DGCR8 is essential for microrna biogenesis and silencing of embryonic stem cell self-renewal

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1 7 Nture Pulishing Group DGCR is essentil for mirorna iogenesis n silening of emryoni stem ell self-renewl Yngming Wng, Rostislv Mevi, Collin Melton, Ruolf Jenish & Roert Blelloh The moleulr ontrols tht govern the ifferentition of emryoni stem (ES) ells remin poorly unerstoo. DGCR is n RNA-ining protein tht ssists the RNse III enzyme Drosh in the proessing of mirornas (mirnas), sulss of smll RNAs. Here we stuy the role of mirnas in ES ell ifferentition y generting Dgr knokout moel. Anlysis of mouse knokout ES ells shows tht DGCR is essentil for iogenesis of mirnas. On the inution of ifferentition, DGCR-efiient ES ells o not fully ownregulte pluripoteny mrkers n retin the ility to proue ES ell olonies; however, they o express some mrkers of ifferentition. This phenotype iffers from tht reporte for Dier knokout ells, suggesting tht Dier hs mirna-inepenent roles in ES ell funtion. Our finings inite tht mirnas funtion in the silening of ES ell self-renewl tht normlly ours with the inution of ifferentition. Emryoni stem ells provie tool for the stuy of the moleulr mehnisms of erly mmmlin evelopment. Differentition of ES ells requires lrge n rpi shifts in their mrna n protein Protein gdna lox flox proe proe proe proe WW srbd srbd Exon Exon. k Exon. k Exon 6 7 Exon.6 k loxp Exon loxp loxp Exon loxp loxp. k 9 Exon Exon proe hcmv HygroTK loxp.6 k Exon Exon. k Exon Exon proe proe Exon Exon proe onstitution. mirnas re well suite to perform this tsk y inhiiting the trnsltion of severl trgets. mirnas re preite to regulte hunres of genes iniviully n simultneously,. An essentil role for smll RNAs in ES ell ifferentition hs een inferre from the phenotype of mouse Dier knokout ES ells. Dier is require for the mturtion of t lest two lsses of smll RNAs: mirnas n short interfering RNAs (sirnas) 7. Thus, it remins unknown whether the lk of mirnas lone is the use of the Dier knokout phenotype. The oule-strne RNA-ining protein DGCR n the RNse III enzyme Drosh form the miroproessor omplex, whih proesses long primry mirnas (pri-mirnas) into short hirpins lle preursor mirnas (pre-mirnas). The resulting hirpins re exporte into the ytoplsm, n re proesse y Dier into mture mirnas,6. Unlike ytoplsmi proessing y Dier, nuler proessing y the miroproessor omplex seems to e speifi to mirnas. By ontrst, Drosh hs een reporte to hve role in riosoml RNA proessing, possily in istint protein omplex 6. Therefore, DGCR my e the only memer of the proessing pthwy tht is speifi to mirnas. To ssess the role of DGCR in mirna proessing n to stuy the glol role of mirnas in erly emryoni evelopment n ES ell ifferentition, we generte mouse Dgr knokout ES ells (Fig. ). To onfirm tht DGCR is essentil for mirna iogenesis, we performe RNA lot nlysis of the ES ells with proes speifi to mir-9, mir-9 n mir-. mir-9 n mir-9 re erive from single gene n re expresse speifilly in ES ells. mir- is inepenently trnsrie n expresse roly in ES ells n ifferentite tissues 7.InDgr knokout ells, neither fully mture nor intermeite pre-mirna prouts were present for ny of these Figure Dgr knokout strtegy. The DGCR protein hs one WW omin n two oule-strne RNA-ining omins (srbds). The WW omin is enoe in exon of the Dgr genomi DNA (gdna); the two srbds re lote in exons 7 9 n, respetively. Exision of