N6-methyladenosine (m6a) is the most prevalent messenger

Transcription

1 m 6 A mrna methyltion regultes tivity to promote the prolifertion n tumorigeniity of enometril ner Jun Liu,,, Mrk A. Ekert,, Bryn T. Hr,,, Song-Mei Liu,, Zhike Lu,, Kngkng Yu,,5, Smnth M. Tien, Agnieszk Chryplewiz, Allen C. Zhu,,6, Ying Yng, Jing-To Hung, Sho-Min Chen, Zhi-Go Xu 7, Xio-Hu Leng 8, Xue-Chen Yu 9, Jie Co, Zezhou Zhng, Jinzho Liu, Ernst Lengyel * n Chun He,, * N 6 -methylenosine (m 6 A) messenger RNA methyltion is gene regultory mehnism ffeting ell ifferentition n prolifertion in evelopment n ner. To stuy the roles of m 6 A mrna methyltion in ell prolifertion n tumorigeniity, we investigte humn enometril ner in whih hotspot R98P muttion is present in key omponent of the methyltrnsferse omplex (METTL). We foun tht out 7% of enometril tumours exhiit reutions in m 6 A methyltion tht re proly ue to either this METTL muttion or reue expression of, nother omponent of the methyltrnsferse omplex. These hnges le to inrese prolifertion n tumorigeniity of enometril ner ells, likely through tivtion of the pthwy. Reutions in m 6 A methyltion le to erese expression of the negtive regultor PHLPP n inrese expression of the positive regultor mtorc. Together, these results revel reue m 6 A mrna methyltion s n onogeni mehnism in enometril ner n ientify m 6 A methyltion s regultor of signlling. N6-methylenosine (m6a) is the most prevlent messenger RNA moifition in humns,. This moifition is reversile, n its iologil effets re mostly meite through writer, erser n reer proteins,. A writer omplex, onsisting of ore METTL m 6 A methyltrnsferse long with regultory suunits 8, tlyses the m 6 A methyltion of mrna. At lest two erser enzymes, FTO n ALKBH5, meite the reversl of this methyltion,9. m 6 A methylte trnsripts re reognize y reer proteins tht regulte pre-mrna proessing, trnsltion 5 9 n egrtion,9,. m 6 A-epenent mrna regultion is essentil in mmmls, n efets in m 6 A methyltion ffet iverse iologil proesses,. In prtiulr, m 6 A mrna methyltion regultes the self-renewl n ifferentition of stem ells y ffeting mrna turnover uring ell ifferentition n plys ritil roles in trnsriptome swithing uring emryoni evelopment 8,. Consistent with these roles, m 6 A mrna methyltion is emerging s pthwy ffeting ner initition n progression in vriety of ners 5. m 6 A mrna methyltion ffets the growth n prolifertion of stem ells n ner ells 8,,,6 5. However, how m 6 A methyltion ffets ell growth n whih unerlying pthwys n mehnisms meite these hnges re still not fully eluite. Herein, we stuy this question in enometril ner, where sequening stuies hve ientifie frequent muttion of the m 6 A methyltrnsferse suunit METTL (ref. 6 ). We foun tht out 7% of enometril tumours exhiit reue m 6 A methyltion when ompre with mthe, norml enometrium. These reutions in m 6 A methyltion were proly use y either muttion of METTL or reue expression of the methyltrnsferse. Reuing m 6 A mrna levels in enometril ner ells through either METTL muttion or ownregultion oul enhne ell prolifertion n tumorigeniity in vitro n in vivo. m 6 A-seq hrteriztion of enometril ner ptient tumours n ell lines revele tht reue m 6 A mrna methyltion oul promote ell prolifertion y ltering the expression of key enzymes tht ffet the signlling pthwy. Inhiition of tivtion reverse the inrese prolifertion use y reue m 6 A methyltion. Together, these results hrterize somti muttion of the m 6 A methyltion mhinery s n importnt ftor promoting ner progression, revel tht reue m 6 A mrna methyltion is most likely n onogeni mehnism unerlying lrge portion of enometril ners n ientify m 6 A methyltion s regultor of the pthwy n ell growth. Results Loss-of-funtion METTL muttions in enometril ner. Sequening stuies hve foun tht the METTL suunit of the ore m 6 A methyltrnsferse omplex is frequently mutte Deprtment of Chemistry n Institute for Biophysil Dynmis, The University of Chigo, Chigo, IL, USA. Howr Hughes Meil Institute, Chigo, IL, USA. Deprtment of Ostetris n Gyneology/Setion of Gyneologi Onology, The University of Chigo, Chigo, IL, USA. Center for Gene Dignosis, Zhongnn Hospitl of Wuhn University, Wuhn, Chin. 5 College of Chemistry, Sihun University, Chengu, Chin. 6 Committee on Cner Biology n Meil Sientist Trining Progrm, The University of Chigo, Chigo, IL, USA. 7 Deprtment of Pthology, Zhongnn Hospitl of Wuhn University, Wuhn, Chin. 8 Huei Key Lortory of Tumor Biologil Behviors & Huei Cner Clinil Stuy Center, Zhongnn Hospitl of Wuhn University, Wuhn, Chin. 9 Deprtment of Ostetris n Gyneology, Zhongnn Hospitl of Wuhn University, Wuhn, Chin. MOE Key Lortory of Mromoleulr Synthesis n Funtionliztion, Deprtment of Polymer Siene n Engineering, Zhejing University, Hngzhou, Chin. Deprtment of Biohemistry n Moleulr Biology, The University of Chigo, Chigo, IL, USA. These uthors ontriute eqully: Jun Liu, Mrk A. Ekert, Bryn T. Hr, Song-Mei Liu. *e-mil: elengyel@uhigo.eu; hunhe@uhigo.eu 7 Nture Cell Biology VOL SEPTEMBER

