A stem cell like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing

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1 A stem cell like chromtin pttern my predispose tumor suppressor genes to DNA hypermethyltion nd heritle silencing Joyce E Ohm, Kelly M McGrvey,, Xioing Yu 3, Linzho Cheng 4, Kornel E Schueel, Leslie Cope 4, Heli P Mohmmd, Wei Chen,5, Vincent C Dniel, Wyne Yu, Dvid M Bermn 6, Thoms Jenuwein 7, Kevin Pruitt, Sul J Shrkis,, D Neil Wtkins, Jmes G Hermn, & Stephen B Bylin, Adult cncers my derive from stem or erly progenitor cells,. Epigenetic modultion of gene expression is essentil for norml function of these erly cells ut is highly norml in cncers, which often show errnt promoter CpG islnd hypermethyltion nd trnscriptionl silencing of tumor suppressor genes nd pro-differentition fctors 3 5. We find tht for such genes, oth norml nd mlignnt emryonic cells generlly lck the hypermethyltion of DNA found in dult cncers. In emryonic stem cells, these genes re held in trnscription-redy stte medited y ivlent promoter chromtin pttern consisting of the repressive mrk, histone H3 methylted t Lys7 (H3K7) y Polycom group proteins, plus the ctive mrk, methylted H3K4. However, emryonic crcinom cells dd two key repressive mrks, dimethylted H3K9 nd trimethylted H3K9, oth ssocited with DNA hypermethyltion in dult cncers 6 8. We hypothesize tht cell chromtin ptterns nd trnsient silencing of these importnt regultory genes in stem or progenitor cells my leve these genes vulnerle to errnt DNA hypermethyltion nd heritle gene silencing during tumor initition nd progression. Epigenetic gene silencing nd ssocited promoter CpG islnd DNA hypermethyltion re prevlent in ll cncer types nd re n lterntive mechnism to muttions tht inctivte tumor suppressor genes within cncer cell 3 5. These epigenetic chnges my precede genetic chnges in premlignnt cells nd foster the ccumultion of dditionl genetic nd epigenetic hits 9. Adult cncers my derive from stem or erly progenitor cells,, nd epigenetic modultion of gene expression is essentil for the norml functioning of these erly cells. We now explore whether DNA hypermethyltion nd heritle silencing of groups of genes in dult tumor initition nd progression might reflect chromtin properties of these genes ssocited with stem or precursor cells. We compred the epigenetic sttus of group of genes frequently hypermethylted nd silenced in dult cncers (Fig. ; ssocited references listed in Supplementry Tle online) in oth norml emryonic stem (ES) cells nd mlignnt counterprts of these cells, emryonl crcinom (EC) cells. Notly, we found tht the genes frequently undergoing promoter CpG islnd DNA hypermethyltion in dult humn cncer cells generlly remined unmethylted in oth ES nd EC cells (Fig. ). Among the genes studied, 3 of 9 (45%) were hypermethylted in single dult colon cncer line, HCT-6, ut none ws hypermethylted in ES cells, nd only 3% nd 7% were completely methylted in the Ter- nd Ter- EC lines, respectively. Thus, the key epigenetic prmeter of promoter CpG islnd hypermethyltion, which is common in lrge group of genes in dult cncer cells, does not seem to e common feture of EC cells. In mouse ES cells, mny developmentl genes re mintined in stte of low trnscriptionl ctivity nd re ville for trnscriptionl increses or decreses when differentition cues re received. The genes we studied in EC cells retined this plsticity of expression, which is lcking in dult cncers when these sme genes re hypermethylted. Both ES nd EC cells cn e induced to differentite towrd neurl linege with ll-trns retinoic cid (ATRA) in vitro. The genes we studied with high sl trnscription stte in EC cells (Fig. ) generlly showed lower expression fter ATRA tretment. However, second, lrger cluster of genes showed low to medium expression in undifferentited Ter- cells (Fig. ), nd the mjority of these (9 of ) showed distinct ctivtion pttern with incresed expression upon or during differentition (Fig. ). Both ES nd EC cells, when grown s xenogrfts in NOD/SCID mice, form tertoms nd tertocrcinoms, respectively, in which there is spontneous differentition nd multilinege commitment to vrying degrees (Fig. ). The chnges in expression of the genes we studied in these tumors were similr to the chnges induced y ATRA in EC cells (Fig. c; for exmple, see (lso known s p6), nd ). E-cdherin nd re notle Cncer Biology Division, The Sidney Kimmel Comprehensive Cncer Center, Progrm in Cellulr nd Moleculr Medicine, 3 Institute for Cell Engineering, 4 Biometry nd Clinicl Trils Division, 5 Progrm in Humn Genetics nd Moleculr Biology nd 6 Pthology Deprtment, The Johns Hopkins University Medicl Institutions, Bltimore, Mrylnd 3, USA. 7 Reserch Institute of Moleculr Pthology, The Vienn Biocenter, Dr. Bohrgsse 7, A-3, Vienn, Austri. Correspondence should e ddressed to S.B.B. (sylin@jhmi.edu). Received Decemer 6; ccepted 4 Jnury 7; pulished online 9 Jnury 7; doi:.38/ng97 NATURE GENETICS VOLUME 39 NUMBER FEBRUARY 7

2 Gene Humn ES Ter- Ter- HCT-6 APC BRCA RBP (CRBP) Cyclin D (CCND) DAP-kinse (DAPK) E-Cdherin () ER (ESR) FANCF FHIT GATA-4 GATA-5 GST-Pi HIC- MLH 6-MGMT CDKN (p4) CDKN (p5) CDKN (p6) TP73 (p73) RAR-β RASSF- SFRP4 SOCS- Thromo (THBS) TIMP-3 TMS- (PYCARD) Unmethylted Prtilly methylted Predominntly methylted Methylted Brin Brest Colon Esophgel Gstric Hed nd neck Hemtopoetic Kidney/Renl Liver Lung Ovrin Pncretic Prostte Uterine Notle methyltion found in literture exceptions, nd differences proly reflect multilinege commitment in vivo versus single-linege differentition in vitro. We found tht the promoter regions of the genes we studied ech contined comintion of ctive (dimethylted H3K4) nd repressive (trimethylted H3K7) histone modifictions in ES cells (Fig. 3). This ivlent stte hs een recently descried in mouse ES cells, for suset of developmentl genes tht re mintined in low expression Figure EC cells retin plsticity of expression tht is lcking in dult cncer cells. () Rel-time quntittive RT-PCR nlysis of genes frequently hypermethylted in dult cncers fter treting EC cells with ATRA ( µm) for (untreted),, 3, 6, 9 nd d. PCRs were performed in triplicte, nd men threshold cycle for ltered gene expression with ATRA tretment ws normlized to GAPDH. The chnge in expression (log scle) for ech gene s multiple of expression in untreted Ter- cells ws clculted using the formul log(c t(tretment) c t(untreted) ). Representtive results of two or more independent experiments re shown. Genes re divided into two groups y threshold cycle: low to medium expression (top) (note high cycle threshold numer for the PCR; men = 3.8) nd genes with high sl expression (note low threshold cycle numer; men =.4). () Immunohistochemistry of tertocrcinom tumors grown in NOD/ SCID mice. Immunostining ws performed y the Johns Hopkins Immunopthology Deprtment ccording to estlished protocols on prffin-emedded section Genes with high sl trnscription stte in undifferentited EC cells BRCA- 3 Cyclin D 6 DAPK 4 GST-pi 3 8 Averge.4 Cytokertin (epithelil cells) x Chromogrnin (neuroendocrine) 4x Figure Genes showing frequent DNA hypermethyltion nd frequently silenced in dult cncers remin unmethylted in emryonl crcinom (EC) nd emryonic stem (ES) cells. We selected pnel of 9 tumor suppressor nd cndidte tumor suppressor genes tht re known to e frequently hypermethylted in vrious cncer cell lines nd primry tumor smples (right) from review of the literture nd from studies in our own lortories (see Supplementry Tle for list of references used; methyltion ws considered notle when >5% of the humn cell lines or ptient smples surveyed were methylted). Methyltion-specific PCR ws used to determine the promoter DNA methyltion sttus of these genes in colon cncer HCT-6 cells, in WA humn ES cells nd in two EC cell lines: Ter- nd Ter- cells. Genes were chrcterized s unmethylted (empty oxes), fully methylted (filled lck oxes) or prtilly methylted (gry