exon from the mrna genertes frme shift, resulting in severl premture stop oons in ownstrem exons. ES ells were sequentilly trgete n trete with Cre reominse to proue Dgr D/flox n D/D ES ells. ells were generte y retrgeting D/D ES ells with the lox onstrut, followe y removl of the HygroTK ssette with Cre reominse. Developmentl n Stem Cell Biology Progrm, Deprtment of Urology, University of Cliforni, Sn Frniso, Cliforni 9, USA. Whitehe Institute of Biomeil Reserh, Cmrige, Msshusetts, USA. Corresponene shoul e resse to R.B. (lellohr@stemell.usf.eu). Reeive July 6; epte Jnury 7; pulishe online Jnury 7; oi:./ng969 VOLUME 9 [ NUMBER [ MARCH 7 NATURE GENETICS

2 7 Nture Pulishing Group mirnas (Fig., n Supplementry Fig. online). The mir-9 proe ientifie two lrge RNA trnsripts (B k) in the Dgr knokout smples, onsistent with the size expete for pri-mir-9 trnsripts. By ontrst, mture n pre-mirna ns, ut not pri-mirna ns, were present in wil-type n Dgr heterozygous ES ells. These results show tht DGCR is solutely require for the proessing of some, if not ll, pri-mirnas to pre-mirnas. To etermine whether DGCR is require for the mturtion of ll mirnas, we performe mirna mirorry nlysis. Using RNA from wil-type ES ells s our referene smple, we oserve glol loss of mirnas in Dgr knokout ells, ut norml expression in Dgr heterozygous ells (Fig., n Supplementry Tle online). The similrity in expression etween wil-type n heterozygous ells suggests tht DGCR is not limiting in the mintenne of steystte levels of mirnas in ES ells. Of the 9 mirna rry proes tht showe signifint signls with wil-type RNA, were gretly reue in the Dgr knokout ells. The remining seven were not signifintly ltere in the Dgr knokout ells, ut signls for t lest four of these seven mirnas seeme to e ue to unvoile ontmintion from RNA from the mouse emryoni firolst (MEF) feeer ells use for ES ell ulture efore isoltion of the RNA. Expression of these mirnas ws high in the MEFs, n erese in the ES ells fter temporry pssge on geltin-ote pltes without feeers (Supplementry Fig. n t not shown). The remining three mirnas showe similr expression in the heterozygous, knokout n MEF feeer ells; therefore, these signls my e smll RNAs tht re not proesse y the miroproessor omplex. Alterntively, they my result from unvoile egrtion prouts in the purifie smll RNA popultion. In either se, our results show tht DGCR is roly require for mirna proessing with little eviene of reunny or ypss mehnisms. The RNse III enzyme Drosh is require for proessing S pre-rrna to.s rrna n my e lso involve in the levge of S pre-rrna to S pre-rrna n S rrna 6. Beuse DGCR ssoites with Drosh to form the miroproessor omplex, we onsiere the possiility tht DGCR is lso involve in ssisting Drosh in rrna proessing. To test this possiility, we performe RNA lot nlyses for the S primry, S, S n S intermeite, n.s terminl rrna trnsripts (Fig. e). We sw no qulittive or quntittive ifferenes in the proessing of riosoml RNAs etween wil-type n knokout ES ells. Although we nnot rule out the possiility tht DGCR hs role Figure Prolifertion efets of Dgr knokout ES ells. () Morphology of Dgr D/flox n D/D ES ell olonies 7 fter plting. The D/flox olony is overgrown, with ifferentition ourring roun its eges. () Men popultion ouling time (n ¼ 6). () Men growth rte of ES ells mesure y MTT ssy (n ¼ 6). () Cell-yle nlysis of Dgr knokout ES ells (n ¼ ). Error rs in represent the s.. mir-9 mir- pri