, - eficient enothelil cells isplye mrkely enhnce Notch ctivity fter overexpression of, s ssesse using ifferent Notch reporter genes (Fig. f n Supplementry Fig. e, f). On the other hn, co-trnsfection of with inhiite Notch ctivity in ose-epenent mnner (Supplementry Fig. g). The inhiitory effect of require its ecetylse ctivity, s ctlyticlly inctive mutnt ( H363Y) ws unle to inhiit Notch responses (Fig. g). Tken together, these results emonstrte tht inhiition of Notch signlling y occurs in enothelil cells tht receive ctivting DLL/Notch signls n inicte regultory mechnism involving s well s the ecetylse ctivity of. Co-immunoprecipittion experiments illustrte tht overexpresse ssocites with Flg n enogenous, ut not with other teste sirtuins (Fig. h n Supplementry Figs 3c). Intriguingly, V levels were consistently lower in Flg overexpressing lystes (Fig. h n Supplementry Fig. 3). This reuction in protein levels ws not oserve when ws co-expresse with mutnt lcking the croxy-terminl PEST omin importnt for protesoml egrtion ((DC)), lthough its ining to ws retine (Fig. i). These t inicte tht might negtively regulte Notch signlling y promoting egrtion. Consistent with this moel, the interction etween enogenous n ws enhnce when enothelil cells were trete with the protesoml inhiitor MG3 (Fig. j). Further chrcteriztion of this protein interction revele tht eletion of the ctlytic omin olishe the ining of to, inicting tht might e sustrte for the ecetylse (Supplementry Fig. 3, e). To ssess whether cts s ecetylse, we ske whether is n cetylte protein. We trnsfecte 93 cells with V n the cetyltrnsferses PCAF n p3, which in to n function s coctivtors t Notch-regulte promoters,6. ws roustly cetylte in cells co-expressing PCAF or p3 (Supplementry Fig., ). In contrst, Tip6, which hs een reporte to cetylte fter ultrviolet rition 7, ws unle to cetylte uner our experimentl conitions (Supplementry Fig. c). Blocking ecetylse ctivity with the sirtuin inhiitor nicotinmie (NAM) inuce cetyltion of overexpresse s well s enogenous in enothelil cells, wheres the clss I/II histone ecetylse inhiitor trichosttin A (TSA) h only minor effect (Fig. k, l). Notly, knockown of increse sl n p3-inuce cetyltion, wheres overexpression prevente its cetyltion Institute for Criovsculr Regenertion, Centre of Moleculr Meicine, Goethe University, D-69 Frnkfurt, Germny. IFOM, the FIRC Institute of Moleculr Oncology, IFOM-IEO Cmpus, 39 Miln, Itly. 3 Vsculr Biology Lortory, Lonon Reserch Institute Cncer Reserch UK, WCA 3LY Lonon, UK. Mx Plnck Institute for Hert n Lung Reserch, Deprtment of Cric Development n Remoeling, D-63 B Nuheim, Germny. Lortory of Protein Signling n Interctions, GxABT, B-3 Gemloux n Interisciplinry Cluster for Applie Genoproteomics (GIGA-R), University of Liege, B- Srt-Tilmn, Belgium. 6 Msschusetts Generl Hospitl Cncer Center, Hrvr Meicl School, Boston, Msschusetts, USA. 7 Moleculr Signl Trnsuction, Institute of Neurology (Einger Institute), Goethe University, D-69 Frnkfurt, Germny. 8 Vsculr Reserch Centre, Institute for Criovsculr Physiology, Goethe University, D-69 Frnkfurt, Germny. 9 Sirtris, GSK Compny, Cmrige, Msschusetts 39, USA. Deprtment of Criology, Internl Meicine III, Goethe University, D-69 Frnkfurt, Germny. Consultnt Group Leer, Vsculr Ptterning Lortory, Veslius Reserch Center, VIB, Cmpus Gsthuiserg, B-3 Leuven, Belgium. {Present ress: Cell Biology n Biophysics, Europen Moleculr Biology Lortory, D-697 Heielerg, Germny. *These uthors contriute eqully to this work. Mcmilln Pulishers Limite. All rights reserve MONTH VOL NATURE

2 RESEARCH LETTER TP-luc Scrmle DLL DBZ CBF CBF-mt -luc -luc c NRARP 6 8 Scrmle Scrmle 6 DLL Fol inuction DLL HEY l IP IgG IP Notch WB AcK WB Input DLL NAM 6 IP IgG IP Notch DLL NAM TP-luc Ctrl SRT 83 DLL e NRARP HEY f g Fol inuction 6 Ctrl SRT 83 DLL 3 TP-luc Scrmle TP-luc pcdna WT H363Y m WB AcK WB V V 3 Scrmle h i j IP IP IgG k IP Flg 7 WB WB Flg WB Myc WB AcK IP Flg WB V WB WB Flg WB V Flg Input Flg V V Myc 7 Input Flg Flg DLL V V (ΔC)Myc MG3 NAM TSA Figure limits enothelil DLL/Notch signlling n trgets for ecetyltion.,, TP-(), 3CBF- or 3CBF mutnt- (mt) luciferse ctivity () in control (scrmle) or --trnsfecte enothelil cells. c, NRARP n HEY mrna levels in scrmle or -trnsfecte enothelil cells., e, TP-luciferse ctivity ()n NRARP n HEY mrna levels (e) in enothelil cells stimulte with SRT83. f, TP- luciferse ctivity in control n -eficient enothelil cells co-trnsfecte with., incresing mounts of. g, TP-luciferse ctivity in enothelil cells trnsfecte with comintions of, n H363Y. h, i, Co-immunoprecipittions from Flg n V or n AcK WT H363Y Myc NAD (DC)Myc co-trnsfecte 93 cells. IP, immunoprecipittion; WB, western lot. j, Co-immunoprecipittion of n in enothelil cells trete with or without MG3. k, Acetyltion of V in 93 cells trete with NAM, TSA, or comintions thereof. AcK, cetyl-lysine. l, Acetyltion of in enothelil cells trete with NAM. m, Acetyltion of V in - -trnsfecte 93 cells. n, Decetyltion ssy with (DC)Myc, recominnt wil-type or H363Y. Reltive quntifictions of cetyltion re shown on the right of the pnel in l, m. DLL ws use to stimulte Notch signlling in enothelil cells in e, j n l. All experiments n $ 3; error rs, men 6 s.. *, P,.; **, P,.; ***, P,.;, not significnt. 