SETDA modultes ell yle progression through mirna network tht regultes p5 trget genes The Hrvrd ommunity hs mde this rtile openly vilble. Plese shre how this ess benefits you. Your story mtters Cittion Tjim, K., T. Ye, S. Jvid, O. Tm, V. Comills, R. Morris, B. S. Wittner, et l. 5. SETDA modultes ell yle progression through mirna network tht regultes p5 trget genes. Nture Communitions (): 857. doi:.8/nomms957. http:// dx.doi.org/.8/nomms957. Published Version doi:.8/nomms957 Citble link http://nrs.hrvrd.edu/urn-:hul.instrepos:99555 Terms of Use This rtile ws downloded from Hrvrd University s DASH repository, nd is mde vilble under the terms nd onditions pplible to Other Posted Mteril, s set forth t http:// nrs.hrvrd.edu/urn-:hul.instrepos:dsh.urrent.terms-ofuse#laa
Reeived Apr 5 Aepted Aug 5 Published Sep 5 DOI:.8/nomms957 OPEN SETDA modultes ell yle progression through mirna network tht regultes p5 trget genes Ken Tjim,, Toshifumi Ye,, Srh Jvid, Oliver Tm, Vlentine Comills, Robert Morris, Ben S. Wittner, Mingzhu Liu, Amnd Engstrom, Fumiyuki Tkhshi, Joshu C. Blk, Sridhr Rmswmy, Toshihiro Shiod, Molly Hmmell, Dniel A. Hber, Johnthn R. Whetstine & Shyml Mheswrn, Expression of the p5-induible ntiprolifertive gene BTG is suppressed in mny ners in the bsene of intivting gene muttions, suggesting lterntive mehnisms of silening. Using shrna sreen trgeting histone lysine methyltrnsferses (KMTs), we show tht SETDA suppresses BTG expression through its indution of severl BTG-trgeting mirnas. This indiret but highly speifi mehnism, by whih hromtin regultor tht medites trnsriptionl tivting mrks n led to the downregultion of ritil effetor gene, is shred with multiple genes in the p5 pthwy. Through suh mirna-dependent effets, SETDA regultes ell yle progression in vitro nd modultes tumorigenesis in mouse xenogrft models. Together, these observtions help explin the remrkbly speifi geneti onsequenes ssoited with ltertions in generi hromtin modultors in ner. Msshusetts Generl Hospitl Cner Center, Hrvrd Medil Shool, Chrlestown, Msshusetts 9, USA. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, New York 7, USA. Deprtment of Surgery, Hrvrd Medil Shool, Chrlestown, Msshusetts 9, USA. These uthors ontributed eqully to this work. Correspondene nd requests for mterils should be ddressed to J.R.W. (emil: johnthn_whetstine@hms.hrvrd.edu) or to S.M. (emil: Mheswrn@helix.mgh.hrvrd.edu). NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
BTG, p5-induible gene, plys ritil role in p5-medited suppression of mouse nd humn fibroblst trnsformtion indued by onogeni-rs. BTG inhibits ell yle progression nd its depletion enhnes tumorigenesis in mouse models. In humn ners, inluding brest nd prostte ners, BTG expression is suppressed. Despite its tumoursuppressor effets, muttions in BTG hve not been deteted, suggesting tht epigeneti mehnisms ould be involved in modulting BTG expression in ells. Chromtin-modifying enzymes re emerging s importnt regultors of tumorigenesis, s well s promising ner therpeuti trgets. Despite their brod effets on hromtin struture, they lso hve striking trget speifiity, prdox tht is poorly understood. Histone methyltion, tlysed by lysine methyltrnsferses (KMT), hs been linked to both trnsriptionl tivtion (HK, HK nd HK79) nd repression (HK9, HK7 nd HK). The Set/COMPASS fmily of HK methyltrnsferses, first identified in Shromyes erevisie, onsists of severl members in humns nd is ssoited with trnsriptionl tivtion. Although HK methyltion is ssoited with trnsriptionl tivtion, the overll hnge in gene expression lndspe within ell my depend on omplex interply between the trget genes indued, whih my led to seondry effets tht repress s well s indue gene expression. Here we onduted n unbised short hirpin RNA (shrna) sreen ginst KMTs to determine whether bseline expression of BTG in proliferting ells is regulted by epigeneti mehnisms. We identified SETDA s KMT whose knokdown strongly indues tivtion of BTG. This prdoxil repressive effet of n tivting hromtin regultor results from its trnsriptionl tivtion of multiple mirornas (mirnas), whih themselves suppress the BTG trnsript. This effet is onsistent with the roles of mirnas in oordinting multiple downstrem trgets tht ply importnt roles in ellulr differentition, prolifertion nd signlling 5,. In ddition to trgeting BTG, SETDA-indued mirnas trget dditionl downstrem effetors of p5, s well s ell yle regultors. These findings illustrte the exquisite trget speifiity exhibited by hromtin-modifying enzymes, nd the omplex network through whih hromtin regultors influene genes involved in tumorigenesis. Results Identifition of SETDA s regultor of BTG expression. To investigte whether epigeneti silening is involved in Reltive expression of BTG nd methyltrnsferses 5 HK Lysine methyltrnsferse / BTG 5 5 5 5 5 7 8 9 5 5 5 5 5 5 5 MLL MLL MLL MLL5 SETDA SETDB ASHL SMYD SMYD WHSCL WHSC SETD7 PRDM9 SETMAR b kd 5 5 5 5 7 shsetda # shsetda # HKMe HKMe HKMe Histone H β-atin Reltive SETDA ( ) nd BTG ( ) expression.5.5.5 Prostte ner shsetda DU5 8 Lung ner shsetda A59 d Reltive SETDA expression ( ) LZ SETDA..8... Reltive BTG expression ( ) e Reltive WDR8 ( ) nd BTG ( ) expression.5.5.5 sint siwdr8 Figure BTG expression is regulted by SETDA. () Knokdown of SETDA speifilly indues BTG. A lentivirl shrna sreen ginst HK KMTs in MDA-MB- ells shows tht SETDA depletion speifilly indues BTG expression ompred with plko-infeted ontrol ells. Fold hnge