mir-9 pre mir-9 mir-9 Rtio of expression level reltive to Rtio of expression level reltive to mir-9- mir-9 ES ell speifi mirnas mirnas mir-9 mir-9-s mir-9-s mir-9-s mir-9-s mir-9 mir-9 mir-9 mir- mir-6- mir- mir- mir-- mir-7 mir- mir- mir- mirnas tht re ES ell speifi S S S S in n unknown proessing pthwy for smll RNAs, our results, together with finings showing the preise ining of DGCR to pri-mirnastemloops, suggest tht DGCR hs speifi role in mirna proessing. Dgr knokout emryos rreste erly in evelopment (Supplementry Tle online). We therefore fouse our stuies on the nlysis of Dgr knokout ES ells. The knokout ES ells h n e Figure DGCR is essentil n my e speifi for mirna iogenesis in ES ells. () RNA lot nlysis of mirnas from wil-type (), heterozygous (D/flox) n two inepenent knokout (D/D) ES ells with proes speifi to mir-9 n mir-. snrna ws use s loing ontrol. () RNA lot nlysis of pri-mirna, pre-mirna n mirna from ES ells with proe speifi to mir-9. () Expression rtios of Dgr heterozygous n knokout ES ells reltive to wil-type ES ell for 9 mirnas. These rtios were lulte from mirna mirorry nlysis. () Expression rtios of Dgr heterozygous n Dgr knokout ES ells reltive to wil-type ES ells for 9 representtive mirnas. The mirnas on the left re ES ell speifi mirnas; those on the right re expresse in ES ells n ifferentite tissues 7.(e) RNA lot nlysis of pre-rrnas. Top, hyriiztion with proe speifi to the ETS sequene of S pre-rrna (ining to S n S trnsripts); ns elow S prerrna were etete owing to non-speifi ining to unnt S rrna. Mile, hyriiztion with proe speifi to the.s rrna sequene (ining to S, S, S n.s trnsripts). Bottom, snrna ws use s loing ontrol. A Time (h) Popultion ouling time (h) Frtion of ells (%) 6 G S G/M S.S NATURE GENETICS VOLUME 9 [ NUMBER [ MARCH 7

3 7 Nture Pulishing Group Ot Fgf T (rhyury) Hnf Krt Gph extene popultion ouling time reltive to their wil-type n heterozygous ounterprts, ut were morphologilly norml n ontinue to express ES ell speifi mrkers (Figs. ). The higher popultion ouling time nnot e expline y n inrese in poptosis euse neither wil-type nor knokout ES ells showe sustntil poptosis (Supplementry Fig. online). The knokout ES ells umulte in the G phse of the ell yle, inresing from pproximtely % of wil-type n heterozygous Dgr ells in G to % of the homozygous Dgr knokout ells (Fig. ). Re-introution of DGCR y homologous reomintion resue the popultion ouling time n G phenotypes. The umultion of DGCR-efiient ES ells in G is reminisent of the result of loss of the mirna-speifi Dier gene, Dr-, in the Drosophil melnogster germ line 9. These results show tht DGCR is require for norml ES ell prolifertion n ell-yle progression. To ssess the ifferentition pity of Dgr knokout ES ells, we ulture them s emryoi oies (EBs) in the sene of leukemi inhiitory ftor (LIF). Uner these onitions, wil-type ES ells spontneously ifferentite into ysti EBs ontining heterogeneous mix of ells representing ll three germ lyers of the erly emryo. Unlike wil-type EBs, Dgr knokout EBs i not form yst (Fig. ). In ition, RT-PCR showe norml expression of vrious mrkers of ifferentition (Fig., n Supplementry Fig. online). Fgf is mrker of primitive etoerm, iret erivtive of the inner ell mss of the lstoyst, tht goes on to mke ll tissues of the emryo proper,. In wil-type EBs, Fgf ws highly expresse y y of ifferentition (Fig. ). By ontrst, expression Dy Dy Dy Dy 6 Dy 6 Dy Dy Dy Dy 6 Dy 6 Figure EB