2 -m 6 A/G. P = 8 wt METTL mu METTL Perentge m 6 A/A in mrna P = 57 P = 5.6 wt METTL mu METTL Tumour Tumour jent Prolifertion.5. wt METTL mu METTL 6 8 Perentge m 6 A/A in mrna Ptient Ptient Ptient e Perentge m 6 A/A in mrna P = 6 Tumour Tumour jent h f Expression in tumour/ tumour jent Norml enometrium P = P =. METTL P = FTO P = ALKBH5 Enometril ner (G) P = 5 5 YTHDF P = 5 YTHDF. g Reltive mrna level.5. P = 5 r =.6 P = 5.. Perentge m 6 A/A 5 µm Proportion of smples Norml Tumour Fig. The METTL(R98P) muttion n reue expression ontriute to erese m 6 A mrna methyltion in enometril ner ptients, The methyltrnsferse tivity of the METTL omplex ontining either the METTL(R98P) mutnt (mu) or wil-type (wt) METTL ws etermine y mesuring the -m 6 A/G rtio y liqui hromtogrphy tnem mss spetrometry (LC-MS/MS) fter inution of the methyltrnsferse omplex with RNA proe. We inepenently purifie two thes of protein n rrie out two inepenent trils per protein preprtion for totl of n = inepenent trils., LC-MS/MS quntifition of the m 6 A/A rtio in poly(a) RNA isolte from HEC--A ells overexpressing wil-type METTL, mutnt METTL or empty vetor ontrol. n = iologil replites., Cell prolifertion of HEC--A ells ws mesure y MTS ssy fter trnsfetion with the inite regents. n = iologil replites. For, error rs inite men ± s.e.m., LC-MS/MS quntifition of the m 6 A/A rtio in poly(a) RNA isolte from three enometril tumours with METTL(R98P) muttion n jent norml enometrium. The r shows the men from n = tehnil replites per ptient. e, Box plot of the reltive m 6 A levels in poly(a) RNA isolte from enometril tumour tissues versus tumour-jent tissues, n = 8 tumour norml pirs. f, Box plot of the expression levels of, METTL, FTO, ALKBH5, YTHDF n YTHDF in tumour tissues reltive to tumour-jent tissues, n = tumour norml pirs for METTL n FTO, n n = 8 tumour norml pirs for the others. For, e n f, the P-vlues were etermine y two-tile t-test. See methos for ox-plot hrteristis. g, Stter plot showing the orreltion of m 6 A methyltion level with the expression of. The liner est fit line is shown in re. The Person orreltion oeffiient (r) n P-vlue (P) from two-tile t-test of r = re shown, n = 8 tumour norml pirs. h, Left: IHC stining of enometril tissue mirorry ores for. Right: Quntifition of IHC stining in norml enometrium (n = ores) n epithelil enometril tumours (n = ores). Stining ws ssesse using utomte softwre 5 n sore on sle of (no stining) to (high stining). Sle rs, 5 μ m. P-vlue etermine y χ -test. in enometril tumours 6, ut the relevne of these muttions n of m 6 A mrna methyltion to the isese hs not yet een estlishe. The preominnt muttion ours t position 98 of METTL, is more prevlent thn other muttions in enometril tumours n ours in out.5% of enometril ner ptients 6. Crystl strutures of the METTL omplex revel tht the Arg 98 resiue lies in the puttive RNA-ining groove t the interfe etween the two suunits 7 9. Consistent with previous oservtions 8, we foun tht the R98P hotspot muttion signifintly reue the RNA methyltion tivity of the writer omplex in vitro (Fig. ). Wheres overexpression of wil-type METTL promote m 6 A methyltion of ellulr poly(a) RNAs in HEC--A enometril ner ells, the mutnt METTL ppere intive upon overexpression (Fig. ). While overexpression of wiltype METTL erese ell prolifertion, overexpression of the mutnt h no notiele effet on ell prolifertion (Fig. ), suggesting tht the METTL muttion is proly loss-of-funtion llele tht shows no eviene of further ominnt negtive effets on m 6 A methyltion or ell prolifertion. To exmine the onsequene of the muttion in tumour tissue, we ientifie three enometril tumour smples ering the METTL(R98P) muttion n purifie mrna from these tumours s well s from jent enign enometril tissues (Methos). Compre with mrna from the wil-type jent Nture Cell Biology VOL SEPTEMBER

3 Perentge m 6 A/A in mrna METTL +/ wt METTL mu METTL P = 5 P = 6 Cell prolifertion 8 METTL +/ wt METTL mu METTL No. of ells ( 5 ). P = 5 P = 9 5 Colonies P = 7 P = e f 6 8 P = 8 g METTL +/ wt METTL mu METTL h METTL +/ wt METTL mu METTL Reltive migrtion istne P = 5 P = METTL +/ wt METTL mu METTL Perentge m 6 A/A in mrna P = 98 sh sh.5 sh.8 Cell prolifertion sh sh.5 sh.8 8 No. of ells ( 5 ).5. P = 7 5 P = sh sh.5 sh.8 i No. of olonies P = P = j Reltive migrtion istne P = 8 P = 8 k Totl tumour weight (g).5. P = No. of peritonel tumours 8 P = 5 5 sh sh.5 sh.8 sh sh.5 sh.8 METTL +/ METTL +/ l P = P = 56 8 P = 5 m P = 5 Totl tumour weight (g) No. of peritonel tumours Totl tumour weight (g) 5 No. of peritonel tumours wt METTL mu METTL wt METTL mu METTL Fig. Reue m 6 A methyltion inreses ell prolifertion, nhorge-inepenent growth, migrtion n in vivo tumour growth., LC-MS/MS quntifition of the m 6 A/A rtio in poly(a) RNA from the inite HEC--A ell lines., Cell prolifertion mesure y MTS ssy of wil-type HEC- -A ells (), METTL +/ knokout ells, n knokout ells resue y stle trnsfetion of wil-type METTL (wt) or METTL(R98P) (mu). Cell numers were normlize to the MTS signl out 5 h fter ell seeing. e, Anhorge-inepenent ell growth (), olony formtion () n ell migrtion in woun-heling experiment (e) were ssesse for wil-type HEC--A ells, METTL +/ knokout ells, n knokout ells resue with wil-type or mutnt METL. f, LC-MS/MS quntifition of the m 6 A/A rtio in poly(a) RNA from the inite HEC--A ell lines. (g) Cell prolifertion mesure y MTS ssy of HEC--A ells stly expressing ontrol shrna (sh) versus shrna trgeting (sh.5, sh.8). Cell numers were normlize to the MTS signl out 5 h fter ell seeing. h j, Anhorge-inepenent ell growth (h), olony formtion (i) n ell migrtion in woun-heling ssy (j) were ssesse for HEC--A ells stly expressing ontrol shrna or shrna trgeting. For j, n = iologil replites. Error rs inite men ± s.e.m. P-vlues etermine y two-tile t-test. k m, Wil-type HEC--A ells n METTL +/ knokout ells (k), knokout ells resue with wil-type or mutnt METTL (l) n HEC--A ells with shrna knokown of or ontrol shrna (m) were injete into mie. The totl tumour weight (left) n the totl numer of tumours (right) were reore fter weeks. For k, n = 8 n for l n m n = mie per group. Error rs inite men ± s.e.m. P-vlues etermine y two-tile t-test. sh sh sh sh 76 Nture Cell Biology VOL SEPTEMBER