oxes). stte; here, we extend these oservtions to genes frequently epigeneticlly silenced in cncer. We hve previously found trimethylted H3K7 to e enriched t ech of the of the promoters of smll pnel of DNA-hypermethylted genes we studied in dult cncer cells 6. This histone modifiction is ctlyzed y, key component of the Polycom group (PcG) complexes (PRC, PRC/3 nd PRC4) (Fig. 3) tht mintin long-term gene silencing in diverse orgnisms nd re essentil for the norml stte of stem nd progenitor cells nd their commitment to vrious tissue types 3,4. Recent studies hve linked with DNA methyltrnsferses nd hve estlished role for this protein during the induction nd trgeting of DNA methyltion 5. These complexes re present in stem nd progenitor cells s well s in tumor cells tht hve similr properties 4,6. We next mtched our list of genes showing frequent DNA hypermethyltion in dult cncers to the full list of PcG-trgeted genes identified y Genes with low to medium sl trnscription stte in undifferentited EC cells RBP 7 GATA-4 35 GATA TP73 3 RARB 7 SFRP TIMP3 9 Averge 3.8 CD34 (endothelil) 4x Glil firillry cidic protein (glil cells) x Expression (multiple of untreted cells), AFP-lph fetoprotein (yolk sc) x Myogenin (muscle) 4x BRCA- Cyclin D DAPK GST-pi MGMT cexpression (multiple of untreted cells),..... CRBP GATA-4 GATA-5 TP73 RARB SFRP4 TIMP3 Dy Dy Dy 3 Dy 6 Dy 9 Dy Tertom ATRA dy CRBP GATA-4 GATA-5 SFRP4 using ntiodies to CD34 ( mrker for endothelil cells), chromogrnin ( mrker for neuroendocrine cells), cytokertin ( mrker for epithelil cells), lph fetoprotein (AFP; mrker for yolk sc development), glil firillry cidic protein (GFAP; mrker for glil cells) nd myogenin ( mrker for muscle). (c) Quntittive RT-PCR ws performed for RNA from 5 6 Ter- cells grown s xenogrphs in NOD/SCID mice until tumors reched pproximtely.5 cm in dimeter. Chnge in expression ws clculted s descried ove, nd results from cells treted with ATRA ( µm) for d re shown for comprison. VOLUME 39 NUMBER FEBRUARY 7 NATURE GENETICS

3 RASSF PRC complex: mintennce of silencing Humn proteins CBX fmily (HPC) HPH fmily (EDR) Ring/ Ring/ Bmi- YY PRC/3 complex: initition of silencing Humn proteins HKMT EED, 3, 4 EZH SUZ PRC4 complex: expressed in cncer nd ES cells Humn proteins HKMT EED EZH SUZ SirT recent studies of humn nd mouse ES 7,8 nd emryonic firolst (EF) cells 9. These genome-wide tiling studies identified etween 8% (PRC; ES cells) 7 nd 4% (PRC/PRC; EF cells) 9 of the nnotted genome to e PcG regulted in these cell types. Notly, we found tht 68% of the genes in Figure were ssocited with PcG in either ES or EF cells, s were 56.5% of 3 genes (Fig. 3) newly identified s hypermethylted in HCT-6 cells (K.E.S. et l., dt not shown). Among emryonic cells, the degree of linege commitment my determine the lnce of chromtin modifictions t gene promoters nd the genes sl expression levels. Similrly, this my e true for comprtments of precursor cells in dult renewing cell systems from which dult cncers derive. We found it interesting tht of the Frequently hypermethylted genes 4.3% 67.9% Polycom (PRC/PRC) (~8-4%) Polycom group ssocited genes: Identified from the nnotted genome of humn emryonic stem cells (ES) (ref. 7) or emryonic firolst cells (EF) (ref. 9) HCT6 hypermethyted genes 34.8% 56.5% Individul genes mrked y PcG Gene homologues or relted fmily memers mrked y PcG Genes not mrked y PcG Figure 3 Genes showing frequent DNA hypermethyltion in cncer re mrked y ivlent chromtin in ES cells, including the Polycom group (PcG) protein ssocited histone modifiction, methylted H3K7 (H3K7me). () Chromtin immunoprecipittion (ChIP) ws performed using ntiodies to dimethylted H3K4 (), trimethylted H3K7 (), dimethylted H3K9 () nd trimethylted H3K9 () on WA9 humn ES cells. PCR ws performed for regions within the CpG islnds nd ner the trnscription strt sites of,,, RASSF, nd. Representtive results of two independent experiments re shown. A negtive control