7 7 (Fig. m n Supplementry Fig., e). Moreover, wil type ut not the inctive H363Y mutnt ecetylte ((DC)) in n in vitro ecetyltion ssy in NAD -epenent mnner (Fig. n). These t emonstrte tht is reversily cetylte n ientify s on fie ecetylse. Using liqui chromtogrphy-tnem mss spectrometry nlysis (LC-MS/MS) we foun cetyltion sites in the trgeting conserve lysine resiues (Fig., n Supplementry Fig., ). Muttion of these lysines to rginine ((KR)) olishe cetyltion inuce y PCAF, p3, or upon NAM tretment or knockown (Fig. c n Supplementry Fig. ce). Compre to wiltype, the (KR) mutnt h similr ility to ctivte Notch reporter genes, ut ws resistnt to chnges in the level of (Fig. n Supplementry Fig. 6, ). These t inicte tht set of lysine resiues confer negtive regultion of Notch signlling y. Intriguingly, conitions tht fvour cetyltion increse protein levels (Fig. k, l n Supplementry Figs, n 7). Becuse unergoes rpi uiquitin-meite egrtion, n cetyltion cn impir uiquitintion, we explore the effects of on protein egrtion n stility. Knockown of increse, wheres overexpression ecrese, protein levels (Fig. e, f n Supplementry Fig. 7). Importntly, protein levels of the (KR) mutnt were not enhnce in - eficient or NAM-trete cells (Fig. f n Supplementry Fig. 7c). Similrly, locking ctivity in enothelil cells y NAM or increse enogenous protein levels, wheres ctivtion of y SRT83 reuce enogenous protein levels (Fig. g, h n Supplementry Fig. 7). Tretment of enothelil cells with MG3 le to n increse in protein levels, which were not further enhnce y NAM tretment (Supplementry Fig. 7e). MG3 lso prevente ownregultion of fter stimultion with SRT83 (Fig. i). Together with the unltere NOTCH mrna expression these t inicte tht ffects the protesoml egrtion of (Supplementry Fig. 7f). Next, we exmine the effect of on the ecy of protein levels in response to DLL stimultion in cycloheximie-trete enothelil cells. Blocking ctivity y NAM or increse, wheres ctivtion y SRT83 ecrese, the mplitue n urtion of enogenous protein levels (Fig. j n Supplementry Fig. 8). Concomitntly, inhiition y NAM or -meite knockown reuce levels of uiquitinte in cells tht co-expresse V n HA-tgge uiquitin (Supplementry Fig. 9c). Together, these t support moel in which cetyltion interferes with uiquitin-epenent protesoml egrtion of, hence enhncing Notch responses. The ecetylse ctivity of countercts the stiliztion of y priming it for uiquitin-meite proteolysis (Supplementry Fig. ). To ssess whether regultes Notch responses in vivo, we inctivte the ctlytic omin of in enothelil cells NATURE VOL MONTH Mcmilln Pulishers Limite. All rights reserve

3 RESEARCH HCD-MS/MS - Mscot score:. Ac RAM ANK PEST N- -C KKKKK K93 K K68 KKKKKK Reltive unnce e y , Flg y 3. y (Supplementry Fig. ) 8 n ssesse postntl retinl ngiogenesis in mice. Recent stuies showe tht DLL/Notch signlling coorintes retinl ngiogenesis y controlling the specifiction of enothelil cells into tip n stlk cells 9. Tip cells express high levels of DLL n guie nscent sprouts, wheres following stlk cells receive DLL/ Notch signls from tip cells to form the vsculr tue. Immunofluorescence stining revele tht Sirt is roustly expresse t the ngiogenic front of the vsculture, prticulrly in stlk cells (Fig. 3, ), in which Notch ctivity is most prominent 9,3. Compre to Sirt flox/ n Tiecre;Sirt flox/ control mice, enothelil-cell-restricte Sirt mutnt mice (Tiecre;Sirt flox/ ) isplye elye expnsion of the vsculr plexus n significnt reuction in vessel ensity n enothelil cell prolifertion (Fig. 3cj n Supplementry Fig. e). These phenotypic chnges resemle those cuse y enhnce Notch ctivity in stlk cells 3 n were mirrore y enhnce expression of the Notch trget genes Nrrp, Hey n Lfng in Dll-trete enothelil cells erive from these mice (Fig. 3k). However, the ility of tip cells to exten filopoi ws unffecte in Sirt mutnt mice (Supplementry Fig. fi). Together with the prominent expression of Sirt in stlk cells, these finings inicte tht Sirt negtively moultes Notch signlling in stlk cells to fcilitte enothelil rnching n prolifertion, therey controlling vsculr growth n ensity. To unerstn further the effects of on the ynmics of tip/ stlk cell ehviour, we use computtionl moelling. Moelling y.9 y V WB AcK WB V g Tuulin Tuulin V V Flg (KR)V h 3 Scrmle i j Tuulin Tuulin DLL MH y y y8 86. y f y 9.6 y 7.7 DLL Figure Destiliztion of y., HCD (higher energy C-trp issocition) MS/MS spectrum showing cetyltion of lysine resiue., Overview of cetylte lysines in the. ANK, nkyrin repets; PEST, proline (P), glutmic ci (E), serine (S) n threonine (T) enriche sequence; RAM, RBPj-ssocite molecule. c, Acetyltion of V wil type or KR in trnsfecte 93 cells trete with NAM., TP-luciferse ctivity in scrmle n --trnsfecte enothelil cells cotrnsfecte with wil-type or (KR). e, protein levels in 93 cells expressing V n Flg. f, V protein levels in 93 cells trnsfecte with scrmle or n V wil type or KR. c 76/77/77 77/78 V m/z (KR)V NAM DLL SRT83 MG3 Tuulin 6/7// 6/6 DLL NAM TSA 3 the increse in levels inuce y Sirt-eficiency revele elye negtive feeck regultion of the Dll/Notch pthwy, resulting in slower selection rte of tip n stlk cells. The tip cells were, however, phenotypiclly norml, proucing similr numers of filopoi (Supplementry Movies n ). The preicte ely in tip n stlk cell selection woul trnslte into oth reuce rnching n elye expnsion of the vsculr plexus, s oserve in vivo. Next, we teste whether Notch inhiition woul restore enothelil cell responses in Sirt mutnts. Tretment of control nimls with the c-secretse inhiitor N-[N-(3,-ifluorophencetyl)-L-lnyl]- S-phenylglycine