in BTG nd KMT expression fter 7 h of virl infetion is shown with ontrols set t. The dotted line mrks twofold indution of BTG ompred with ontrol. KMTs nd BTG re shown s red nd blue/green brs, respetively. (b) Totl proteins isolted from ontrol nd SETDA-depleted MDA-MB- ells were nlysed for HKMe, HKMe, HKMe, totl histone H nd -tin protein expression. () SETDA suppresses BTG in prostte nd lung ner ells. SETDA nd BTG expression in SETDA-depleted ells represents the verge derived from ells individully infeted with the two different shsetda onstruts (shsetda# nd shsetda#), with the -infeted ontrol set t. (d) Expression of lentivirlly expressed SETDA in MCFA ells dereses BTG expression (white br). Fold expression of SETDA in ontrol nd SETDA-infeted ells is shown (blk br). (e) WDR8, non-tlyti subunit of the SET/COMPASS omplex, regultes BTG expression. WDR8 nd BTG expression in WDR8-depleted MDA-MB- ells represents the verge derived from ells individully trnsfeted with the two different siwdr8 sequenes, with the sint (non-trgeting, NT)- trnsfeted ontrol set t. Dt re represented s men±s.d. of the verge of three experimentl replites. Asterisks indite P vlues of Po.5 for, e by Student s t-test. NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
Fold hnge in mir. MDA-MB- DU5.8... mir 59 59 59 59 shsetda shsetda A59 59 59 shsetda b..8... Antisense Reltive mir expression NT 59 Fold hnge in BTG mrna 5 Antisense NT 59 Fold hnge in BTG mrna MDA-MB-.5.5 DU5 d % men fluoresene intensity 8 e % men fluoresene intensity 8 mir NT NT 59 NT NT 59 shsetda shsetda mir NT 59 mir NT 59 Figure SETDA regultes BTG through indution of mirnas. () Regultion of BTG expression by SETDA is medited through mirnas. SETDA regultes mirna- nd -59-5p, whose expression ws nlysed using qpcr in nd shsetda-infeted MDA-MB-, DU5 nd A59 ells. Expression of mirnas in ontrols ws set t. Expression of the mirnas in SETDA-depleted ells represents the verge derived from ells individully infeted with two shsetda onstruts. (b) Suppression of mirna- nd -59-5p with Anti-miRs indues BTG expression. Expression of the two mirnas indited ws suppressed with Anti-miRs, nd eh mirna (left) nd BTG mrna (right) ws mesured using qpcr. The level of eh mirna nd BTG in ells trnsfeted with non-trgeting (NT) sequenes ws set t. () Expression of mirna- nd -59-5p brogtes BTG indution in SETDA-depleted ells. BTG expression in -infeted ontrol ells trnsfeted with NT sequenes ws set t. (d) mirna- nd -59-5p, trget the -UTR of BTG. A plsmid ontining GFP linked to -UTR of BTG ws o-trnsfeted with the two mirna sequenes into 9Tells, nd the intensity of GFP expression ws monitored by fluoresene-tivted ell sorting. NT sequenes were used s ontrol nd the intensity of GFP in this smple ws set t %. (e) Deletion of the region hrbouring binding sites for mirna- nd -59 from the -UTR of BTG brogtes mirna-- nd -59-5p-medited suppression of the -UTR of BTG. Dt re represented s men±s.d. of the verge of three experimentl replites. Asterisks indite P vlues of Po.5 for ll figures by Student s t-test. For the relevnt figures, NT represents NT, nd 59 indites mirna-59-5p. suppressing BTG expression in proliferting ells, we mesured BTG indution in the humn brest ner ell line MDA- MB-, following infetion with n rryed lentivirl shrna librry ontining shrnas ginst KMTs. Four to ten independent shrna onstruts were tested, trgeting eh KMT implited in hromtin mrks, HK, HK9, HK7, HK, HK79 nd HK methyltion, s well s unknown substrtes (Supplementry Fig. ). Bseline BTG mrna is undetetble in MDA-MB- ells, filitting mesurement of its indution dys fter infetion. Suessful knokdown of KMT expression (%) with t lest two shrnas, ompred with ontrol plko vetor-infeted ells, ws observed for of the trgeted KMTs (Fig. nd Supplementry Fig. b). Among these, knokdown of SETDA by eight of nine shrnas ws unique in leding to.5-fold inrese in BTG mrna (Fig. ). Knokdown of no other KMT led to signifint nd robust inrese or derese in BTG expression (Fig. nd Supplementry Fig. b). Depletion of SETDA, KMT whose expression is inresed in brest, prostte nd lung ners 7,8 (Supplementry Fig. ), did not lter totl HKMe, HKMe nd HKMe nd histone H levels in ells (Fig. b nd Supplementry Fig. 8). BTG mrna indution (up to -fold) on SETDA depletion ws observed in multiple brest (n ¼ ), prostte (n ¼ ), lung (n ¼ ) nd olon (n ¼ ) ner ell lines, nd it ws independent of the muttionl sttus of p5, known regultor of BTG (Fig. nd Supplementry Fig. b; Po.5, Student s t-test). In ddition to mrna quntifition, SETDA-medited BTG regultion ws vlidted using western blot nlysis (Supplementry Fig. nd Supplementry Fig. 8b). Further, etopi overexpression of SETDA in MCFA brest epithelil ells, whih express BTG t bseline, led to redued expression of the endogenous trnsript (Fig. d; Po.5, Student s t-test). Depletion of WDR8, non-tlyti subunit speifi to the SET/ COMPASS omplexes (SETDA nd SETDB) nd not shred by the MLL/COMPASS omplexes, lso indued BTG expression (Fig. e). Consistent with these findings, nlysis of lrge dt set onsisting prostte tumours 9, showed signifint inverse orreltion between BTG nd SETDA mrna expression in tumours, n effet tht ws most pronouned in lower-grde prostte tumours (Gleson sore r). No suh orreltion ws present in norml prostte tissue (Supplementry Fig. ); similr negtive orreltion ws lso observed in brest nd lung, ners (Supplementry Fig. b). SETDA suppresses BTG expression through mirna network. Hving identified pronouned effet of KMT hromtin regultor on BTG expression, we first nlysed the hromtin mrks t the BTG promoter. Tiled hromtin immunopreipittion quntittive PCR (ChIP qpcr) ross 5-kb region spnning the BTG trnsriptionl strt site (Supplementry Fig. ) demonstrted peks of HKMe, HKMe nd HKMe mrks in the proximity of the trnsription strt site (Supplementry Fig. d). SETDA depletion led to signifint deline in the HKMe peks s well s NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