ifferentition n tertom formtion. () Morphology of EBs from wil-type, D/D n resue ES ells. EBs were ulture for. () RT-PCR nlysis of pluripoteny (Ot) n ifferentition mrkers (Fgf, T (rhyury), Hnf, Krt) fter EB ifferentition. Gph ws use s referene. () Quntittive RT-PCR for the mrkers shown in. The-tin gene ws use s referene. For eh gene, t were normlize to the mrna level t y of wil-type EB ifferentition. () Tertom formtion y wil-type n Dgr knokout ES ells. Arrow in D/D tumor ientifies region of epithelil ifferentition Dy /flow Fgf Hnf Dy Dy of Fgf in Dgr knokout ells ws elye: it peke t y of ifferentition n i not reh wil-type levels. To ssess lter steps in EB ifferentition, we use mrkers of enoerm (Hnf n Afp), mesoerm (T; lso known s rhyury), etoerm (Sox), epithelium (Krt), trophetoerm (Eomes) n the germ line (Dx, lso known s Vs; Fig., n Supplementry Fig. ). With the exeption of Krt, Eomes n Dx, expression of the mrkers ws either sent or severely reue in the Dgr knokout EBs. Expression of Krt ws elye, ut y y 6 of ifferentition it showe levels similr to those of wil-type ells. Eomes ws wekly expresse in oth wil-type n Dgr knokout ells. Dx expression erese on EB ifferentition of wil-type ells, ut not Dgr knokout ells. All expression efets in the EBs were resue y the re-introution of Dgr (Supplementry Fig. online). Tertoms, like EBs, normlly onsist of heterogeneous mix of ifferentite ell types. We generte tertoms y suutneously injeting Dgr knokout n wil-type ES ells into immunoefiient mie. Consistent with the efets in EB ifferentition, the tertoms proue with the Dgr knokout ells ppere lrgely unifferentite prt from some glnulr-like strutures (Fig. ). Together, the EB n tertom ifferentition experiments show grossly norml, ut not solute loss of, expression of ifferentition mrkers in the Dgr knokout popultion. To ssess further the ifferentition efet of Dgr knokout ES ells, we trete the ells with retinoi i, strong inuer of ifferentition. When ulture in retinoi i, oth wil-type n Ot T (rhyury) Krt Dy 6 VOLUME 9 [ NUMBER [ MARCH 7 NATURE GENETICS

4 7 Nture Pulishing Group Ot Rex Sox Nnog Gph Dy Dy Dy Dy Dy Dy Dy Dy Dgr knokout ES ells took on morphology onsistent with mesenhyml ifferentition (t not shown). Even in the presene of retinoi i, however, the pluripoteny mrkers Ot, Rex, Sox n Nnog were not silene ompletely in Dgr knokout ells (Fig.,). We returne the retinoi i trete ells to ES ell ulture onitions n mesure their ility to reform ES ell like olonies. The frequeny of olony formtion ws out -fol greter for Dgr knokout ells thn for wil-type or Dgr heterozygous ells (Fig. ). In ition, ll of the olonies were lkline phosphtse positive n h the stereotypil olony morphology of ES ells. To evlute further this pprent filure of Dgr knokout ES ells to turn off the stem ell progrm, we performe lonl ssy of ifferentition. n Dgr knokout ES ells were plte t lonl ensity without MEFs in the presene or sene of LIF. With or without LIF, the wil-type olonies egn to ifferentite, s ssesse y flttening roun the eges n loss of lkline phosphtse stining (Fig. n Supplementry Fig. online). Differentition ws more omplete in the sene of LIF. By ontrst, Dgr knokout olonies i not fltten roun the eges n ontinue to express lkline phosphtse even in the sene of LIF. Together, these t show tht Dgr knokout ES ells nnot effiiently silene the ES ell progrm even uner stringent ifferentition onitions. Our results show