t-utyl ester (DAPT) cuse hyperense n hyperrnche vessel network, s reporte 9,,3, (Fig. 3l n Supplementry Fig. m). In response to DAPT tretment, vessel ensity in Sirt mutnts increse to similr extent s in DAPT-trete controls, inicting tht the reuction in vessel ensity is consequence of increse Notch ctivity (Fig. 3l n Supplementry Fig. jm). Likewise, knockown of sirt in zerfish y ntisense morpholino-moifie oligonucleoties resulte in efects of the trunk vsculture similr to those cuse y increse Notch ctivity in stlk cells 3,6,7 n were chrcterize y thin, misguie, poorly connecte n hypocellulr intersomitic vessels 8 (Fig.,, e n Supplementry Fig., ). Tretment of sirt morphnts with DAPT or knockown of the rtery-specific Notch lign ll, t lest in prt, normlize the ptterning n cellulrity of intersomitic vessels (KR) 3 TP-luc Scrmle DLL NAM TSA Control SRT83 NAM CHX DLL Time (h).... g, h, protein expression in enothelil cells trete with NAM n/or TSA (g) or trnsfecte with scrmle or (h). Reltive quntifictions of protein levels re shown on the right of the pnel. i, protein levels in enothelil cells pre-trete with or without MG3 n/or SRT83. j, Enothelil cells were pre-trete with CHX n NAM or SRT83 n protein levels nlyse t the inicte time points. DLL ws use to stimulte Notch signlling in enothelil cells in gj. protein levels in gi were ssesse fter 6 h of DLL stimultion. All experiments n $ 3; error rs, men 6 s.. *, P,.; **, P,.; ***, P,.;, not significnt. Mcmilln Pulishers Limite. All rights reserve MONTH VOL NATURE 3

4 RESEARCH LETTER c IsoB / Sirt flox/ V A μm μm IsoB / Sirt V A g flox/ f Cre flox/ Cre flox/ e Cre flox/ A V Cre flox/ h * * μm μm r Cre flox/ N Cre flox/ H flox/ ey l Dll flox/ Cre flox/ Cre flox/ flox/ Cre flox/ DAPT ng k flox/ Cre flox/ Cre flox/ Fol inuction 3 Cpillry plexus BrU/IsoB nuclei per fiel No. of rnch-points Vsculr front Enothelil cell coverge per fiel (%) j No. of rnch-points i rp * Lf ** * Figure 3 Inctivtion of enhnces enothelil Notch responses in mice., Sirt locliztion (re) in the retinl enothelium (isolectin B, green)., Higher mgnifiction of inset in is shown. Asterisks inicte Sirt expression in stlk enothelil cells; rrowhes inicte some tip cells with wek or sent Sirt expression. ce, Imges of P Sirt control n mutnt retins stine with IsoB. Blue n green oxes inicte the vsculr front n cpillry plexus, respectively. A, rteries; V, veins. fh, IsoB (re) n BrU (green) lelling in P retins of the respective genotypes. i, j, Sttisticl summry of the numer of vessel rnch points n the numer of BrU/IsoB-positive cells of the respective genotypes. k, qpcr of Hey, Nrrp n Lfng mrna expression in Dll-stimulte enothelil cells erive from mice of the respective genotypes. l, Sttisticl summry of the percentge of IsoBpositive vessel coverge in vehicle n DAPTtrete retins of control n Sirt mutnt mice. All experiments n $ ; error rs, men 6 s.. *, P,.; **, P,.; ***, P,.;, not significnt. (Fig. e n Supplementry Fig. c), inicting tht misregultion of Notch ctivity in enothelil cells contriutes to the vsculr ptterning efects in sirt morphnts. To issect further the effects of on Notch-controlle cell responses, we monitore sprout formtion n prolifertion in isolte humn enothelil cells. Control enothelil cell spherois extene multicellulr sprouts (Supplementry Fig., ). In contrst, knockown of impire sprout formtion n elongtion8 (Supplementry Fig., ). Importntly, these efects were restore y c-secretse inhiition (DBZ) (Supplementry Fig., ). Likewise, inhiition of enothelil cell growth y DLL/Notch signlling ws enhnce in -eficient enothelil cells n rescue y DBZ (Supplementry Fig. c). To test the cell-utonomous effect of Sirt in moulting Notch responses, we use clusters of ifferentiting mouse emryonic stem cells (emryoi oies (EBs)), which form rnche vsculr networks in response to VEGF-A stimultion8. Control EBs expressing the fluorophore DsRe-MST (DsRe) evelope extensively rnche vessel networks, wheres genetic inctivtion of Sirt (SirtDex/Dex) impire vsculr outgrowth n ensity (Fig. f, g). Recent work on geneticlly mosic EBs illustrte tht Control sirt MO c f PECAM g h SirtΔ e/δ e DsRe:SirtΔ e/δ e l m sirt MO DAPT DAPT μm e Control Numer of ISV enothelil cells t 8 h.p.f. 3 levels: pixel intensity ( 6) DAPT ll MO i sirt MO DsRe 3 k μm DsRe SirtΔ e/δ e DsRe Tip position in : chimers (%) j 8 DAPI 6 Merge PECAM DsRe SirtΔ e/δ e μm N AT U R E VO L M O N T H Mcmilln Pulishers Limite. All rights reserve Figure Inctivtion of les to cellutonomous increse in Notch signlling n efective enothelil cell sprouting., Lterl trunk views of 8 h post fertiliztion Tg(fli:EGFP)y zerfish emryos. Control emryos or sirt morphnts were trete with DAPT n DMSO. e, Enothelil cell nuclei counts in the intersomitic vessels (ISVs) of trnsgenic Tg(fli:nEGFP)y7 emryos t 8 h post fertiliztion (h.p.f.) trete with or without DAPT, or injecte with ll-specific morpholinos. fh, Overview of vsculr sprouts from EBs of DsRe (WT) (f), SirtDe/De (g) ES cells n : chimers (h). i, Quntifiction of totl pixel intensity of the immunostining per nucleus in DsRe n Sirt mutnt enothelil cells. j, Quntifiction of tip-cell contriution of ech ES cell genotype in vsculr sprouts. km, immunostining (green) of iniviul sprouts of EBs from the respective genotypes. DsRe cells (re), Sirt mutnt cells (non-lelle), DAPI (lue), PECAM (grey). All experiments n $. Error rs, men 6 s.. **, P,.; ***, P,..