TMEM5 shsetda v HKMe HKMe HKMe.. mir- mir-. SETDA HKMe 8 shsetda v...8.8..8........ Primer # 5 5 5 Primer # Primer # % input / totl H b % input % input / totl H % input / totl H EIFH HKMe...8. HKMe HKMe. 8 shsetda v.8. mir-59. SETDA. Primer # Primer # Primer # d % input mir-59. HKMe % input / totl H.8 shsetda v. Figure SETDA regultes the promoters of mir- nd mir-59-5p. () HKMe, HKMe nd HKMe mrks on the promoter region of TMEM5 were nlysed using primers tht spn the region. The results show tht SETDA does not regulte these hromtin mrks on the TMEM5 promoter. (b) SETDA binding nd HKMe on the puttive promoter region of mir- were nlysed using two primers tht spn the region. The results show tht SETDA depletion suppresses HK methyltion nd SETDA binding to this domin. () HKMe, HKMe nd HKMe on the promoter region of EIFH were nlysed using four primers tht spn the region. The results show tht SETDA does not regulte these hromtin mrks on the EIFH promoter. (d) SETDA binding nd HKMe mrks on the puttive promoter region of mir-59 ws nlysed using single primer tht spns the region. The results show tht SETDA depletion suppresses HK methyltion nd SETDA binding to this domin. For ll experiments, shsetda dt points represent the verge derived from ChIP ssys performed with ells individully infeted with two different shsetda onstruts. Dt re represented s men±s.d. of the verge of three experimentl replites. Asterisks indite P vlues of Po.5 for relevnt figures by Student s t-test. SETDA oupny within this re (Supplementry Fig. d,e; Po.5, Student s t-test) suggesting tht SETDA mintins the tivting HK methyltion mrks on the BTG promoter. As ontrol, depleting MLL did not ffet HK mrks or MLL oupny on the BTG promoter, suggesting tht BTG is speifi trget of SETDA (Supplementry Fig. f,g). However, the presene of tivting hromtin mrks on the BTG promoter would hve suggested tht SETDA depletion should result in deresed BTG expression, rther thn the observed BTG indution. To better understnd this pprent prdox, we investigted whether SETDA might lso indue negtive regultors of BTG, nmely mirnas. We sreened for mirnas differentilly expressed in shsetda or -infeted MDA-MB- ells. SETDA knokdown using two independent shrnas led to n upregultion of 7 mirnas (more thn twofold) nd suppression of 8 mirnas (B%; Supplementry Tble ). Three trget predition lgorithms, TrgetSn, mirnd 5 nd PiTr, identified the downregultion of five BTG-trgeting mirnas, mir-59-5p, mir-7, mir-8, mir-b nd mir-, in SETDA-depleted ells. We further vlidted nd hrterized two BTG-trgeting mirnas: mir-59-5p, whih shows the highest levels of suppression (B75%) in SETDA-depleted ells, nd the previously identified BTG-trgeting mir- (ref. 7). Depletion of SETDA suppressed both mirnas in multiple ell lines (Fig. nd Supplementry Fig. 5; Po.5, Student s t-test). Suppressing the mirnas with orresponding nti-mirs inresed BTG mrna two- to fourfold (Fig. b nd Supplementry Fig. 5b; Po.5, Student s t-test), wheres restortion of either mirnas individully into SETDA-depleted ell lines bolished BTG indution (Fig. nd Supplementry Fig. 5; Po.5, Student s t-test). Expression of green fluoresent protein (GFP) onstrut linked to the -untrnslted repet (UTR) of BTG demonstrted tht both mirnas suppress GFP expression in ells (Fig. d). Deletion of the region hrbouring the two mirna--binding sites nd the single mirna-59-binding site from the -UTR of BTG brogted this effet (Fig. e nd Supplementry Fig. 5d). Thus, the repression of mirna- nd -59-5p expression by SETDA depletion my ontribute to the inrese in BTG mrna levels. Interestingly, expression of both mir- nd -59-5p is inresed in brest ner smples ompred with norml tissue (P ¼.e for mir-, P ¼.8e 9 for mir-59-5p, Student s t-test; Supplementry Fig. 5e), nd n inverse orreltion is observed between BTG expression nd mirna- nd -59-5p, supporting the relevne of these findings in linil speimens (or ¼., P ¼.e for BTG/miR-; or ¼., P ¼.e for BTG/miR- 59-5p, Person s orreltion; Supplementry Fig. 5f). Together, these results suggest tht SETDA-indued mirnas suppress BTG expression. SETDA regultes the promoters of BTG-trgeting mirnas. We then determined whether SETDA diretly regultes the expression of BTG-trgeting mirnas. mir- resides within intron of the host gene TMEM5, whose promoter is B9 kb wy from the mirna (Supplementry Fig., green ovl). HKMe, HKMe nd HKMe levels of the TMEM5 NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