tht DGCR is require for the iogenesis of mirnas n tht mirnas re essentil for silening ES ell selfrenewl. A similr efet in silening the stem ell progrm hs een esrie for knokout moel of the NuRD omplex, lrge omplex with nuleosome remoeling n histone eetyltion tivities. In ition, the G9 histone methyltrnsferse n the Dnmt n Dnmt DNA methyltrnsferses hve een reporte to e essentil for mintining ifferentition of ES ells fter retinoi i tretment. Therefore, thedgr knokout phenotype ples DGCR mong unique group of glol regultors of ellulr ifferentition. We propose tht the filure to silene ES self-renewl upon ifferentition in Dgr mutnt kgroun is seonry to loss of mirnas tht normlly inhiit trnsltion of proteins tht iretly or iniretly mintin ES ell pluripoteny. The Dgr knokout ES ell phenotype iffers from tht reporte for the Dier knokout. Dier knokout ES ells, like Dgr knokout ES ells, re efetive in ifferentition. Unlike Dgr knokout..... Dys in RA... Dys in RA Figure Monolyer ifferentition of ES ells in the presene of retinoi i. () RT-PCR nlysis of pluripoteny mrkers. n Dgr knokout ES ells were plte s monolyer n trete with retinoi i (RA) in the sene of LIF to inue mesenhyml ifferentition. Gph ws use s loing ontrol. () Quntittive PCR nlysis of the pluripoteny mrkers Ot n Nnog (n ¼ ). The -tin gene ws use s referene. For eh smple, t were normlize to the mrna level t y. () ES ell olony formtion of ifferentite ells. After vrying urtions of monolyer ifferentition, ells were returne to ES ell ulture onitions n ssye for their ility to form lkline phosphtse positive olonies. Error rs inite the rnge of mesurements (n ¼ ). () Clonl nlysis of ES ell ifferentition in the sene of MEF feeers n in the presene or sene of LIF, s inite. Shown re perentges of unifferentite, mixe n ifferentite olonies uner the inite onitions. D inites ; D inites. Ot Nnog Numer of ES ell olonies (,) per, ells plte Perentge of olonies 9 D D D D + LIF LIF D D D D ells, however, Dier knokout ells o not express ifferentition mrkers uring EB ulture. Dier knokout EBs stop growing fter pproximtely of ulture, wheres Dgr knokout ells ontinue to grow n to ifferentite even fter 6 (t not shown). In ition, Dier knokout ES ells seem to hve more profoun initil prolifertion efet tht is overome over time, presumly ue to itionl geneti events. By ontrst, Dgr knokout ES ells show stle n more sutle prolifertion efet. These ifferenes in the phenotypes of Dier n Dgr knokout ES ells suggest tht Dier hs mirna-inepenent roles in ES ell funtion. Dgr knokout ES ells provie mens to ientify these roles. Dgr knokout ells n e lso use to isset the funtion of iniviul mirnas y reintrouing speifi mirnas n testing their ility to resue ientifie knokout phenotypes. Thus, the Dgr knokout moel shoul provie powerful mens to stuy the funtion of smll RNAs in ES ell self-renewl n prolifertion n in the silening of stem ell potentil uring the proess of linege ommitment. METHODS Trgeting strtegy. Dgr knokout ES ells were generte y removing exon, resulting in the formtion of severl premture stop oons ownstrem of the trgete region (Fig. ). We generte n rms y PCR (High Fielity Tq, Invitrogen) from teril rtifiil hromosome (Chilren s Hospitl n Reserh Center, Okln). A loxp-hiniii frgment ws inserte etween the rms. A HygroTK seletion ssette flnke y loxp sites ws inserte t BglII site ownstrem of exon. V6. ES ells were trnsfete with the lox onstrut n selete in mg/ml of