4 Counts 5,,5 log (E tumour /E norml ) Regultion of poptoti proess Regultion of phosphorus metolism Non-nonil Wnt signling pthwy Woun heling Protein kinse B signling Wnt signling pthwy Regultion of response to stimulus NIK/NF-kppB signling Regultion of ellulr protein Cell migrtion MAPK se Metolism Regultion of ell ommunition Regultion of ell yle proess Trnsport Posttrnsriptionl regultion of Regultion of ell yle gene expression Regultion of immune system proess Regultion of ell hesion Regultion of growth Cell hesion Regultion of ell prolifertion Cell-ell hesion 8 log (enrihment tumour ) 6 pthwy 6 8 log (enrihment norml ) GF Cytokine ECM RTK IRS CytokineR JAK IGTA FAK IGTB rf- MEK ERK CTMP PPA enos NO O PKD PIK PIP p CDK O p7 Cylin HSP9 mtorc FOXO -- BAD CCND PHLPP TCL Csp9 CREB Cell prolifertion Angiogenesis DNA repir Cell yle Apoptosis Fig. m 6 A-seq of tumours with reue m 6 A methyltion., Histogrm showing the hnges in m 6 A enrihment etween norml n tumour smples of ll peks showing enrihment in the norml tissue. The hnge in enrihment is the mein of n = 5 tumour norml pirs., GO term nlysis of trnsripts with reue m 6 A in tumour tissues versus jent norml tissues., Stter plot of the m 6 A enrihment in norml tumour-jent n tumour tissue for m 6 A peks in genes involve in the PIK/ pthwy. The re line is the y = x line. 65/765 of the m 6 A peks exmine show greter enrihment in the norml smple thn the tumour smple. The enrihment vlues re the mein of n = 5 ptient smples., Digrm of the PIK/ pthwy with genes ffete y m 6 A mrke in re. The igrm is se on KEGG nnottions 5. norml tissues, mrna from the three mutnt tumours h reue overll m 6 A methyltion (P =, pire two-tile t-test), suggesting tht the METTL(R98P) muttion inhiits m 6 A mrna methyltion in tumours (Fig. ). Enometril ner is ssoite with low levels of m 6 A mrna methyltion. Intriguingly, out 7% of ll tumours we exmine (inluing mjority of tumours with wil-type METTL) exhiite reue totl m 6 A mrna methyltion when ompre with jent, norml enometril tissues (Fig. e). Thus, we hypothesize tht enometril ner oul e more roly ssoite with the ltere expression of ftors tht regulte m 6 A mrna methyltion. To test this hypothesis, we evlute the expression of m 6 A writers, ersers n reers in tumour n jent norml enometril tissues y quntittive PCR with reverse trnsription (RT-qPCR) (Fig. f n Supplementry Fig. ). We foun tht mjority of enometril ners exhiite signifintly reue expression of the m 6 A methyltrnsferse when ompre with jent norml tissues. Derese expression orreltes with reue m 6 A methyltion in these tumour tissues (Fig. g). Immunohistohemistry of tissue mirorry with oth norml enometrium n epithelil enometril ner speimens revele signifint erese in expression in tumour tissue t the protein level (Fig. h). METTL muttion n erese expression pper to e mutully exlusive, s ll three of the tumours with the METTL muttion h norml expression of reltive to jent norml tissues. Anlysis of the TCGA enometril ner tset i not revel ny signifint orreltion etween the muttion sttus of frequently mutte genes in enometril ner n low expression (Supplementry Fig. ). Tken together, these results suggest tht lrge proportion of humn enometril tumours re hrterize y reue m 6 A mrna methyltion, through either METTL loss-of-funtion muttion or erese expression. Reue m 6 A methyltion promotes enometril ner ell prolifertion. Consiering tht out % of ll mrnas re methylte insie most mmmlin ells,, the oserve glol erese in m 6 A mrna methyltion oul hve signifint effets on ellulr physiology, in prtiulr if the methyltion of key trnsripts is ffete 5. Anlysis of ptients from the TCGA tset showe tht tumours with low expression re ssoite with slight inrese in mortlity, though this ifferene is not sttistilly signifint (Supplementry Fig. ). However, nlysis of other relte ner types (high-gre serous ovrin ner n pnreti enorinom ) foun sttistilly signifint inreses in mortlity ssoite with erese expression (Supplementry Fig. ). Therefore, we next exmine whether the reue m 6 A methyltion oserve in the humn enometril tumour tissue smples ffets funtions ssoite with tumour progression in humn tumour ells. To investigte the effets of METTL loss of funtion in enometril ner ell lines, we use CRISPR tehnology to elete METTL from HEC--A ells. We otine lones exhiiting only heterozygous knokout of METTL, refleting the essentil nture of the writer omplex in mmmls. Heterozygous knokout ws onfirme y western lot Nture Cell Biology VOL SEPTEMBER

5 METTL +/ wt METTL mu METTL p-(s7) p-(t8) sh sh.5 sh.8 shmettl 75 kd 75 kd 75 kd METTL +/ wt METTL mu METTL p-tuerin(t6) p-pras(t6) p-foxo(s56) sh sh.5 sh.8 shmettl 5 kd 7 kd 75 kd Re ensity 5 Tumour input PHLPP Tumour IP Norml input Norml IP 5 5 PRR5 5 5 mtor PHLPP p-mtor(s8) 5 kd 5 kd p-p7(t57) 5 kd 7 kd 5 5 PRR5L 7 kd 5 Reltive fol enrihment Reltive mrna level METTL +/.5 P = P = P = 8 P <. P < P = P = PHLPP PRR5 PRR5L mtor.5. wt METTL mu METTL sh sh.5 P < P = P = P = 5 P < P = 7 P = 8 P =. * * P < P < P < PHLPP PRR5 PRR5L P < P = P = mtor PHLPP PRR5 PRR5L mtor e Norml enometrium 5 µm f Norml enometrium 5 µm Enometril ner (G) Enometril ner (G) Proportion of smples Proportion of smples.. P = 7 Norml Tumour P = 9 Norml Tumour Fig. Reue m 6 A methyltion tivtes., Immunolot nlysing levels of phosphoryltion n expression of proteins tht regulte phosphoryltion in HEC--A ells with the inite perturtions to m 6 A methyltion., Immunolot exmining the phosphoryltion of trget proteins in HEC--A ells with the inite perturtions to m 6 A methyltion. Quntifition of the immunolots