MDA-MB- (brest) % (85) / Trnsripts upregulted in SETDA-depleted ells (n=897) Trnsripts regulted by SETDA-indued mirnas (n=85) 8% (7) b NUAK EGR ULK SIPAL ID RHOB HIC LBH DPYSL EPHB FAMA FGF8 IHH NTN SMAD7 TMEM CXCR7 DCLK SIDT MAPK CHAC LMBRL MFHAS NEDD9 TP5INP BTG CDKNB ADAMTS7 Corf87 CADM CXADR EOMES FRL GLCCI GPCPD HIPR INSM SLCA ZNF7 CLU BAMBI JAG TMCC Corf5 ST8SIA CSRNP ELAVL SUVH STC CPEB FAMA GPR7B PGML EGLN KLF7 DUSP8 COLA HMCN IGFBP5 LDOC C9orf DNM MAPRE SLCA TBCD9 VLDLR RPSKC ADAM7 BACH CLCN TSCD SCHIP BMF CLDN NTN MAPK COL5A AQP ARRDC AXIN HMGCS NRC PPIC PRKAB SERPINE TMEMB TNIK VSTML ZBTB7 EREG PLK ILA ProSAPiP ARIDB EZH IPK KHNYN PLEKHM RNF RRAGD SIK TSC Gene set nme TP5 Trgets (n=9) TP Trgets (n=) ESR Trgets dn (n=9) EZH Trgets up (n=) Photodynmi therpy Stress up (n=9) TP5 AND TP Trgets (n=8) FDR (.E-) (7.9E 9) (.8E ) (.E ) (.E ) (.9E ) e MDA-MB- (brest) Reltive mrna expression 5 # mirna Trget genes ACVRB, ADAMTS7, AQP, ARIDA, ARRDC, BACH, FAMA, JPH, MAPRE, NLGN, PPPRB, PRKAB, RNF, SBK, SESN, SLCA, SLC5A7, TMOD ACHE, BMF, DPP, DYRKB, LONRF, PGML, SESN, USP DUSP8 WDTC 5 BTG, ProSAPiP, TP5INP ARIDA.5 SESN TP5INP 5.5 8.5.5 MDA-MB- DU5 A59 MDA-MB- DU5 A59 MDA-MB- DU5 A59 shsetda A59 (lung) d shsetda shsetda Figure SETDA-indued mirnas re enrihed for regultors of the p5 downstrem pthwy nd ell yle-regultory genes. () Shemti representtion of the genes upregulted in SETDA-depleted MDA-MB- ells. Twenty per ent of the genes (85 out of 897 mrnas) upregulted in SETDA-depleted ells re high-onfidene trgets (green) of SETDA-regulted mirnas. (b) The six top tegories of enrihed gene signtures in the GSEA dtbse re shown for the 85 genes regulted by SETDA-indued mirnas. Genes ontributing to the enrihment re highlighted in green. The number of genes ontributing to the enrihed signtures within eh tegory is given in prentheses. FDR ws determined with P vlue by the gene set enrihment hypergeometri test. () Shemti representtion of SETDA-indued mirna trget genes tht re ommon to both MDA-MB- nd A59 ells. Comprison of the 85 trget genes regulted by SETDA-indued mirnas ginst 7 genes indued in SETDA-depleted A59 ells identified ommon genes. (d) List of the ommon genes trgeted by SETDA-indued mirnas. The number of mirnas regulting eh gene is listed on the left. (e) Expression of p5 downstrem trget genes shown were nlysed using qpcr in - nd shsetda-infeted MDA-MB-, DU5 nd A59 ells. Expression in SETDA-depleted ells represents the verge derived from ells individully infeted with the two shsetda onstruts, with the -infeted ontrol set t. Dt re represented s men±s.d. of the verge of three experimentl replites. Asterisks indite P vlues of Po.5 by Student s t-test. promoter do not hnge on SETDA knokdown (Fig. ). Thus, the suppression of mir- is not due to TMEM5 host gene regultion. The UCSC genome dtbse reveled proximl HKMe pek B kb wy from mir- (Supplementry Fig., red ovl). ChIP nlysis of this region demonstrtes tht both SETDA binding nd HKMe re signifintly redued on SETDA depletion (Fig. b; Po.5, Student s t-test). We similrly evluted mir-59, whih lolizes within the host gene EIFH, whose promoter is B5 kb upstrem of the mirna (Supplementry Fig. b, green ovl). Agin, HK methyltion of the EIFH promoter does not hnge on SETDA knokdown (Fig. ). Anlysis of the region oiniding with the mir-59 promoter previously desribed 8 (Supplementry Fig. b, red ovl) demonstrtes signifint derese in both SETDA binding nd HKMe on SETDA knokdown (Fig. d; Po.5, Student s t-test). These results revel tht BTG-trgeting mirnas, mir- nd mir-59, re both diret trgets of SETDA nd tht they re indued independently of their host trnsripts by this hromtin regultor. SETDA-indued mirnas re enrihed for p5 trget genes. The effet of SETDA on BTG expression my illustrte broder mirna network by whih this hromtin regultor modultes genes involved in tumorigenesis. To evlute the full effet of SETDA-indued mirna trgets, we identified mirorna mrna intertions mong ll genes nd mirnas differentilly expressed in SETDA-depleted MDA-MB- ells using TrgetSn, mirnd nd PiTr. Only those identified by ll three lgorithms were seleted to form NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions 5
Cells in ell yle phse (%) 9 G S G/M b DU5 MDA-MB- A59..8 9.. 9 8.7.. 9.77.55 9. 8.7.9.8 77.7 75.97 8. 9. 5.79 shsetda shsetda shsetda Cells in ell yle phse (%) MDA-MB- G S G/M 8.59. 8.. 7. 55.5 sint siwdr8 Cells in ell yle phse (%) 9 DU5 G S G/M 8.. 7.5.9 8.9 7.5.59 7.8.79 sint sint sibtg shsetda d Cells in ell yle phse (%) 9 DU5 G S G/M.55 5..78. 5.8.8.8 8..5 7.9.9 7.5 NT NT mir- mir-59 shsetda Figure 5 SETDA regultes ell yle progression. () SETDA depletion inreses the G frtion. Cell yle nlysis of MDA-MB-, A59 nd DU5 ells infeted with nd shsetda onstruts. The per ent ell yle distribution of SETDA-depleted ells represents the verge derived from ells individully infeted with the two different shsetda onstruts. (b) WDR8 depletion inreses the G frtion. Cell yle nlysis of MDA-MB- ells trnsfeted with siwdr8 nd sint sequenes. The per ent ell yle distribution of WDR8-depleted ells represents the verge derived from ells individully trnsfeted with the two different siwdr8 onstruts. () Depletion of BTG prtilly reverses the inrese in the G frtion exhibited by SETDA knokdown ells. Cell yle nlysis of shsetda-infeted ells trnsfeted with sibtg nd NT sirna (sint). -infeted ells trnsfeted with sint re shown s ontrol. As previously seen, knokdown of SETDA (sint) inreses the frtion of ells in G ompred with (sint)-infeted ells. Suppression of BTG with sibtg in shsetda ells reverses this effet. (d) mirnas reverse the G inrese in SETDA-depleted ells. Cell yle nlysis of shsetda-infeted ells trnsfeted with mirna- nd -59 mimis. Knokdown of SETDA (sint) inreses the frtion of ells in G ompred with (sint)-infeted ells. The mimis ginst mirna- nd -59 prtilly suppress this effet. Dt re represented s men±s.d. of the verge of three experimentl