5 RESEARCH the ility of iniviul cells to gin the tip position is ynmiclly n competitively regulte y Dll/Notch signlling 9. Aing DsRe cells to Sirt mutnt cells in : rtio prtilly restore sprouting n rnching (Fig. fh), s DsRe cells (wil type for Sirt) effectively forme tip cells, wheres Sirt mutnt cells, exhiiting increse levels, preferentilly forme stlk cells (Fig. im). Furthermore, inhiition of Sirt y NAM in Dll heterozygote EBs (Dll /lcz ), which re chrcterize y reuce Notch signlling n excessive vessel rnching 9, restore norml rnching ehviour (Supplementry Fig. 3), inicting tht inhiition cn re-just equte levels of Notch signlling in Dll /lcz cells. Our stuies unrvel novel role for s cell-utonomous negtive moultor of Notch signlling n highlight reversile cetyltion of s key moleculr mechnism to just the ynmics of Notch responses. Negtive regultion of Notch signlling y might, however, involve itionl components of the Notch pthwy such s the co-repressor NCOR n trnscription fctors of the HES/HEY n FOXO fmily, which hve een shown to e regulte y (refs ). By coopertive regultion of these fctors tht function t ifferent steps in the Notch pthwy, might synergisticlly moulte Notch signlling to chieve roust regultion of Notch-controlle cellulr responses. Notly, the regultory function of in the Notch pthwy seems to exten eyon NOTCH n to inclue other fmily memers such s NOTCH (Supplementry Fig. ), inicting generl mechnism for Notch regultion y tht my pply to severl Notch-controlle iologicl processes. However, we cnnot rule out tht might lso inirectly ffect Notch signlling in other tissues or cellulr environments, s shown in geing neuronl cells, where hs een foun to increse Notch signlling through RXR-epenent regultion of ADAM (ref. ). However, in enothelil cells ADAM expression ws not ffecte y (Supplementry Fig. ), inicting tht the inirect moultion of Notch signlling through RXR is not opertionl in enothelil cells. Becuse requires NAD for its ctlytic ctivity, it is responsive to chnges in the metolic n reox stte of the cell 6 n might therefore function s cellulr sensor coupling energy n oxygen homeostsis to Notch-epenent control of rnching vessel morphogenesis. METHODS SUMMARY Humn umilicl vein enothelil cells (HUVECs) were from Lonz n culture s escrie 8. Mouse lung enothelil cells were isolte, purifie n culture s escrie 8. The culture of ES cells n the genertion of EBs were performe s escrie 8. Enothelil-restricte Sirt mutnt mice were on C7BL/6 genetic ckgroun n generte s escrie 8. Tg(fli:nEGFP)y7 n Tg(fli:EGFP)y zerfish lines were use, mintine n re uner stnr conitions. Antisense morpholino oligonucleoties trgeting sirt n ll were use s escrie 9,9.Mss spectrometric experiments were performe on nno-flow HPLC system (Proxeon) connecte to LTQ-Oritrp Velos instrument (Thermo Fisher Scientific) equippe with nnoelectrospry source (Proxeon). Full Methos n ny ssocite references re ville in the online version of the pper t Receive Octoer 9; ccepte Ferury. Pulishe online 7 April.. Kopn, R. & Ilgn, M. X. The cnonicl Notch signling pthwy: unfoling the ctivtion mechnism. Cell 37, 633 (9).. Roc, C. & Ams, R. H. Regultion of vsculr morphogenesis y Notch signling. Genes Dev., (7). 3. Phng, L. K. & Gerhrt, H. Angiogenesis: tem effort coorinte y notch. Dev. Cell 6, 968 (9).. Milne, J. C. et l. Smll molecule ctivtors of s therpeutics for the tretment of type ietes. Nture, 776 (7).. Kurook, H. & Honjo, T. Functionl interction etween the mouse Notch intrcellulr region n histone cetyltrnsferses PCAF n GCN. J. Biol. Chem. 7, 77 (). 6. Oswl, F. et l. p3 cts s trnscriptionl coctivtor for mmmlin Notch-. Mol. Cell. Biol., (). 7. Kim, M. Y. et l. Tip6 histone cetyltrnsferse cts s negtive regultor of Notch signling y mens of cetyltion. Mol. Cell. Biol. 7, 6669 (7). 8. Potente, M. et l. controls enothelil ngiogenic functions uring vsculr growth. Genes Dev., 668 (7). 9. Hellström, M. et l. Dll signlling through Notch regultes formtion of tip cells uring ngiogenesis. Nture, (7).. Rigwy, J. et l. Inhiition of Dll signlling inhiits tumour growth y eregulting ngiogenesis. Nture, 8387 (6).. Suchting, S. et l. The Notch lign Delt-like negtively regultes enothelil tip cell formtion n vessel rnching. Proc. Ntl Ac. Sci. USA, 333 (7).. Loov, I. B. et l. Delt-likelign(Dll) isinuce yvegfs negtive regultor of ngiogenic sprouting. Proc. Ntl Ac. Sci. USA, 393 (7). 3. Phng, L. K. et l. Nrrp coorintes enothelil Notch n Wnt signling to control vessel ensity in ngiogenesis. Dev. Cell 6, 78 (9).. Beneito, R. et l. The notch ligns Dll n Jgge hve opposing effects on ngiogenesis. Cell 37, 3 (9).. Bentley, K., Mriggi, G., Gerhrt, H. & Btes, P. A. Tipping the lnce: roustness of tip cell selection, migrtion n fusion in ngiogenesis. PLoS Comput. Biol., e9 (9). 6. Siekmnn, A. F. & Lwson, N. D. Notch signlling limits ngiogenic cell ehviourin eveloping zerfish rteries. Nture, 7878 (7). 7. Leslie, J. D. et l. Enothelil signlling y the Notch lign Delt-like restricts ngiogenesis. Development 3, 8398 (7). 8. Jkosson, L. et l. Heprn sulfte in trns potentites VEGFR-meite ngiogenesis. Dev. Cell, 663 (6). 