in n re presente in Supplementry Fig. f. Not ll pnels shown re from the sme immunolot, n rw gel imges with the pproprite loing ontrols re provie in Supplementry Figure 8. ) The verge re ensity from m 6 A-seq experiments on n = 5 tumour norml pirs showing the m 6 A peks ientifie in the PHLPP, PRR5, mtor n PRR5L trnsripts., m 6 A-IP omine with RT-qPCR ws use to quntify the reltive m 6 A level (top) n mrna levels (ottom) of PHLPP, PRR5, PRR5L n mtor trnsripts in the wil-type, METTL +/, wil-type METTL, mutnt METTL, sh n sh HEC--A ells. Error rs inite men ± s.e.m. from n = iologil replites. P-vlues etermine y two-tile t-test. e,f, Left: IHC stining of tissue mirorry ores for PHLPP (e) n PRR5 (f). Right: Quntifition of IHC stining in norml enometrium (n = ) n enometril tumours (n = ). Stining ws ssesse using utomte softwre 5 n sore on sle of (no stining) to (high stining). Sle rs, 5 μ m. The P-vlue ws etermine y χ -test. (Supplementry Fig. ) n sequening (Methos). As expete, the METTL +/ ells exhiite reue m 6 A mrna methyltion, n the reutions in m 6 A methyltion re similr to those oserve in the tumour smples (Fig. ). Consistent with role for METTL loss of funtion in enometril ner, heterozygous knokout of METTL inrese ell prolifertion, nhorgeinepenent growth, olony formtion, ell migrtion n invsion (Fig. e n Supplementry Fig. ). The reue m 6 A methyltion n hnges to the ner ell physiology oul e prtilly resue y stle expression of wil-type METTL ut not mutnt METTL (Fig. e n Supplementry Fig. ). We oserve similr effets fter short hirpin RNA (shrna) knokown of METTL versus ontrol shrna (Supplementry Fig., -h). To etermine the effets of reue expression in enometril ner ells, ws stly knoke own y shrna in HEC--A ells using two ifferent shrna sequenes (Supplementry Fig. i). Similr to the expression of mutnt METTL, knokown of erese the overll levels of m 6 A mrna methyltion n promote ell prolifertion, nhorge-inepenent growth, olony formtion, migrtion n invsion reltive to ontrol ells (Fig. f j n Supplementry Fig. j). To orroorte the oservtions tht reue m 6 A methyltion stimultes ggressive phenotypes of ner ells in vitro, we investigte the roles of METTL n in tumour growth in vivo. Wil-type HEC--A ells n METTL +/ knokout ells were injete into the peritonel vity of nue mie, n tumour numers n totl mss were evlute fter weeks. METTL +/ knokout ells showe rmtilly lrger tumours n n inrese numer of metstses reltive to wil-type HEC- -A ells (Fig. k). Similr trens were oserve when METTL +/ ells resue with wil-type n mutnt METTL were ompre (Fig. l) n when ompring knokown HEC--A ells with ontrol (Fig. m). Tken together, these results revel tht the reue m 6 A mrna methyltion oserve in the ptient enometril tumour smples, whether inue y METTL(R98P) muttion or reue expression, oul promote the tumorigeniity of enometril ner ells n might ply ritil roles in the progression of enometril ner. m 6 A-seq ientifies trnsripts with ltere methyltion in enometril tumours. Next, we performe m 6 A-seq nlysis of humn enometril tumour tissues versus norml, tumour-jent tissues from five ptients. All five tumours exhiite low totl m 6 A levels; one rrie the METTL muttion n the four others exhiite low expression (Supplementry Fig.,). Consistent with previous m 6 A-seq results,, the m 6 A peks we ientifie were enrihe ner the strt n stop oons n were hrterize y the nonil GGACU motif (Supplementry Fig.,). In the norml tissue smples, we ientifie on verge out, signifint m 6 A peks (flse isovery rte < 5) in out 8, trnsripts, n the ientifie trnsripts show goo greement etween smples (Supplementry Fig. e). Among the m 6 A peks etete in 78 Nture Cell Biology VOL SEPTEMBER

6 5 kd 5 kd 5 kd 7 kd Log(rtio) si siythdf siythdf si PHLPP p-mtor(s8) mtor Input IP Flowthrough PHLPP PRR5 PRR5L mtor e Reltive mrna level Log(rtio). 8 PHLPP P = 5 5 PRR5 P = P = 5 P = 5 PRR5L P = P = 5 mtor si si siythdf siythdf Input IP Flowthrough PHLPP PRR5 PRR5L mtor PHLPP normlize to input f Remining mrna fter Tl.6. si siythdf Nonriosome -6S t / =.86 h t / =.7 h R =.998 R = Time fter Tl (h) si siythdf Polysome g Remining mrna fter TI. si siythdf t / = 7.69 h t / =. h R =.98 R =.98 6 Time fter TI (h) h Remining mrna fter Tl. t / = 5.56 h t / = 7.7 h R =.99 R =.97 si siythdf 6 Time fter TI (h) i Remining mrna fter Tl. t / = 6.67 h t / =. h R =.99 R =.866 si siythdf 6 Time fter TI (h) Fig. 5 Regultion of pthwy genes y m 6 A reer proteins., Immunolot nlysing the levels of PHLPP, mtor n p-mtor(s8) in HEC- -A ells upon trnsient sirna knokown of YTHDF, YTHDF or. Quntifition of this immunolot is shown in Supplementry Fig. 5. Rw gel imges re provie in Supplementry Fig. 8., RT-qPCR ws use to quntify the reltive levels of PHLPP, PRR5, PRR5L n mtor upon trnsient sirna knokown of YTHDF, YTHDF or in HEC--A ells. Error rs inite men ± s.e.m. from n = iologil replites. P-vlues etermine y twotile t-test., Polysome profiling ws use to exmine the istriution of PHLLP trnsripts mong non-riosoml, riosome-ssoite n polysomessoite frtions. n = iologil replites.,e, YTHDF () n YTHDF (e) were immunopreipitte n RNA IP-qPCR ws use to ssess the ssoition of the inite trnsripts with eh protein. n = iologil replites. Error rs inite men ± s.e.m. Horizontl line inites no hnge for IP or flowthrough ompre to input. f i, RNA lifetime for PHLPP (f), PRR5 (g), PRR5L (h) n mtor (i) in HEC--A ells trnsfete with ontrol sirna or sirna trgeting YTHDF ws etermine y monitoring trnsript unne fter trnsription inhiition (TI). n = iologil replites. Error rs inite men ± s.e.m. For etils of the etermintion of the ey hlf-lives, see