replites. Asterisks indite P vlues of Po. for,b nd Po.5 for,d by Student s t-test. high-onfidene set of onsensus mirorna trgets. Of the 897 genes indued in SETDA knokdown MDA-MB- ells, only 85 (%) genes were mirna trgets (Fig. nd Supplementry Tble ). Gene set enrihment nlysis (GSEA) of these 85 genes identified gene signtures (Po7.e 7, flse disovery rte (FDR) q-vlue (FDR) o.5e 5, hypergeometri test; Fig. b nd Supplementry Tble ), with enrihment for genes indued by p5 being the top hit (P ¼.e, FDR ¼.e, hypergeometri test 9 ). Additionl pthwys signifintly enrihed were TP (P ¼.e, FDR ¼ 7.9e 9, hypergeometri test 9 ) nd ESR (P ¼.e 9, FDR ¼.8e, hypergeometri test ) trgets (Fig. b nd Supplementry Tble ). Comprison of these 85 mirna trgets indued in SETDAdepleted MDA-MB- ells ginst the set of genes omprbly indued in SETDA-depleted A59 ells identified onsensus genes (Fig.,d). The ommon SETDA-regulted genes, thus identified, were eh found to be trgeted by one to five SETDA-indued mirnas (Fig. d nd Supplementry Tble ). BTG is one of three genes (BTG, ProSAPiP nd TP5INP) trgeted by the highest number of SETDA-regulted mirnas (Supplementry Tble ). GSEA of the genes lso showed signifint enrihment for nonil p5 downstrem pthwy genes (P ¼.e, FDR ¼.95e, Benjmini Hohberg method). Among the downstrem omponents most signifintly enrihed, nd independently onfirmed in multiple ell lines using qpcr, re ARIDA, SESN nd TP5INP (Fig. e). Additionl omputtionl nlyses (Gene, Disese Fetures Ontology-bsed Overview System) showed tht of the genes ommon between MDA-MB- nd A59 ells (trgeted by SETDA-indued mirnas) were signifintly relted to the p5 pthwy, neoplsms nd ell yle regultion (Supplementry Fig. 7); StrBse enrihment nlysis, of these SETDA-indued mirnas shows tht they re signifintly enrihed for omponents ssoited with negtive regultion of ell prolifertion, nd p5 nd ner-ssoited pthwys (Supplementry Fig. 7b). Tken ll together, these results show tht SETDA-regulted mirnas trget multiple genes involved in ell yle regultion, nd p5 downstrem pthwys, inluding BTG. SETDA depletion inhibits the ell yle nd tumour growth. To determine the funtionl onsequene of SETDA, we nlysed ell yle distribution in SETDA-depleted ells. Consistent with the ell yle-regultory effets of BTG (ref. ), depletion of SETDA modultes ell yle progression in ner ells. In MDA-MB- s well s in A59 lung ner ells nd in DU5 prostte ner ells, SETDA depletion inresed the G frtion (Fig. 5; Po., Student s t-test). A similr inrese in the G frtion ws lso observed in WDR8-depleted MDA-MB- ells (Fig. 5b). The inrese in G indued by SETDA knokdown ws prtilly reversed using sirnamedited inhibition of BTG (Fig. 5; Po.5, Student s t-test). NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
Fold hnge in tumour volume DU5 (n=) shsetda v (n=) 8 Dys 8 5 MDA-MB- (n=) shsetda v (n=) Consistent with this effet, etopi expression of mir- nd -59-5p mimis redued the G frtion in SETDA knokdown ells (Fig. 5d; Po.5, Student s t-test). The effet of SETDA on ell prolifertion ws even more striking in vivo, where SETDA knokdown brogted tumorigenesis of DU5 nd MDA-MB- ells (Fig. ; Po.5, Student s t-test). Disussion SETDA-medited regultion of BTG revels new dimension in the omplex regultory pthwys medited by hromtinmodifying enzymes. Although SETDA regultes HKMe methyltion of the BTG promoter, suggesting diret trnsriptionl regultion of BTG, it onurrently indues the expression of severl BTG-trgeting mirnas. The ultimte onsequene of these opposing signls is the suppression of BTG expression, refleting the dominne of the mirna-medited effets. Anlysis of SETDA-regulted mirna/mrna trgets ross multiple ell lines shows tht SETDA-indued mirna network brodly suppresses the expression of p5 downstrem trgets nd ell yle-regultory genes in ddition to BTG. Of note, the suppression of p5 downstrem trgets, ARIDA, SESN, TP5INP nd BTG, by SETDA-indued mirnas is independent of the ellulr p5 sttus. These findings identify subset of p5 trget genes speifilly indued by hromtin modifier through p5-independent regultory mehnism, nd b 5 Dy Dy Dy 8 Dys 7 8 shsetda Figure SETDA regultes tumorigenesis. () DU5 ells infeted with - nd shsetda lentiviruses were inoulted subutneously into immunoompromised mie. Fold hnge in tumour volume nd the number of nimls in eh group re shown. The volume of SETDA-depleted tumours represents the verge derived from ells individully infeted with the two different shsetda onstruts. Dt re represented s men±s.d. of the verge of eh group. Asterisks indite P vlues of Po.5 by Student s t-test. (b) Luiferse expressing MDA-MB- ells infeted with - nd shsetda lentiviruses were inoulted into the mmmry ft pd of immunoompromised mie. Tumour growth ws monitored by bioluminesene imging. The fluoresene intensity of SETDA-depleted tumours represents the verge derived from tumour ells individully infeted with the two different shsetda onstruts. The number of nimls in eh group re shown. Dt re represented s men±s.d. of the verge of eh group. Asterisks indite P vlues of Po.5 by Student s t-test. () Seril bioluminesene imging of nd shsetda-mda-mb- tumour-bering mie from experiment desribed bove (Fig. b) is shown. illustrte the exquisite speifiity nd omplexity involved in SETDA-regulted gene expression. Consistent with the indution of the p5 downstrem trgets, BTG, TP5INP nd SESN, ll of whih hve tumoursuppressor funtions on their own,,, SETDA depletion suppresses