9. Jkosson, L. et l. Enothelil cells ynmiclly compete for the tip cell position uring ngiogenic sprouting. Nture Cell Biol., 9393 ().. Picr, F. et l. Sirt promotes ft moiliztion in white ipocytes y repressing PPAR-gmm. Nture 9, ().. Hishr, S. et l. Histone ecetylse moultes neuronl ifferentition y its nucler trnsloction. Proc. Ntl Ac. Sci. USA, 996 (8).. Tkt, T. & Ishikw, F. Humn Sir-relte protein ssocites with the HLH repressors HES n HEY n is involve in HES- n HEY-meite trnscriptionl repression. Biochem. Biophys. Res. Commun. 3, 7 (3). 3. Prozorovski, T. et l. Sirt contriutes criticlly to the reox-epenent fte of neurl progenitors. Nture Cell Biol., 3839 (8).. Kitmur, T. et l. A Foxo/Notch pthwy controls myogenic ifferentition n fier type specifiction. J. Clin. Invest. 7, 778 (7).. Donmez, G., Wng, D., Cohen, D. E. & Gurente, L. suppresses -myloi prouction y ctivting the lph-secretse gene ADAM. Cell, 333 (). 6. Finkel, T., Deng, C. X. & Mostoslvsky, R. Recent progress in the iology n physiology of sirtuins. Nture 6, 879 (9). Supplementry Informtion is linke to the online version of the pper t Acknowlegements We re thnkful to F. W. Alt, R. Kopn, Z. Lou, E. Seto, S. L. Berger, S. Dine Hywr, S. McMhon, G. Thurston n N. D. Lwson for regents n to I. Dikic for comments. This work ws supporte y grnts from the DFG (PO36/-, SFB 83/A6 n Exc 7/). F.D. ws supporte y the Interuniversity Attrction Poles ProgrmBelgin Science Policy (IUAP-BELSPO PVI/8). R.M. is supporte y the Siney Kimmel Cncer Reserch Fountion, New Investigtor Grnt from the Msschusetts Life Sciences Center, n AFAR Reserch Grnt n NIH grnts (RDK889-A nrgm937-). H.G. is supporte y Cncer Reserch UK, the Europen Moleculr Biology Orgnistion Young Investigtor Progrmme, n The Lister Institute of Preventive Meicine. H.G. n K.B. re supporte y the Fontion Leucq Trnstlntic Network of Excellence ARTEMIS. C.A.F. is supporte y the Mrie Curie FP7 People inititive. G.D. n M.M. thnk F. Pezzimenti for fish cre n technicl help, n AIRC (Associzione Itlin per l Ricerc sul Cncro) for finncil support. Author Contriutions V.G. n M.P. esigne n guie reserch. V.G., G.D., C.A.F., M.K., L.-K.P., K.B., L.T., F.D., M.H.H.S., B.Z., R.P.B., M.M., H.G. n M.P. performe reserch. V.G., G.D., C.A.F., M.K., L.-K.P., K.B., L.T., F.D., M.M., H.G. n M.P. nlyse t. R.M., C.H.W. n T.B. provie regents n/or technicl support. T.B., A.M.Z. n S.D. gve conceptul vice. V.G., H.G. n M.P. wrote the pper. All uthors commente on the mnuscript. Author Informtion Reprints n permissions informtion is ville t The uthors eclre no competing finncil interests. Reers re welcome to comment on the online version of this rticle t Corresponence n requests for mterils shoul e resse to M.P. (potente@em.uni-frnkfurt.e). Mcmilln Pulishers Limite. All rights reserve MONTH VOL NATURE

6 RESEARCH LETTER METHODS Cells n cell culture. Poole humn umilicl vein enothelil cells (HUVECs) were purchse from Lonz n culture s escrie 7. HEK93T cells were purchse from ATCC n Invitrogen n culture s recommene. Mouse lung enothelil cells (MLECs) were isolte, purifie n culture s escrie 8. DLL stimultion of enothelil cells. Lyophilize recominnt humn or mouse DLL ws purchse from R&D Systems n reconstitute t mgml in PBS contining.% ovine serum lumin. For stimultion of culture enothelil cells, DLL ws immoilize y coting culture ishes with mgml DLL in PBS for h t room temperture or overnight t uc. Regents n phrmcologicl in vitro cell tretments. HUVECs or HEK93 cells were phrmcologiclly trete with mm trichosttin A (TSA; Cliochem), mm nicotinmie (NAM; Sigm), mm MG3 (Cliochem) or mgml cyclohexemie (CHX; Cliochem). SRT83 ws use t concentrtion of mm (Sirtris Phrmceuticls). Control groups were trete with the respective vehicles. To inhiit Notch signlling in vitro, HUVECswereincutewith.8mM DBZ ((S,S)--[-(3,-Difluorophenyl)cetylmino]-N-(-methyl-6-oxo-6,7-ihyro-Hienzo[,]zepin-7-yl)propionmie). When HUVECs were co-stimulte with DLL, cells were pre-trete for t lest h efore eing replte on DLL-cote ishes. Plsmis n trnsfections. The intrcellulr omin of murine Notch (p,3 to p 7,93) n Notch (p,7 to p,89) ws clone in-frme into the mmmlin expression vectors pcdna3./nv-dest or pdest s well s pcdna3. N-Flg n pdest9 hrouring V or Flg tgs t the N or C terminus, respectively. Humn, SIRT, SIRT3 n SIRT were suclone in erivtive of the pcdna3.