hygromyin (Rohe). Sulones were pike n nlyze y DNA lotting. The resulting wil-type/lox sulones were trnsfete with Cre reominse expressing plsmi n selete in. nm gnylovir (Sigm). The seon llele of Dgr ws remove y seon roun of trgeting. /flox ES ells were trgete n expose to Cre reominse to proue Dgr D/D n D/flox ES ells. For the resue experiment, D/D ES ells were retrgete with the lox onstrut to proue lox/d ells, n the HygroTK seletion ssette ws then remove. Tissue ulture. ES ells were ulture on MEF feeer ells in ES ell meium 6. For miorrry nlysis, EB n monolyer ifferentition experiments, feeer ells were remove y ifferentil herene to unote pltes for. h, followe y of ulture on geltinize pltes. Alterntively for mirorry nlysis, ES ells were wene from MEFs for five pssges to minimize feeer ontmintion. For EB ifferentition, ells were gently trete with trypsin Unifferentite Dys in RA Mixe Differentite NATURE GENETICS VOLUME 9 [ NUMBER [ MARCH 7

5 7 Nture Pulishing Group n ulture in suspension in Petri ishes or low-tthment ulture ishes in ES ell meium without LIF. For tertom formtion, 6 to 6 ES ells were injete into n immunoefiient mouse y suutneous injetion. Tertoms were llowe to evelop to out m in imeter efore fixtion n stining with hemotoxylin n eosin. For monolyer ifferentition, ES ells were ulture on geltinize pltes in ES ell meium without LIF n in the presene of nm retinoi i. The reltive numers of Dgr knokout ES ells were oule to ompenste for their prolifertion efiieny. Totl RNA ws ollete (Trizol, Invitrogen) t vrious time points n nlyze y RT-PCR. For the ES ell olony formtion ssy, ells trete with retinoi i were returne to ES ell onitions. Cells were ulture in sixwell plsti plte for, stine for lkline phosphtse (Vetor Lortories) n ounte mnully. For lonl ifferentition of ES ells, ES ells were wene off MEF feeer ells n plte t ells per well of six-well plte in ES ell meium supplemente with LIF. The next y, mei ws reple with mei with or without LIF supplement. Meium ws hnge ily. After or, olonies were fixe in % prformlehye n stine with,6-imiino--phenylinole (DAPI) n Vetor Re Alkline Phosphtse Sustrte Kit I (Vetor Lortories). Colonies were ounte n sore for morphology (unifferentite, mixe n ifferentite) y using fluoresene mirosope t mgnifition. Colonies ontining only lkline phosphtse positive ells re efine s unifferentite. Colonies ontining only lkline phosphtse negtive ells re efine s ifferentite. Mixe olonies re efine s olonies with positive ore n surrouning negtive outgrowth. Between n olonies were ounte for eh ell type per time point. RNA lot nlysis. Anlysis of mture mirnas ws performe s previously esrie 7. For nlysis of pri n pre mir-9, mg of totl RNA ws resolve in.% enturing grose n formlehye gel. Anlysis of prerrna ws one s esrie 6. All lots were strippe in % SDS t 7 C for h n then proe for the smll nuler RNA (snrna) s loing ontrol. The proes for mir-9 (lso for pri n pre-mir 9), mir-9 n mir- were DNA oligonuleoties omplementry to the respetive mture mirnas. See Supplementry Tle online for the proes use in RNA lot nlysis. Mirorry nlysis. We resolve mg of totl RNA from wil-type, Dgr heterozygous n Dgr knokout ES ells on % enturing polyrylmie gel. The gel portion orresponing to B 6 nt ws ut out, extrte in TEN uffer ( mm Tris, ph., mm EDTA, mm NCl) n preipitte with ethnol. Before PAGE seprtion, tre mount of n unrelte -nt smll RNA riolele with Ptthe en ws e to eh totl RNA