tumour growth in vivo. This estblishes new funtionl role of SETDA, whih hs previously been identified s developmentlly required for gstrultion, but dispensble for mouse embryo implnttion 5. SETDA is responsible for mintenne of the mjority of HK methyltion in embryoni stem ells. Our dt, however, demonstrte tht SETDA is not required for overll mintenne of HK methyltion in tumour ells (Fig. b), suggesting tht SETDA tivity is dependent on the ellulr ontext. Protein protein intertions defining ontext-dependent HK trimethyltion by Set/COMPASS hs been reported in yest. Nonetheless, depletion of SETDA in both tumour ells nd embryoni stem ells interferes with ell yle progression by inresing G (ref. 5). Whether SETDA-medited ell yle regultion in embryoni stem ells involves mirna intermedites remins to be determined. In onlusion, the regultion of BTG by SETDA serves s model to illustrte () the exquisite trget speifiity exhibited by histone lysine methyltrnsferses, () mirna network-medited mehnism through whih SETDA, positive hromtin regultor of trnsription, suppresses the expression of severl genes involved in ell yle regultion nd the p5 pthwy nd () new role for SETDA in regulting tumour growth. Given tht the misregultion of HK methyltion resulting from MLL fusion proteins, mutnt MLL nd MLL proteins, nd berrnt demethylse tivity hs been implited in ner progression 7 9, our observtions linking the HK regultor SETDA in tumorigenesis supports the importne of this hromtin modifier nd the potentil pplition of smll moleules trgeting the enzymti tivity of KMTs in ner. Methods Cell ulture. Humn brest, prostte nd lung ner ell lines MDA-MB-, MDA-MB-8, MCF7, BT59, DU5, LNCP, A59 nd H99 were grown in Dulbeo s modified medium. Humn olon ner ell lines HCT8, HCT, H, DLD, SW nd HT9 were grown in RPMI medium. Both growth medi were supplemented with % fetl bovine serum nd peniillin/streptomyin (Pen/Strep). The non-tumorigeni MCFA ells were ultured in DMEM/F medium with 5% horse serum, epiderml growth ftor, Hydroortizole, Choler Toxin, Insulin nd Pen/Strep. All ell lines were purhsed from the Amerin Type Culture Colletion (ATCC, Mnsss, VA, USA). All ell lines were mintined in 5% CO t 7 C. Sreening for KMTs tht modulte BTG expression. Lentivirl shrna onstruts were obtined from the RNAi Consortium shrna Librry t the Brod Institute. Conditioned medium ontining infetive lentivirl prtiles ws generted by o-trnsfeting mg of lentivirl vetor (plko), mg of pcmv d8.9 nd mg phcmv-g into 9T humn embryoni kidney ells using FuGENE trnsfetion regent (Rohe Applied Siene). Superntnts were olleted 8 h fter trnsfetion nd were filtered through.5-mm membrne (Millipore). Cells were infeted using 8 mgml polybrene nd seleted with mgml puromyin. RNA ws isolted 7 h fter infetion nd nlysed using qpcr for expression of BTG nd eh histone lysine methyltrnsferse. The primer sequenes used to nlyse expression using qpcr re provided in Supplementry Tble. Chromtin immunopreipittion ssy. SETDA binding nd HK methyltion of BTG, TMEM5, EIFH, mir- nd -59 were nlysed using ChIP. The genomi regions to be nlysed were hosen on the bsis of the HKMe profiles obtined from the UCSC dt. Cells were grown to 8% onfluene, rosslinked with % formldehyde for 5 min t 7 C nd quenhed with formldehyde ontining 5 mm glyine. After wshing twie with old PBS, ells were olleted into ml of lysis buffer (5 mm PIPES, 85 mm KCl nd.5% NP-, Protese Inhibitors). Nulei were olleted nd inubted in nuler lysis buffer (5 mm Tris (ph 8.), mm EDTA (ph 8.),.% EDTA, Protese Inhibitors). The NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions 7
rosslinked hromtin ws sonited into DNA frgments of B 5 bp in length. Protein G beds were first inubted on rottor t C for h with mg eh of the following ntibodies ginst Histone H (Abm, b79), Histone H monomethyl K (Abm, b8895), Histone H dimethyl K (Abm, b9), Histone H trimethyl K (Abm, b858), SETDA (BETHYL, A 89A), MLL (BETHYL, A 7A) nd ontrol IgG (Cell Signling, 79S). Chromtin solution ( mg) ws inubted with protein G mgneti beds onjugted with ntibodies overnight on rottor t C. After six wshes, the beds were eluted with elution buffer (5 mm NHCO, mm NCl, % SDS). Following both RNseA nd proteinse K tretments, nd reverse rosslinking, DNA ws purified with the PCR len-up kit (Qigen) nd nlysed with qpcr using primers ginst the relevnt trgets listed in Supplementry Tble 5. Western blot nlysis. The ntibodies used for western blot nlysis were rbbit nti-set polylonl ntibody (:,, BETHYL, A 89A), rbbit nti-btg polylonl ntibody (:,, GenWy Bioteh, GWB-D5FE7), rbbit nti-histone H ntibody (:,, Abm, b79), rbbit nti-histone H monomethyl K ntibody (:5, Abm, b8895), Got nti-histone H dimethyl K ntibody (:,, Abm, b9), rbbit nti-histone H trimethyl K ntibody (:,, Abm, b858) nd mouse nti-atin monolonl ntibody (:5,, BD Biosiene, 5). Cells were lysed in RIPA buffer ( mm Tris, ph 8., 5 mm NCl, mm NF,.% SDS, % Nonidet P- nd protese inhibitor mixture (Rohe)), proteins were seprted on 5% polyrylmide grdient SDS gel (Bio-Rd), trnsferred on polyvinylidene difluoride membrnes (Millipore) nd bloked in TBST (Tris-buffered sline nd Tween ; 5 mm Tris, ph 7., mm NCl, 5 mm KCl nd.