() vector to generte C-terminl Flg-tgge fusions s escrie 8. The seril eletions mutnts of were provie y Z. Lou 9, the (DC)Myc y R. Kopn, the p3 expression plsmi y R. Eckner, PCAFFlg y S. L. Berger n TIP6Flg y S. McMhon. The Notch-regulte luciferse reporter genes TP n 3CBF were from U. Zimer-Strol n D. Hywr, respectively. Trnsient trnsfections of HEK93 cells were crrie out with Lipofectmine trnsfection regent (Invitrogen). HUVECs were grown to 67% confluence n trnsfecte with the Trns Pss V regents (New Engln Biols) s recommene. RNA interference. To silence gene expression, cells were trnsfecte with specific synthesize y Eurogentec (9-GAAGTTGACCTCCTC ATTG-39) or vlite pool of uplexes irecte ginst humn (On-Trget plus SMART pool), which ws purchse from Dhrmcon. A scrmle ws use s control (9-TTCTCCGAACGTGGCACGA-39). HUVECs n HEK93 cells were trnsfecte with the inicte s ( nm) using the GeneTrns II regent (MoBiTec) or Lipofectmine (Invitrogen), respectively. Luciferse ssys. Reporter ssys in HUVECs were performe with the Dul- Luciferse Reporter Assy System (Promeg) n LUMAT LB 97 luminometer (BERTHOLD Technologies). Briefly, h fter co-trnsfection with the Notch luciferse reporters n the constitutive Renill luciferse reporter pgl.7hrluc/tk (Promeg) HUVECs were lyse n reporter ssys performe ccoring to the mnufcturers protocols. For experiments, cells were trnsfecte with s n fter h trnsfecte with the Notch luciferse reporters n the constitutive Renill luciferse reporter. For experiments in which Notch ctivity ws inuce y DLL, trnsfecte HUVECs were replte on DLL-cote ishes 6 h fter plsmi trnsfections. Luciferse ctivity ws mesure fter n itionl h. Reporter ctivity ws juste for the internl Renill luciferse control n is expresse reltive to control. RNA nlysis y rel-time quntittive PCR. RNAfromHUVECsorMLECs ws isolte with the RNesy Kit (Qigen) s recommene. TqMn Gene Expression Assys for HEY/Hey (Hs36_m; Mm698_m), NRARP/ Nrrp (Hs_m; Mm89_m), Lfng (Mm68_m), NOTCH (Hs6_m) n ADAM/Am (Hs383_m; Mm7_m) were otine from Applie Biosystems n qpcr ws crrie out using the StepOnePlus rel-time PCR system (Applie Biosystems). Co-immunoprecipittions. For co-immunoprecipittions of overexpresse proteins, HEK93 cells were lyse h post-trnsfection in IPLS uffer ( mm Tris-HCL ph7., mm NCl,. mm EDTA n,% Noniet P-). After pre-clering, immunoprecipittions were performe using irect-conjugte immuno-ffinity grose es ginst Flg (Sigm), Myc (Sigm) or V (Invitrogen) t uc with gentle rottion over night. After western lotting, the presence of the co-immunopurifie proteins ws nlyse with ntioies ginst Flg (:, Sigm), Myc (:, Snt Cruz) n V (:, Invitrogen). For co-immunoprecipittion of enogenous with enogenous, HUVECs were pretrete with mm MG3 (Cliochem) for h n then re-plte on DLL-cote culture ishes in the presence of MG3 for 6 h. Cells were lyse in IPLS uffer. After pre-clering, ws immunopurifie with mouse nti-humn monoclonl ntioy (Snt Cruz) for h t uc followe y incution with A/G grose es (Snt Cruz) for n itionl h. Immune complexes were nlyse y western lotting using (:,, Cell Signling Technology) n (:,, Cell Signling Technology) ntioies. Detection of cetyltion. For etection of cetylte in HEK93 cells, V-trnsfecte HEK93 cells were trete with NAM, TSA, the respective vehicles or comintions thereof for 6 or 6 h. Acetyltion ws lso mesure in trnsfecte cells h fter V overexpression. For oth pproches, cells were lyse in RIPA uffer supplemente with mm TSA n mm NAM (AcRIPA). After preclering cell extrcts were sujecte to immunoprecipittion using grose es couple to V ntioy. The immune complexes were nlyse y western lotting with polyclonl nti-cetylte lysine ntioies (AcK, :3, Cell Signling or :,, Acm). For etection of enogenous cetylte, HUVECs were plte on DLL-cote culture ishes n stimulte for 6 h in the presence or sence of NAM. HUVECs were lyse in AcRIPA uffer. After pre-clering, ws purifie from totl cell lystes with Notch-specific ntioy (Cell Signling) for h t uc followe y incution with protein A/G grose es (Snt Cruz) for n itionl h. Western lotting ws performe with n ntioy trgeting cetylte lysines (:3, Cell Signling). In vitro ecetyltion ssy. (DC)Myc ws expresse in HEK93 cells lone or together with p3 n immunopurifie using n nti-myc ntioy. Immunoprecipitte (DC)Myc ws four times in IPLS uffer n once in ST uffer ( mm Tris-HCL, ph 9, mm MgCl n. mm DTT). Purifie (DC)Myc ws then incute with recominnt wil-type or ecetylse efective (H363Y) in 3 ml of ST uffer contining 8 nm TSA n supplemente or not with mm ofnad. Rections were incute t 3 uc uner roust gittion for h n stoppe y ition of Lemmli uffer. Acetyltion of wsthen ssesse y western lotting using n nti-cetylte lysine ntioy. Uiquitintion ssy. For etection of uiquitinte in HEK93 cells, V ws overexpresse in HEK93 cells together with HA-tgge uiquitin. The effect of on uiquitintion ws ssesse fter knockown of y or fter NAM tretment. Cells were lyse in RIPA uffer n sujecte to immunoprecipittion using nti-v-couple grose es. The immune complexes were nlyse y western lotting with nti-ha ntioies (HA, :, Roche). For quntifiction of uiquitintion the nti-ha signl ws normlize to the V levels. Western lot nlysis n ntioies. SDSPAGE n western lot nlyses were performe ccoring to stnr proceures n etecte with the ECL etection kit (GE Helth Cre Bio-Sciences). Quntifiction of n intensities y ensitometry ws crrie out using the Imge J softwre. Mss spectrometric nlysis. HEK93 cells were trnsfecte with V n PCAFFlg n h fter trnsfection, trete with nicotinmie for 6 h. After V immunoprecipittion proteins were seprte y one-imensionl SDSPAGE (% Novex-gels, Invitrogen) n stine with colloil Coomssie. V gel ns were excise n sujecte to in-gel igest with trypsin 3. The resulting tryptic pepties were extrcte with