smple to monitor the effiieny of gel extrtion. After gel extrtion, n equl mount of positive ontrol RNA (Amion) ws e to eh smple. Susequently, smll RNAs were polyenylte, ligte with pture sequenes using n NCoe mirna leling system (Invitrogen) n hyriize to mirorrys spotte with the Amion mmmlin mirna proe set (Whitehe Institute Center for Mirorry Tehnology). Mirorrys were snne on GenePix B snner (Axon Instruments) n nlyze with Genepix Pro version.. In ll nlyses, smll RNAs from wil-type ES ells were use s referene. The rtios for positive ontrol RNAs were use for normliztion. Mirorry nlyses were one twie for MEFs, three times for Dgr heterozygous knokout ES ells, n twie for two inepenent Dgr-null ES ell lines. The orreltion oeffiients of reverse ye experiments for two inepenent ell lines were.99 n.97. The rtios shown in Figure, re the verge of ll experiments (Supplementry Tle ). RT-PCR. We trete mg of totl RNA with DNse (Promeg) to remove genomi DNA. DNA ws synthesize with SuperSriptIII First Strn DNA Synthesis RT-PCR kit (Invitrogen) from mg of trete RNA. For semi-quntittive PCR, the primers for Ot, Rex, Gph, T (rhyury) n Krt were gifts (see Aknowlegments). The primers for Hnf n Fgf hve een esrie,7. Quntittive PCR ws one on 7 Rel Time PCR System (Applie Biosystems). For eh ell smple, PCR ws one in triplite to minimize the vrition. -Atin mrna ws use s n enogenous ontrol. Primers were esigne with Primer Express. (Applie Biosystems). See Supplementry Tle for the primers use in quntittive rel-time PCR. Cell prolifertion ssy n ell-yle nlysis. To etermine the ell popultion ouling time,. 6 ES ells were plte onto 6-mm plsti ishes n ounte fter. The ell popultion ouling time ws otine with the eqution Y en ¼ Y strt (t/t),wheret is the ell popultion ouling time, Y strt is the strting numer of ES ells plte, n Y en is the ening numer of ES ells fter growth for perio of time, t. Eh ell line ws ounte minimum of five times. For the MTT ssy, ells were plte t, ells per well in 96-well plte n nlyze,, 6 n 9 h fter plting. At the inite time points, ES ell meium ws reple with ml mg/ml - (,-imethylthizol--yl)-.-iphenyltetrzolium romie (MTT) (Moleulr Proes) in DMEM. After inution t 7 C for h, the MTT solution ws remove, n ml DMSO ws e to issolve preipitte. Asorne ws reore t nm using Spetrmx M miroplte reer (Moleulr Devies). For ell-yle nlysis, ells were fixe in ethnol, lele with propiium ioine n nlyze y flow ytometry. For poptosis nlysis, ells were lele with propiium ioine n FITC Annexin V (BioVision), n nlyze y flow-ytometry. Perentge of propiium ioine negtive n Annexin V positive ells in the totl popultion ws lulte to trk erly stges of poptosis. Aession oe. Gene Expression Omnius: GSE66. Note: Supplementry informtion is ville on the Nture Genetis wesite. ACKNOWLEDGMENTS We thnk C. Ber n the Anit Sil Lortory for tehnil vie; J. Dusmn for tehnil help; J. Reiter n N. Shomron for regents n M. Rmlho- Sntos, J. Reiter n memers of the Blelloh Lortory for ritilly reing the mnusript. The primers for Ot, Rex, Gph, T (rhyury) n Krt were gifts from J. Reiter (University of Cliforni Sn Frniso) This work ws supporte y grnts to R.B. from the University of Cliforni Sn Frniso Urology Deprtment, the Snler Fountion, the Lne Armstrong Fountion n the Ntionl Institutes of Helth (K NS). Y.W. is supporte y the Cliforni Institute of Regenertive Meiine postotorl fellowship progrm. C.M. is supporte y the Ntionl Siene Fountion grute reserh fellowship progrm. AUTHOR CONTRIBUTIONS Y.W. i the experiments esrie in Figs. n n quntittive PCR. C.M. i the experiments esrie in Fig.. R.M. together with R.B. performe other experiments, inluing onstrution of knokout ES ells. R.J. provie regent n infrstruture support for trgeting experiments. R.B. ws involve in esigning the experiments n wrote the pper with help from Y.W. n C.M. COMPETING INTERESTS STATEMENT The uthors elre tht they hve no ompeting finnil interests. Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions. Vleni-Snhez, M.A., Liu, J., Hnnon, G.J. & Prker, R. Control of trnsltion n mrna egrtion y mirnas n sirnas. Genes Dev., (6).. Lewis, B.P., Burge, C.B. & Brtel, D.P. Conserve see piring, often flnke y enosines, inites tht thousns of humn genes re mirorna trgets. Cell, ().. 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6 7 Nture Pulishing Group 9. Denli, A.M., Tops, B.B., Plsterk, R.H., Ketting, R.F. & Hnnon, G.J. Proessing of primry mirornas y the Miroproessor omplex. Nture, ().. Gregory, R.I. et l. The Miroproessor omplex meites the genesis of mirornas. Nture, ().. Hn, J. et l. The Drosh-DGCR omplex in primry mirorna proessing. Genes Dev., 6 7 ().. Lnthler, M., Ylin, A. & Tushl, T. The humn DiGeorge synrome ritil region gene n its D. melnogster homolog re require for mirna iogenesis. Curr. Biol., 6 67 ().. Hn, J. et l. Moleulr sis for the reognition of primry mirornas y the Drosh- DGCR omplex. Cell, 7 9 (6).. Yi, R., Qin, Y., Mr, I.G. & Cullen, B.R. Exportin- meites the nuler export of pre-mirornas n short hirpin RNAs. Genes Dev. 7, 6 ().. Lun, E., Guttinger, S., Clo, A., Dhlerg, J.E. & Kuty, U. Nuler export of mirorna preursors. Siene, 9 9 (). 6. Wu, H., Xu, H., Mirgli, L.J. & Crooke, S.T. Humn RNse III is 6-kD protein involve in preriosoml RNA proessing. J. Biol. Chem. 7, (). 7. Houviy, H.B., Murry, M.F. & Shrp, P.A. Emryoni stem ell-speifi mirornas. Dev. Cell, ().. Houviy, H.B., Dennis, L., Jenish, R. & Shrp, P.A. Chrteriztion of highly vrile eutherin mirorna gene. RNA, 7 (). 9. Htfiel, S.D. et l. Stem ell ivision is regulte y the mirorna pthwy. Nture, ().. Heert, J.M., Boyle, M. & Mrtin, G.R. mrna loliztion stuies suggest tht murine FGF- plys role in gstrultion. Development, 7 (99).. Rthjen, J. et l. Formtion of primitive etoerm like ell popultion, EPL ells, from ES ells in response to iologilly erive ftors. J. Cell Si., 6 6 (999).. Pelton, T.A., Shrm, S., Shulz, T.C., Rthjen, J. & Rthjen, P.D. Trnsient pluripotent ell popultions uring primitive etoerm formtion: orreltion of in vivo n in vitro pluripotent ell evelopment. J. Cell Si., 9 9 ().. Kji, K. et l. The NuRD omponent M is require for pluripoteny of emryoni stem ells. Nt. Cell Biol., 9 (6).. Felmn, N. et l. G9-meite irreversile epigeneti intivtion of Ot-/ uring erly emryogenesis. Nt. Cell Biol., 9 (6).. Murhison, E.P., Prtrige, J.F., Tm, O.H., Cheloufi, S. & Hnnon, G.J. Chrteriztion of Dier-efiient murine emryoni stem ells. Pro. Ntl. A. Si. USA, (). 6. Blelloh, R.H. et l. Nuler loning of emryonl rinom ells. Pro. Ntl. A. Si. USA, 9 99 (). 7. Lu, N.C., Lim, L.P., Weinstein, E.G. & Brtel, D.P. An unnt lss of tiny RNAs with prole regultory roles in Cenorhitis elegns. Siene 9, 6 ().. Di Cristofno, A., Pese, B., Coron-Cro, C. & Pnolfi, P.P. Pten is essentil for emryoni evelopment n tumor suppression. Nt. Genet. 9, (99). NATURE GENETICS VOLUME 9 [ NUMBER [ MARCH 7