% Tween) ontining 5% milk. The ntibodies were used t eh dilution, s desribed bove, in % milk/tbst before use. Blots were inubted with the primry ntibodies for h t room temperture. Blots were wshed (three times) with % milk/tbst nd were inubted with the pproprite horserdish peroxidse-onjugted ntibodies. Bound ntibodies were deteted with enhned hemiluminesene (ECL) (Amershm Phrmi Bioteh). Vlidtion of SETDA-regulted mirnas. Briefly, RNA isolted from nd shsetda (shsetda# nd shsetda#)-infeted MDA-MB- ells, dys fter infetion, ws ssyed with n A & B rd onsisting of probes ginst totl of 75 mirnas. The effet of SETDA knokdown on mirna expression ws lulted s the rtio of mirna expression in shsetda-mda-mb- nd -MDA-MB- ells. An rbitrry threshold of % suppression nd twofold inrese in expression were used to determine the mirna popultion tht ws regulted by SETDA. For further evlution of seleted trgets, totl RNA inluding mirna ws isolted from ells using the mirvn mirna Isoltion Kit (Ambion). mirnas were deteted using two-step qrt PCR, with the first step being reverse trnsription (RT) of DNA nd the seond step being rel-time qpcr. All regents were supplied by Applied Biosystems. The RT retion ws performed in 5 ml volume, ontining.5 ml Tqmn RT Buffer ( ),.5 ml mm dntps ( mm),. ml Reverse Trnsriptse,.9 ml RNse inhibitor ( U ml ),. ml speifi mirna primer, ng totl RNA nd nulese-free wter. Rel-time qpcr ws performed in ml retion volume using the stndrd protools on n Applied Biosystems 79HT System. Briefly,.5 ml of DNA ws mixed with ml TqMn universl PCR mster mix ( ),. ml TqMn mirna ssy nd.5 ml nulese-free wter. The retion onditions were the sme s bove for rel-time PCR in rry experiments. For eh RNA smple, the trget mirna nd RNU8 retions were run in triplite. Mimis of mir- nd -59-5p or ontrol (5 nm) were trnsfeted into ells using the Lipofetmine RNAiMAX (Invitrogen) ording to the mnufturer s protool. Anti-miRs ginst mir- nd -59-5p merury LNA or ontrol (5 nm) were similrly introdued into ells. The sequenes of mimis nd the nti-mirs re given below. miridian mirorna Mimis hs-mir--5p: 5 -UAUUGCACAUUACUAAGUUGCA- hs-mir-59-5p: 5 -GAGCUUAUUCAUAAAAGUGCAG- Negtive ontrol: 5 -UUGUACUACACAAAAGUACUG- mircury LNA inhibitor hs-mir--5p: 5 -CAACTTAGTAATGTGCAAT- hs-mir-59-5p: 5 -CTGCACTTTTATGAATAAGCT- Negtive ontrol: 5 -AGAGCTCCCTTCAATCCAAA- -UTR plsmid reporter ssy. plenti-utr-gfp plsmid, in whih the -UTR of BTG is inserted s the -UTR of GFP, ws purhsed from Applied Biologil Mterils In. The -UTR of BTG lking the binding sites for mir- nd -59-5p ws derived from this plsmid using the QuikChnge Lightning Site-Direted Mutgenesis Kit (Agilent Tehnologies) ording to the mnufturer s protool. 9T humn embryoni kidney ells were trnsfeted with both the wild-type nd the mir-binding site-deleted plenti-utr plsmids using X-tremeGENE HP DNA trnsfetion regent (Rohe Applied Siene). Cells were trnsfeted with mirnas (mir-, mir-59-5p or ontrol) using Lipofetmine RNAiMAX, 8 h fter trnsfetion, with plenti-utr-gfp nd were nlysed with MACS Flow Cytometry (Miltenyl Biote) for the men fluoresent intensity 7 h fter trnsfetion of the mir. RNA interferene ssy. sibtg sequene (5 -CAGAGCACUACAAACACCA CUGGUU- ), siwdr8# sequene (5 -UCUCAUCUGUAGUUUCCAATT- ), siwdr8# sequene (5 -GAUCCAGAAGGGUUAAUUUTT- ) or ontrol (5 nm) were trnsfeted into ells using the Lipofetmine RNAiMAX (Invitrogen) ording to the mnufturer s protool. Cell yle nlysis. Cells were hrvested, wshed twie in PBS, resuspended in.% sponin/pbs (Sigm) ontining mgml RNse (Sigm) nd 5 mgml propidium iodide (Sigm) nd inubted for min t 7 C. Smples were nlysed with flow ytometry using the FACSlibur nd CellQuest softwre (Beton Dikinson). In vivo tumorigenesis ssy. DU5 ells ( ) infeted with nd shsetda lentiviruses (in ml of : PBS nd mtrigel (BD Biosienes)) were seprtely inoulted subutneously into the flnks on either side of - to 8-week-old mle thymi nude mie (n ¼ for ontrol; n ¼ for experimentl group). Tumours were mesured every week nd tumour volume ws lulted s length width.5. For MDA-MB- xenogrfts, -week-old femle thymi nude mie (n ¼ for ontrol group, n ¼ for experimentl group) were nesthetized with isofluorne, nd luiferse-tgged tumour ells ( ) infeted with nd shsetda lentiviruses ( ) in ml of : PBS nd Mtrigel (BD) were injeted into the fourth right mmmry ft pd. Tumour-derived signls were monitored by in vivo live imging using IVIS Lumin II (PerkinElmer). D-Luiferon substrte (PerkinElmer) ws injeted intrperitonelly t 5 ml per niml; mie were nesthetized fter 5 min; nd bioluminesent imges were tken. All mie were red for nd experiments were performed under the AAALAS guidelines using protools pproved by the institutionl review bord nd the institutionl niml re nd use ommittee of the Msshusetts Generl Hospitl. Arryed gene expression nlysis. RNA expression in SETDA-depleted A59 nd MDA-MB- ells ws nlysed using the Humn Gene Expression 5 K Arrys (GPL75, Rohe Nimblegen). Briefly, RNA ws extrted from A59 nd MDA-MB- ells infeted with or two independent shsetda onstruts (shsetda# nd shsetda#) using the RNesy Mini kit (Qigen). DNA synthesis ws performed using the Rohe DNA synthesis system ( 7 8 ). DNA ws hybridized to the Humn Gene Expression 5 K Arrys in duplite ording to the mnufturer s protool. The mirorrys were snned on Nimblegen MS t mm resolution. Sns were onverted to robust mirorry verge (RMA)-normlized expression vlues using the Rohe NimbleSn. softwre. The list of upregulted genes ommon to MDA-MB- ells infeted with shsetda# nd shsetda# ws ompiled. The 897 genes indued in SETDA-depleted ells thus identified were nlysed ginst the 8 mirnas ommonly suppressed in MDA-MB- ells infeted with shsetda# nd shsetda#. The 85 mirna mrna intertions