Methos. over hlf of the ptient smples, we foun tht their m 6 A mrna methyltion ws reue glolly in the tumour omprtment ompre with jent, norml ontrol tissues (Fig. ). The trnsripts exhiiting erese m 6 A methyltion were firly onsistent etween tissue smples (Supplementry Fig. f), n the trnsripts showing erese m 6 A methyltion in t lest two smples were enrihe for gene ontology (GO) terms relte to ell migrtion, prolifertion, growth, hesion n ell eth (Fig. ). m 6 A-seq experiments revele similr glol ereses in m 6 A methyltion n GO term enrihment in HEC--A knokown n mutnt METTL ells reltive to ontrols (Supplementry Fig. e-g n Supplementry Fig. ). m 6 A methyltion regultes tivtion of. The GO term nlysis ientifie the /protein kinse B signlling pthwy s eing signifintly ltere y reue m 6 A methyltion in oth the ptient smples (P =.5 8 ) n the enometril ner ell lines (P =. 8 ) (Fig. n Supplementry Fig. ). Beuse the signlling pthwy promotes ell survivl n growth n is frequently tivte through onogeni muttions in enometril ner n other ners 5, we hypothesize tht reue m 6 A methyltion might promote tumour growth through tivtion of the pthwy. Inee, mny of the genes involve in the pthwy showe reue m 6 A methyltion in tumours when ompre with tumour-jent tissues (Fig.,). We evlute suset of these trnsripts in the mutnt METTL n knokown ells y m 6 A immunopreipittion (m 6 A-IP) followe y RT-qPCR, whih onfirme their reue m 6 A methyltion (Supplementry Fig. -e). We next etermine if reue m 6 A methyltion in enometril ner ells ffets signlling y investigting the phosphoryltion sttus of. Our METTL loss-of-funtion HEC--A ell lines (METTL +/, mutnt METTL resue n shmettl) showe inrese phosphoryltion of t Ser-7 when ompre with the relevnt ontrol ell lines (wil-type HEC--A ells, wil-type METTL resue n sh, respetively) (Fig. ). Similr inreses in (S7) phosphoryltion were seen in the knokown HEC--A ell lines reltive to ontrol knokown ells (Fig. ). In ontrst, phosphoryltion t Thr-8 n the totl protein expression remine unhnge (Fig. ). To ssess whether these hnges in phosphoryltion stimulte signlling, we ssesse the phosphoryltion sttus of ownstrem effetors of (Fig. ). Both FOXO n p7 showe inrese phosphoryltion in the METTL loss-of-funtion n Nture Cell Biology VOL SEPTEMBER

7 Perentge m 6 A/A in mrna f.6 P = P = 76 P = P = 8 5 kd 5 kd 5 kd 5 kd 75 kd 5 kd 7 kd METTL si si simettl O/E O/E O/E METTL Prolifertion.5. p-(s7) PHLPP p-mtor(s8) p-foxo(s56) p-p7(t57) METTL 6 8 si si simettl g Prolifertion 5 kd 5 kd 5 kd 5 kd si si simettl.5. Meium Meium 6 8 Strve Strve si Colonies EGF ( ng ml ) / / 8 h simettl EGF ( ng ml ) / / 8 h P = 56 P = 9 METTL P = P = 8 p-(s7) p-(s7) si si simettl Meium Strve Intensity normlize to e Reltive migrtion istne P = P = P = P = 56 METTL si si si simettl EGF ( ng ml ) / / 8 h si si simettl 6 8 Fig. 6 Effets of m 6 A methyltion on non-trnsforme T-HESC enometril ell line. Effets of ltertions to m 6 A methyltion on non-trnsforme T-HESC enometril ells were exmine fter trnsient trnsfetion with ontrol sirna, sirna trgeting, sirna trgeting METTL, empty vetor, plsmi enoing or plsmi enoing METTL., LC-MS/MS quntifition of the m 6 A/A rtio in poly(a) RNA on trnsient trnsfetion of T-HESC ells fter the inite tretments.,, Cell prolifertion mesure y MTS ssy of T-HESCs trnsfete with the inite regents. Cell numers were normlize to the MTS signl out 5 h fter ell seeing., Colony formtion of T-HESCs trnsforme with the inite regents. e, Migrtion in wounheling ssy. For e, n = iologil replites n error rs inite men ± s.e.m. P-vlues etermine y two-tile t-test. f, Immunolot showing the effets of the inite perturtions to m 6 A methyltion on the expression n phosphoryltion of proteins involve in the pthwy in T-HESCs. O/E, overexpression. Three inepenent experiments hve een repete with similr results. g, Immunolots showing the time ourse of (S7) phosphoryltion fter EGF stimultion in T-HESCs trete with ontrol sirna or sirnas trgeting or METTL for 8 h. Plots quntifying the time ourse of EGF tivtion show men ± s.e.m. from n = iologil replites. Rw gel imges for f,g re provie in Supplementry Fig. 8. knokown ells reltive to ontrol. Two other sustrtes, tuerin n PRAS, showe no onsistent hnges in phosphoryltion, ongruent with previous reports tht only suset of trgets re ffete y hnges to Ser-7 phosphoryltion without ny ifferene in Thr- phosphoryltion 6. These results suggest tht reuing m 6 A methyltion tivtes the pthwy. m 6 A methyltion ontrols the expression of regultors of tivtion. To etermine the mehnisms unerlying inrese tivtion upon reue m 6 A methyltion, we exmine PHLPP, phosphtse regulting (S7) phosphoryltion 7, n mtorc, kinse tht phosphoryltes (S7) 8. Trnsripts enoing PHLPP n three omponents of the mtorc omplex (PRR5, PRR5L n mtor) showe erese m 6 A methyltion in ptient smples (Fig. ). These trnsripts lso showe erese m 6 A methyltion in the METTL loss-offuntion n knokown HEC--A ell lines (Fig. ). In these ell lines, we oserve erese expression of PHLPP protein, while its mrna levels were not notiely ltere; in ontrst, we oserve inrese mrna expression of PRR5, PRR5L n mtor in ition to inrese protein levels of p-mtor(s8), mrker for mtorc 9 (Fig.,). These hnges re onsistent with inrese phosphoryltion n tivity. To investigte whether these hnges in protein expression our in tumour tissues, we performe immunohistohemil (IHC) stining in norml enometrium n enometril tumours in tissue mirorry (Fig. e,f n Supplementry Fig g,h). PHLPP ws inee ownregulte in humn enometril tumours when ompre with enign enometril glns. We oserve inrese stining