cetonitrile, n eslte with reverse phse C8 STAGE tips 3. Mss spectrometric experiments were performe on nno-flow HPLC system (Proxeon) connecte to LTQ- Oritrp Velos instrument (Thermo Fisher Scientific) equippe with nnoelectrospry source (Proxeon). The mss spectrometer ws operte in the t epenent moe to monitor MS n MS/MS spectr. Survey full-scn MS spectr (from m/z 3,) were cquire in the Oritrp with resolution of R 6, t m/z fter ccumultion of,, ions. The five most intense ions from the preview survey scn elivere y the Oritrp were sequence y collision-inuce issocition (CID) in the LTQ. For higher C-trp issocition (HCD) 3, ions were ccumulte in the C-trp n MS/MS spectr were etecte in the oritrp t resolution of 7, (ref. 3). Mss spectr were nlyse using MxQunt softwre 33 n utomte tse serching (Mtrix Science). All tnem mss spectr were serche ginst the mouse Interntionl Protein Inex protein sequence tse (IPI version 3.) n conctente with reverse copies of ll sequences. The require flse positive rte ws set to % t the protein level, n mximum llowe mss evition ws set to p.p.m. in MS moe n. D for MS/MS peks. Cysteine crmiomethyltion ws serche s fixe moifiction n N-cetyl protein, oxiize methionine n cetyltion of lysine ws serche s vrile moifictions. A mximum of three misse clevges ws llowe. Site-irecte mutgenesis. The cetyltion-efective (KR)V mutnt ws generte y site-irecte mutgenesis replcing the cetylte lysines y rginine using the QuikChnge Lightning Multi Site-Directe Mutgenesis Kit (Agilent technologies). Primer sequences re ville upon request. In ition, we generte synthetic gene encoing for the murine sequence hrouring the lysine-to-rginine sustitutions t the sites ientifie y mss spectrometry Mcmilln Pulishers Limite. All rights reserve

7 RESEARCH (Genert). Using Gtewy cloning the synthesize gene ws clone in frme into the mmmlin expression vector pdest9 contining V tg t the C terminus. Enothelil cell prolifertion n three-imensionl sprouting ssy. Cell growth ws ssye y colorimetric proceure with the cell counting kit-8 (Dojino Lortories), moifie from metho tht uses 3-(,-imethylthizol- -yl)-,-iphenyltetrzolium romie s recommene. HUVECs were culture in DLL or solvent n trete with DBZ (.8 mm) or DMSO (solvent). The cell counting kit-8 regent ws e to the cultures ccoring to mnufcturer s instructions n growth ssesse fter,, 8, 7 n 96 h in culture. Enothelil cell spherois of efine cell numer were generte s escrie previously 7 n sprouting ssesse fter h. Mouse emryonic stem cells n emryoi oies ssy. DsRe-MST ES cells n Dll /lcz ES cells were otine from A. Ngy n G. Thurston (Regeneron Phrmceuticls), respectively, n hve een escrie previously 3,9. The genertion of Sirt mutnt ES cells ws previously escrie 3. The lck of exon in the Sirt gene genertes truncte protein tht is ctlyticlly inctive n oth cells n mice exhiit the sme phenotype s the full Sirt knockout, s previously escrie 3. ES cell culture n genertion of EBs were performe s escrie 8. The following primry ntioies were use for etection: rt ntimouse CD3 ntioy (BD Biosciences), rit nti-mouse cleve Notch (Acm). Seconry ntioies: Alex 67 onkey nti-rt IgG, Alex 88 onkey nti-rit, (Moleculr Proes), n DAPI (Sigm) for stining of nuclei. Quntifiction nlysis of tip cells n levels in EB sprouts. Complete high-resolution three-imensionl renering of EBs ws cquire using Lser Scnning Microscope 7 (Zeiss) equippe with motorize stge. For quntifiction of tip cells, tip cells per EB were mnully score n mrke for genotypic origin using the Imris 7.. softwre. Tip regions were efine s the regions from the very tips to the secon or thir vsculr loop. Quntifiction ws one using Prism.c softwre. For the quntifiction of protein levels, complete, 3,, it imges of vsculr sprouts were collecte with 363 ojective lens using Lser Scnning Microscope 7 (Zeiss), keeping the sme settings for ll imges (Supplementry Fig. 6). Imges were processe using the Imris 7.. softwre. To ientify nucler spots we use Surfce Imris function. Mesurements of numer of spots, volume n totl pixel intensity of ech spot were tken n sttistics were nlyse using Prism.c softwre. Zerfish strins, mintennce n rug tretment. Zerfish strins were mintine n re uner stnr conitions. AB wil type strin, Tg(fli:nEGFP)y7 n Tg(fli:EGFP)y 36 lines were use. Fish were rise/mintine ccoring to EU regultions on lortory nimls. DAPT (Sigm) ws issolve in DMSO n e to E3 mei to give finl concentrtion of mmdaptplus.%dmso. Emryos were echorionte n incute t 8. uc in E3 contining mm DAPT n.