predited by the three mirna trget preditor progrmmes, TrgetSn, mirnd nd PiTr, were then ompred with the list of ommon genes upregulted in A59 ells infeted with shsetda# nd SETDA#. This nlysis identified genes to be ommon trgets of SETDA-regulted mirnas. These 85 nd genes were then subjeted to pthwy enrihment nlysis (GSEA). The gene set enrihment hypergeometri tests between the 85 mirna trgets nd the urted gene lists (C) of MSigDB were omplished using the investigte gene sets tool (http://www.brodinstitute. org/gse/msigdb/nnotte.jsp; top gene sets; FDR ¼.5). When noted, GSEA ws performed by the Brod Institute (Cmbridge, MA, USA). P vlues were generted using hypergeometri GSEA pplied to the eh subset of Brod s MSigDB gene set dtbse. The Benjmini Hohberg method ws used to ompute FDR q-vlues from the P vlues. Onomine mirorry dt sets. Onomine dt sets were nlysed for differenes in SETDA mrna between ner nd norml tissues (gene rnk top %; P vlueo.5, Student s t-test). Representtive box plot from Onomine (www.onomine.org) is shown to illustrte the differene in SETDA mrna levels. All dt re log-trnsformed nd medin-entred, nd the 5th 75th perentiles re indited by the losed box. Sttistil nlysis. Sttistil nlysis ws performed with two-tiled Student s t-test or s desribed in eh method by the Brod Institute. The differenes between the mens were onsidered to be sttistilly signifint t Po.5. Referenes. Boiko, A. D. et l. A systemti serh for downstrem meditors of tumor suppressor funtion of p5 revels mjor role of BTG in suppression of Rs-indued trnsformtion. Genes Dev., 5 (). 8 NATURE COMMUNICATIONS :857 DOI:.8/nomms957 www.nture.om/ntureommunitions
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Androgen-regulted mir- trgets BTG nd is overexpressed in strtion-resistnt prostte ner. Onogene, 7 (). 8. Monteys, A. M. et l. Struture nd tivity of puttive introni mirna promoters. RNA, 95 55 (). 9. Perez, C. A., Ott, J., Mys, D. J. & Pietenpol, J. A. p onsensus DNA-binding site: identifition, nlysis nd pplition into pmh lgorithm. Onogene, 7 77 (7).. Gozgit, J. M. et l. PLD is overexpressed in n ER-negtive MCF-7 ell line vrint nd subset of phospho-akt-negtive brest rinoms. Br. J. Cner 97, 89 87 (7).. Yng, J. H. et l. strbse: dtbse for exploring mirorna-mrna intertion mps from Argonute CLIP-Seq nd Degrdome-Seq dt. Nulei Aids Res. 9, D D9 ().. Li, J. H., Liu, S., Zhou, H., Qu, L. H. & Yng, J. H. strbse v.: deoding mirna-erna, mirna-nrna nd protein-rna intertion networks from lrge-sle CLIP-Seq dt. Nulei Aids Res., D9 D97 ().. Budnov, A. V. & Krin, M. p5 trget genes sestrin nd sestrin onnet genotoxi stress nd mtor signling. Cell, 5 (8).. Okmur, S. et l. p5dinp, p5-induible gene, regultes p5-dependent poptosis. Mol. Cell 8, 85 9 (). 5. Bledu, A. S. et l. The HK methyltrnsferse Setd is first required t the epiblst stge, wheres Setdb beomes essentil fter gstrultion. Development, 5 ().. Thornton, J. L. et l. Context dependeny of Set/COMPASS-medited histone H Lys trimethyltion. Genes Dev. 8, 5 (). 7. Amente, S., Lni, L. & Mjello, B. The histone LSD demethylse in stemness nd ner trnsription progrms. Biohim. Biophys. At 89, 98 98 (). 8. Herz, H. M., Hu, D. & Shiltifrd, A. Enhner mlfuntion in ner. Mol. Cell 5, 859 8 (). 9. Krivtsov, A. V. & Armstrong, S. A. MLL trnslotions, histone modifitions nd leukemi stem-ell development. Nt. Rev. Cner 7, 8 8 (7). Aknowledgements We thnk Drs Motmedi nd Dyson for helpful disussions nd ritil reding of this mnusript. This work ws supported by Susn G. Komen for the Cure Grnt KG9 nd the NCI Federl Shre Progrm nd Inome to MGH Mrtin Reserh Prize (to S.M.); The Lilly Onology Fellowship from The Jpnese Respirtory Soiety, Overses Study Grnt from Knzw Medil Reserh Foundtion nd Mohid Memoril Foundtion for Medil nd Phrmeutil Reserh (to T.Y.); nd ACS (RSG--5- -CCG to J.R.W.) nd NIH (RGM97 to J.R.W.); J.W. is Leukemi nd Lymphom Sholr nd Tepper Fmily MGH Reserh Sholr. The Jne Coffin Childs Memoril Fund for Medil Reserh (to J.B.) nd MGH ECOR Tosteson postdotorl fellowship (to J.B.). Author ontributions S.M. oneived projet, designed the experiments, nlysed dt nd wrote the mnusript; K.T., T.Y. nd S.J. designed nd performed experiments, nlysed dt nd mde the figures; V.C., M.L., A.E., T.S. nd F.T. performed experiments nd nlysed dt, O.T., B.S.W., R.M., M.H., S.R. nd T.S nlysed dt; D.H. nlysed dt nd wrote the pper; J.B. nd J.W. nlysed dt nd edited pper. All uthors disussed the results nd ommented on the mnusript. Additionl informtion Aession odes. Mirorry dt hve been deposited in the NCBI Gene Expression Omnibus dtbse under ession ode GSE 798. Supplementry Informtion ompnies this pper t http://www.nture.om/ ntureommunitions Competing finnil interests: The uthors delre no ompeting finnil interests. Reprints nd permission informtion is vilble online t http://npg.nture.om/ reprintsndpermissions/ How to ite this rtile: Tjim, K. et l. SETDA modultes ell yle progression through mirna network tht regultes p5 trget genes. Nt. Commun. :857 doi:.8/nomms957 (5). This work is liensed under Cretive Commons Attribution. Interntionl Liense. The imges or other third prty mteril in this rtile re inluded in the rtile s Cretive Commons liense, unless indited otherwise in the redit line; if the mteril is not inluded under the Cretive Commons liense, users will need to obtin permission from the liense holder to reprodue the mteril. 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