for PRR5, PRR5L n phospho-mtor(s8) in suset of tumours, though the inreses were not lwys sttistilly signifint, suggesting tht itionl ftors my e influening mtorc expression in tumours when ompre with our ell lines. We next explore the mehnism for how m 6 A methyltion regultes the expression of PHLPP n mtorc. Beuse m 6 A methyltion ppere to promote the expression of PHLPP, we hypothesize tht PHLPP trnsripts re trgets of YTHDF, the m 6 A reer protein tht promotes trnsltion of m 6 A methylte trnsripts 6. Consistent with this hypothesis, short interfering RNA (sirna) knokown of YTHDF in HEC--A ells erese expression of PHLPP to similr extent to knokown of (Fig. 5). These hnges in PHLPP expression re ue not to hnges in the unne of the PHLPP trnsript ut to the ssoition of the PHLPP trnsript with tively trnsriing riosomes (Fig. 5, n Supplementry Fig. 5,). mtorc, on the other hn, ppere to e ownregulte y m 6 A methyltion, suggesting tht it is trget of YTHDF, the m 6 A reer protein tht promotes the ey of m 6 A methylte trnsripts. Consistent with this hypothesis, sirna knokown of YTHDF inrese the unne of the PRR5, PRR5L n mtor trnsripts n these trnsripts showe erese RNA ey rtes upon knokown of YTHDF (Fig. 5,g i). Knokown of YTHDF lso resulte in higher protein levels of mtor n p-mtor(s8) (Fig. 5). The effets of the reer proteins ppere to e speifi to these sets of trnsripts, s knokown of YTHDF i not ppreily ffet the expression or ey of PHLPP (Fig. 5,,f), while knokown of YTHDF i not ppreily ffet the expression of mtor or p-mtor(s8) or the ssoition of PRR5, PRR5L or mtor trnsripts with riosomes (Fig. 5 n Supplementry Fig 5-f). The speifiity for reer funtion proly ours t the level of 8 Nture Cell Biology VOL SEPTEMBER

8 sh sh.5 wt METTL PHLPP mu METTL sh sh.5 wt METTL sirictor mu METTL 5 kd p-(s7) 5 kd p-(s7) 5 kd 5 kd 7 kd Flg 5 kd 5 kd Ritor 7 kd Cell prolifertion Cell prolifertion sh/ sh/phlpp sh/ sh/phlpp sh/si sh/sirictor sh/si sh/sirictor Cell prolifertion Cell prolifertion.5.5. wt METTL/ wt METTL/PHLPP mu METTL/ mu METTL/PHLPP 6 8 wt METTL/si wt METTL/siRICTOR mu METTL/si mu METTL/siRICTOR YTHDF m 6 A Trnsltion of PHLPP PHLPP P METTL AAA Cner METTL muttion ownregultion Ativity YTHDF m 6 A AAA PRR5, PRR5L, MTOR PRR5, PRR5L, MTOR ey Fig. 7 The pthwy meites the hnges in ell prolifertion from reue m 6 A methyltion.,, Immunolots nlysing the effet of Flg PHLPP overexpression () or RICTOR knokown () on phosphoryltion in HEC--A ells. Three inepenent experiments hve een replite with similr results. Rw gel imges re provie in Supplementry Fig. 8., Prolifertion mesure y MTS ssy of knokown versus ontrol knokown ells (left) or wil-type METTL versus mutnt METTL HEC--A ells (right). Cells were trnsiently trnsfete with PHLPP overexpression plsmi versus empty vetor (top) or sirnas trgeting RICTOR versus negtive ontrol sirnas (ottom), n = iologil replites; error rs inite men ± s.e.m., Moel showing how reue m 6 A methyltion lters signlling to ontriute to tumour progression. RNA ining, s PHLPP trnsripts interte more strongly with YTHDF thn YTHDF, while PRR5, PRR5L n mtor trnsripts interte more strongly with YTHDF thn YTHDF (Fig. 5,e). To etter unerstn the effets of m 6 A-meite hnges to PHLPP n mtorc expression on tivtion, we exmine the time ourse of tivtion upon stimultion of HEC--A ells. Consistent with previous stuies 7, we oserve trnsient phosphoryltion of in our knokown ontrol ells fter epierml growth ftor (EGF) stimultion, whih erese fter h; however, in the knokown ell line, tivtion persiste for muh longer perios of time (Supplementry Fig 6), onsistent with erese PHLPP expression. Similr results were oserve for the wil-type METTL versus mutnt METTL HEC--A ells (Supplementry Fig. 6), though the kinetis of ephosphoryltion were slower in the wil-type METTL ell line thn in the knokown ontrol ell line, perhps refleting inomplete resue of the HEC--A METTL +/ ells. To onfirm tht our results exten eyon the HEC--A enometril ner ell line, we teste the effets of knokown n overexpression of n METTL in seon enometril ner ell line, RL95-, s well s htert-immortlize humn enometril stroml ells (T-HESCs), norml non-trnsforme ell line, n foun similr m 6 A-meite hnges to ell prolifertion n signlling (Fig. 6 n Supplementry Fig. 6-f). Thus, erese m 6 A methyltion les to iminishe YTHDF-promote trnsltion of PHLPP, negtive regultor of tivtion, n prelues the YTHDF-promote ey of mrnas enoing the mtorc omplex, positive regultor of. Altogether, these experiments revel tht reue m 6 A mrna methyltion ffets multiple pthwy omponents to stimulte tivtion. Inrese signlling meites the effets of reue m 6 A methyltion on ell prolifertion. To etermine if enhne tivtion unerlies the inrese prolifertion oserve upon reuing m 6 A methyltion in enometril ner ells, we ttempte to resue this phenotype y either overexpressing PHLPP or y inhiiting mtorc through knokown of the mtorc-speifi suunit gene RICTOR. Consistent with previous results 7,8, overexpression of PHLPP (Fig. 7 n Supplementry Fig. 7) n knokown of RICTOR (Fig. 7 n Supplementry Fig. 7) oth erese the levels of p-(s7) in knokown, METTL mutnt n METTL +/- HEC--A ells. PHLPP overexpression or RICTOR knokown in knokown ells reue ell prolifertion rtes, wheres these tretments h muh smller effets on the ontrol ells (Fig. 7). Importntly, the ell prolifertion rtes of knokown ells fter PHLPP overexpression or RICTOR knokown were omprle to those of the ontrol ells with norml expression. Similr results were oserve in the METTL mutnt ells (Fig. 7) s well s the METTL +/ ells (Supplementry Fig. 7,). Similr results were lso seen when using smll-moleule inhiitor of the enzyme (Supplementry Fig. 7e,f). Thus, geneti or phrmologi Nture Cell Biology VOL SEPTEMBER