% DMSO from the en of gstrultion until 8 h.p.f. E3 contining DMSO lone ws use s control. Antisense morpholino oligonucleoties, fluorescence microscopy n cell counts. Antisense morpholino oligonucleoties ginst the exonintron splice site, 9-CGAACCAAACTCACCAATCTGTGGC-39, specificlly trgeting zygotic sirt (MO-sirt, splice locking MO) 8, n the 9-GTTCGAGCTTACCGG CCACCCAAAG-39, specificlly trgeting zygotic ll (MO-ll, splice locking MO) 6 were use. For cell counting experiments, 8 h.p.f. Tg(fli:nEGFP)y7 emryos were fixe in % prformlehye in PBS contining CCl. mm n % sucrose overnight t uc n were then wshe in PBS t room temperture. Emryos were permeilize for 3 h t room temperture n then mounte in 8% glycerol solution. Using confocl microscope, enothelil cells of Tg(fli:nEGFP)y7 were counte from projections of Z-series of opticl sections. The region counte in ech specimen spnne two somites (three intersegmentl vessels). Immunofluorescence n nlysis of postntl ngiogenesis in the mouse retin. Enothelil-restricte Sirt mutnt mice were on C7BL/6 genetic ckgroun n generte s escrie 8. Mice were kille t postntl y (P) n their eyes collecte for nlysis. To inhiit Notch signlling in mice, DAPT (Cliochem) ws issolve in % ethnol n 9% corn oil. The DAPT solution ws then injecte intrperitonelly t ose of mg kg per mouse. Control mice were injecte with the sme mount of vehicle. Mice were trete for 8 h. Injections were performe once y, t P3 n P n eyes hrveste t P. For whole-mount nlysis of retinl loo vessels, retins were fixe in % PFA for h t uc, or in MeOH t uc. Antioies were ilute in PBS contining.% BSA n.% Tween except for isolectin-b, which ws ilute in PBlec (PBS (ph 6.8), % Triton X-, mm CCl, mm MgCl, mm MnCl ). Primry ntioies use: iotinylte-isolectin-b (Vector Lortories n Moleculr Proes), BrU (Moleculr Proes) n (Upstte). Alex-88- or 68- conjugte seconry ntioies were use (Moleculr Proes). For lelling of proliferting cells, mg kg of BrU (BD Biosciences) per pup ws injecte intrperitonelly 3 h efore they were kille. Retins were fixe for h in % PFA n thn incute for h in N HCl. Retins were locke with % BSA/.% Tween in PBS n incute overnight t uc with mouse monoclonl nti-bru ntioy. Seconry etection ws performe with got nti-mouse Alex Fluor-couple seconry ntioy. After BrU stining, retins were fixe for 3 min in % PFA, wshe n thn stine with irectly conjugte Isolectin- B. Retins were flt mounte (Dko fluorescent mounting meium) n confocl lser scnning microscopy ws performe using Zeiss LSM microscope. All quntifictions were one with high-resolution confocl imges using Z-sections of the smple. For quntifying vessel migrtion, the istnce of vessel growth from the optic nerve to the periphery ws mesure using the ImgeJ softwre. For vessel ensity, the numer of vessel rnch points ws counte from five fiels per retin size 3 mm, from t lest retin smples per group. The numer of filopoil extensions were quntifie t the ngiogenic front n counte in t lest 8 fiels (size mm, 6 retins per group). The totl numer of filopoi ws normlize to mm of vessel length t the ngiogenic front. BrUlelle isolectin B-positive enothelil cells were counte in 3 fiels (size 3 mm, 6 retins per group). Computtionl moelling of enothelil tip-cell selection. A vessel segment comprising seven cells, with one cell per vessel cross-section, ws use with ll prmeters set s escrie. Sirt loss ws moelle y extening the numer of time steps tht the current ctive Notch level, which represents in the moel, ffecte ownstrem trgets. In the selection moel, Notch ctivity les to ownregultion of VEGFR receptors. It ws only effective for one time step when moelling wil-type vessels (one time step representing s in the moel). To mtch the oserve.-fol increse in ue to the loss of Sirt (referre to s Sirt null vessels), the time the current ctive Notch level ws effective for ws extene to three time steps; however, on the thir time step only hlf of tht level continue to ffect VEGFR ownregultion. To investigte the sensitivity n effect of incresing the lifetime, further simultions were performe where it ws set to hypotheticl.- n then 9.-fol increse, using similr pproch. Sttisticl nlysis. Sttisticl nlysis ws performe y ANOVA followe y Bonferroni s multiple comprison test, or Stuent s t-test using Prism (GrphPAD Softwre Inc.). P vlues of,. were consiere significnt. 7. Potente, M. et l. Involvement of Foxo trnscription fctors in ngiogenesis n postntl neovsculriztion. J. Clin. Invest., 3839 (). 8. North, B. J. et l. The humn Sir ortholog, SIRT, is n NAD-epenent tuulin ecetylse. Mol. Cell, 37 (3). 9. Kim, J. E., Chen, J. & Lou, Z. DBC is negtive regultor of. Nture, 8386 (8). 3. Shevchenko, A. et l. In-gel igestion for mss spectrometric chrcteriztion of proteins n proteomes. Nture Protocols, 8686 (6). 3. Rppsiler, J., Mnn, M. & Ishihm, Y. Protocol for micro-purifiction, enrichment, pre-frctiontion n storge of pepties for proteomics using StgeTips. Nture Protocols, (7). 3. Olsen, J. V. et l. A ul pressure liner ion trp Oritrp instrument with very high sequencing spee. Mol. Cell. Proteomics 8, (9). 33. Cox, J. & Mnn, M. MxQunt enles high peptie ientifiction rtes, iniviulize p.p..-rnge mss ccurcies n proteome-wie protein quntifiction. Nture Biotechnol. 6, (8). 3. Vintersten, K. et l. Mouse in re: re fluorescent protein expression in mouse ES cells, emryos, n ult nimls. Genesis, 6 (). 3. Cheng, H. L. et l. Developmentl efects n p3 hypercetyltion in Sir homolog ()-eficient mice. Proc. Ntl Ac. Sci. USA, (3). 36. Lwson, N. D. & Weinstein, B. M. In vivo imging of emryonic vsculr evelopment using trnsgenic zerfish. Dev. Biol. 8, 3738 (). Mcmilln Pulishers Limite. All rights reserve