9 suppression of reverses the inrese prolifertion oserve in knokown n METTL loss-of-funtion ells. Disussion The PIK/ pthwy plys importnt roles in vriety of iologil proesses, n ysfuntionl signlling ontriutes to iseses suh s ner, ietes n utoimmune isese,5. In this stuy, we isovere tht m 6 A mrna methyltion regultes the pthwy to ontrol ell prolifertion in enometril ner (Fig. 7). m 6 A methyltion normlly ttenutes tivity in the enometrium y promoting the m 6 A-epenent trnsltion of PHLPP n m 6 A-epenent egrtion of trnsripts enoing suunits of mtorc. Upregultion of the PHLPP phosphtse n ownregultion of the mtorc kinse oth ontriute to the inhiition of tivity y mintining ephosphoryltion of (S7). Reue m 6 A methyltion isrupts the regultion of these trnsripts, leing to erese PHLPP expression, inrese mtorc expression n inrese tivity (Fig. 7). This mehnism proly ontriutes to lrge frtion of enometril tumours, s out 7% of tumour smples from enometril ner ptients exhiite erese m 6 A levels ue to either erese expression of or loss-of-funtion muttion in METTL. Using ulture enometril ner ells, we revele tht either muttion of METTL or ownregultion of reue m 6 A mrna methyltion n enhne prolifertion n tumorigeniity. Our m 6 A-seq results from enometril tumours n mthe norml tissue long with our mehnisti stuies in enometril ner ells revel regultion of tivtion s n importnt meitor of these hnges to ell prolifertion. Inrese tivtion is proly one of the min meitors of inrese prolifertion in ells with reue m 6 A methyltion, s inhiition of is suffiient to resue the hnges in ell prolifertion. However, we nnot rule out the involvement of other signlling pthwys tht oul e ltere iretly or iniretly y hnges to m 6 A methyltion. Beuse is known to e n importnt regultor of ell prolifertion, growth n survivl in mny ners, these finings my e pplile eyon enometril ner to other ners riven y inrese signlling. Other types of tumour oul exploit errnt RNA methyltion to gin survivl n growth vntges vi tivtion in ition to other propose mehnisms 5. Inee, others hve oserve inrese prolifertion of stem ells n ner ells with reue m 6 A methyltion,,6, n while this pper ws uner review m 6 A methyltion ws reporte to ffet tivity in AML, renl ell rinom n T-ell ifferentition 5. Although our results suggest tht erese m 6 A methyltion promotes tumorigenesis in the enometrium, other ners re ssoite with high expression n inrese m 6 A methyltion n oul involve ifferent mehnisms,. Nevertheless, our results suggest tht regultion of tivity through m 6 A methyltion oul e generl growth ontrol mehnism tht ffets rnge of other iologil proesses, whih will e new iretion to explore in the future. Methos Methos, inluing sttements of t vilility n ny ssoite ession oes n referenes, re ville t org/8/s Reeive: 8 August 7; Aepte: 9 July 8; Pulishe online: 7 August 8 Referenes. Liu, N. & Pn, T. N 6 -methylenosine-enoe epitrnsriptomis. Nt. Strut. Mol. Biol., 98 (6).. Zho, B. S., Rountree, I. A. & He, C. Post-trnsriptionl gene regultion y mrna moifitions. Nt. Rev. Mol. Cell Biol. 8, (7).. Ji, G. et l. N 6 -methylenosine in nuler RNA is mjor sustrte of the oesity-ssoite FTO. Nt. Chem. Biol. 7, ().. Bokr, J. A., Rth-Shmugh, M. E., Luwizk, R., Nryn, P. & Rottmn, F. Chrteriztion n prtil purifition of mrna N 6 -enosine methyltrnsferse from HeL ell nulei. Internl mrna methyltion requires multisuunit omplex. J. Biol. Chem. 69, (99). 5. Liu, J. et l. A -METTL omplex meites mmmlin nuler RNA N6-enosine methyltion. Nt. Chem. Biol., 9 95 (). 6. Ping, X.-L. et l. Mmmlin AP is regultory suunit of the RNA N 6 -methylenosine methyltrnsferse. Cell Res., (). 7. Shwrtz, S. et l. Perturtion of m 6 A writers revels two istint lsses of mrna methyltion t internl n 5' sites. Cell Rep. 8, 8 96 (). 8. Wng, Y. et l. N 6 -methylenosine moifition estilizes evelopmentl regultors in emryoni stem ells. Nt. Cell Biol. 6, 9 98 (). 9. Zheng, G. et l. ALKBH5 is mmmlin RNA emethylse tht impts RNA metolism n mouse fertility. Mol. Cell 9, 8 9 ().. Dominissini, D. et l. Topology of the humn n mouse m6a RNA methylomes revele y m6a-seq. Nture 85, 6 ().. Zho, X. et l. FTO-epenent emethyltion of N6-methylenosine regultes mrna spliing n is require for ipogenesis. Cell Res., 9 ().. Liu, N. et l. N 6 -methylenosine-epenent RNA struturl swithes regulte RNA protein intertions. Nture 58, (5).. Xio, W. et l. Nuler m 6 A reer YTHDC regultes mrna spliing. Mol. Cell 6, (6).. Rountree, I. A. et l. YTHDC meites nuler export of N 6 - methylenosine methylte mrnas. elife 6, e (7). 5. Meyer, K. D. et l. 5' UTR m 6 A promotes p-inepenent trnsltion. Cell 6, 999 (5). 6. Wng, X. et l. N 6 -methylenosine moultes messenger RNA trnsltion effiieny. Cell 6, (5). 7. Zhou, J. et l. Dynmi m 6 A mrna methyltion irets trnsltionl ontrol of het shok response. Nture 56, (5). 8. Li, A. et l. Cytoplsmi m 6 A reer YTHDF promotes mrna trnsltion. Cell Res. 7, 7 (7). 9. Shi, H. et l. YTHDF filittes trnsltion n ey of N6- methylenosine-moifie RNA. Cell Res. 7, 5 8 (7).. Wng, X. et l. N 6 -methylenosine-epenent regultion of messenger RNA stility. Nture 55, 7 ().. Geul, S. et l. Stem ells. m 6 A mrna methyltion filittes resolution of nïve pluripoteny towr ifferentition. Siene 7, 6 (5).. Btist, P. J. et l. m 6 A RNA moifition ontrols ell fte trnsition in mmmlin emryoni stem ells. Cell Stem Cell 5, ().. Zho, B. S. et l. m 6 A-epenent mternl mrna lerne filittes zerfish mternl-to-zygoti trnsition. Nture 5, (7).. Lin, S., Choe, J., Du, P., Trioulet, R. & Gregory, R. I. The m 6 A methyltrnsferse promotes trnsltion in humn ner ells. Mol. Cell 6, 5 5 (6). 5. Zhng, C. et l. Hypoxi inues the rest ner stem ell phenotype y HIF-epenent n ALKBH5-meite m 6 A-emethyltion of NANOG mrna. Pro. Ntl A. Si. USA, E7 E56 (6). 6. M, J. Z. et l. METTL suppresses the metstti potentil of HCC y moulting m 6 A-epenent primry mirna proessing. Heptology 65, 59 5 (7). 7. Li, Z. et l. FTO plys n onogeni role in ute myeloi leukemi s N 6 -methylenosine RNA emethylse. Cner Cell, 7 (7). 8. Zhng, S. et l. m 6 A emethylse ALKBH5 mintins tumorigeniity of gliolstom stem-like ells y sustining FOXM expression n ell prolifertion progrm. Cner Cell, 6 (7). 9. Cui, Q. et l. m 6 A RNA methyltion regultes the self-renewl n tumorigenesis of gliolstom stem ells. Cell Rep. 8, 6 6 (7).. Li, X. et l. The m 6 A methyltrnsferse : ting s tumor suppressor in renl ell rinom. Onotrget 8, (7).. Wng, X. et l. Reue m 6 A mrna methyltion is orrelte with the progression of humn ervil ner. Onotrget 8, (7).. Chen, M. et l. RNA N 6 -methylenosine methyltrnsferse promotes liver ner progression through YTHDF epenent posttrnsriptionl silening of SOCS. Heptology 67, 5 7 (7).. Vu, L. P. et l. The N 6 -methylenosine (m 6 A)-forming enzyme ontrols myeloi ifferentition of norml hemtopoieti n leukemi ells. Nt. Me., (7).. Weng, H. et l. METTL inhiits hemtopoieti stem/progenitor ifferentition n promotes leukemogenesis vi mrna m 6 A moifition. Cell Stem Cell, 9 5 (8). 5. Su, R. et l. R-HG exhiits nti-tumor tivity y trgeting FTO/m(6)A/ MYC/CEBPA signling. Cell 7, 9 5 (8). 6. Knoth, C. et l. Integrte genomi hrteriztion of enometril rinom. Nture 97, 67 7 (). 7. Slez, P. & Jinek, M. Struturl insights into the moleulr mehnism of the m 6 A writer omplex. elife 5, e8 (6). 8 Nture Cell Biology VOL SEPTEMBER

10 8. Wng, P., Doxter, K. A. & Nm, Y. Struturl sis for oopertive funtion of Mettl n Mettl methyltrnsferses. Mol. Cell 6, 6 7 (6). 9. Wng, X. et l. Struturl sis of N 6 -enosine methyltion y the -METTL omplex. Nture 5, (6).. Meyer, K. D. et l. Comprehensive nlysis of mrna methyltion revels enrihment in UTRs n ner stop oons. Cell 9, ().. Cner Genome Atls Reserh Network, Integrte genomi nlyses of ovrin rinom. Nture 7, ().. The Cner Genome Atls Reserh Network, Integrte genomi hrteriztion of pnreti utl enorinom. Cner Cell, 85 (7).. Le Gllo, M. & Bell, D. W. The emerging genomi lnspe of enometril ner. Clin. Chem. 6, 98 ().. Mnning, B. D. & Toker, A. /PKB signling: nvigting the network. Cell 69, 8 5 (7). 5. Vivno, I. & Swyers, C. L. The phosphtiylinositol -kinse pthwy in humn ner. Nt. Rev. Cner, 89 5 (). 6. Jinto, E. et l. SIN/MIP mintins ritor-mtor omplex integrity n regultes Akt phosphoryltion n sustrte speifiity. Cell 7, 5 7 (6). 7. Brognr, J., Siereki, E., Go, T. & Newton, A. C. PHLPP n seon isoform, PHLPP, ifferentilly ttenute the mplitue of Akt signling y regulting istint Akt isoforms. Mol. Cell 5, 97 9 (7). 8. Srssov, D. D., Guertin, D. A., Ali, S. M. & Stini, D. M. Phosphoryltion n regultion of Akt/PKB y the ritor-mtor omplex. Siene 7, 98 (5). 9. Copp, J., Mnning, G. & Hunter, T. TORC-speifi phosphoryltion of mmmlin trget of rpmyin (mtor): phospho-ser8 is mrker for intt mtor signling omplex. Cner Res. 69, 8 87 (9). 5. Li, H. B. et l. m 6 A mrna methyltion ontrols T ell homeostsis y trgeting the IL-7/STAT5/SOCS pthwys. Nture 58, 8 (7). 5. Vrghese, F., Bukhri, A. B., Mlhotr, R. & De, A. IHC Profiler: n open soure plugin for the quntittive evlution n utomte soring of immunohistohemistry imges of humn tissue smples. PLoS ONE 9, e968 (). 5. Knehis, M., Sto, Y., Kwshim, M., Furumihi, M. & Tne, M. KEGG s referene resoure for gene n protein nnottion. Nulei Ais Res., D57 D6 (6). Aknowlegements We thnk Mrtin Aryee (Msshusetts Generl Hospitl) for initil isussions n Angel Anersen for eiting the mnusript. This work ws supporte y Mrsh Rivkin Fountion wr (M.A.E.); University of Chigo Institute for Biophysil Dynmis Yen Fellowship (B.T.H.); Ntionl Nturl Siene Fountion of Chin grnts 87 n 8799 (S.L.); The Ntionl Bsi Reserh Progrmme grnts CB76 n CB765 (S.L.); the Ntionl Key Reserh n Development Progrmme of Chin 7YFA568 (Jz.L.); the Thousns Young Tlents Pln of Chin n Hunres Tlents Progrmme of Zhejing University (Jz.L.); Ntionl Cner Institute grnts CA88 (E.L.) n F CA7 (B.T.H.); Ntionl Institutes of Helth grnts R HG8688 n RM HG895 (C.H.); n University of Chigo Cner Center Support Grnt PCA599. M.A.E. thnks the Hrris Fmily Fountion for their generous support. C.H. is n investigtor of the Howr Hughes Meil Institute. Author ontriutions Jun L., M.A.E., B.T.H., E.L. n C.H. esigne the experiments. M.A.E., S.M.T., S.L., Y.Y., J.H., S.C., Z.X, X.L., X.Y. n E.L. ollete the ptient smples with ssistne from J.C., Z.Z. n Jz.L. Jun L., M.A.E. n B.T.H. rrie out the experiments with help from K.Y., S.M.T., A.C. n A.C.Z. Jun L., M.A.E., B.T.H., E.L. n C.H. nlyse the t n interprete the finings. Z.L. ie with nlysis of the sequening t. Jz.L ie in the erly esign of experiments. Jun L. n B.T.H. wrote the mnusript with input from M.A.E., E.L. n C.H. Competing interests C.H. is sientifi founer of Aent Therpeutis n memer of its sientifi visory or. All other uthors elre no ompeting interests. Aitionl informtion Supplementry informtion is ville for this pper t s Reprints n permissions informtion is ville t Corresponene n requests for mterils shoul e resse to E.L. or C.H. Pulisher s note: Springer Nture remins neutrl with regr to jurisitionl lims in pulishe mps n institutionl ffilitions. Nture Cell Biology VOL SEPTEMBER