without ntiodies is included for comprison. () Left pnel: Polycom repressive complexes, /3 nd 4 re shown. Right pnel: genes showing frequent DNA hypermethyltion in dult cncers re trgeted y PcG trget genes in humn ES cells nd EF cells. We compiled list of genes mrked y PcG proteins nd estimted in two recent studies to represent etween 8% (PRC; ES cells) 7 nd 4% (PRC/PRC; EF cells) 9 of the nnotted genome. In these lists, we identified our studied genes showing frequent DNA hypermethyltion in dult cncers (top left); 68% were ssocited with PcG in either ES or EF cells in the ove studies. An dditionl 4% hd relted proteins under Polycom control. Similr results were seen using 3 genes newly identified y microrry studies (dt not shown) to e DNA hypermethylted in HCT-6 cells (top right). Figure 4 Genes studied in EC cells shre the ivlent chromtin pttern seen in ES cells ut dd dditionl repressive mrks chrcteristic of these sme genes when DNA demethyltion is induced in dult cncers. () ChIP ws performed using ntiodies to,, nd on Ter- cells. PCR ws performed for regions within the CpG islnds nd ner the trnscription strt sites of,,,, CKDNA,,, nd RASSF. Representtive results of two independent experiments re shown. A negtive control without ntiodies is included for comprison. () Quntifiction of representtive gels shown in showing the rtio of (ctive mrk) nd (repressive mrk) to input DNA in Ter- cells for,,,,, nd, nd s men for four of these genes (,, nd ) from previous studies 6 of HCT-6 DKO nd wild-type cells (fr right). Error rs indicte s.e.m. (c) Rtio of nd to input DNA in Ter- cells for,,,,, nd, nd s n verge for four of these genes (,,, ) from previous studies 6 in HCT6 wild-type cells, where ech of the genes shows DNA hypermethyltion nd lcks ny sl trnscription, nd in HCT6 DKO cells, where ech gene hs ecome fully DNA demethylted nd is re-expressed 6 (fr right). (d) Protein lot nlysis for the repressive histone modifictions, dimethylted H3K9 nd trimethylted H3K9 in cells nd the humn ES cell line WA. Lmin B ws used s loding control. RASSF c Bound/input genes from Figure tht were identified s PcG trgets, pproximtely 5% were listed s PcG trgets in oth ES nd EF cells. However, the remining 5% were listed s unique PcG trgets in either ES or EF cells. This might explin how different ptterns of hypermethylted genes in dult cncers might, then, reflect their chromtin sttus in cells from which cncers develop. We found tht lthough the genes studied in EC cells shred the ivlent chromtin pttern seen in ES cells (Fig. 4,), they lso cquired two dditionl key repressive mrks chrcteristic of dult cncers in EC cells. Thus, trimethylted H3K9, which is chrcteristic of silenced trnscription in pericentromeric regions, nd to lesser nd more vrile extent, dimethylted H3K9, were enriched t the Bound/input DKO (Avg.) HCT6 (Avg.) d Lmin B Humn ES cells DKO (Avg.) HCT6 (Avg.) NATURE GENETICS VOLUME 39 NUMBER FEBRUARY 7 3

4 c Protein locliztion (multiple of control) Suz SirT Expression (multiple of control) (log scle) promoters (Fig. 4,c). Both of these H3K9 mrks re chrcteristic of DNA-hypermethylted genes in dult cncers 6. In oth Neurospor nd Aridopsis thlin, muttions in genes encoding histone methyltrnsferses tht ctlyze H3K9 methyltion cuse significnt loss of genomic DNA methyltion 3. We found it interesting tht in the EC cells, glol levels of oth of the H3K9 repressive mrks were considerly higher thn in ES cells (Fig. 4d), suggesting permissive ckground for the promoter chnges in the neoplstic cells. Although the chromtin pttern identified ove in EC cells is similr to tht for DNA-hypermethylted genes in dult cncers, severl importnt differences exist. First, in dult cncer cells, the ctivting mrk, methylted H3K4, is diminished, nd these genes generlly hve fully repressive rther thn ivlent chromtin 6,4. However, this ctivting mrk is enriched gin when expression nd DNA demethyltion of these genes in dult cncer cells re induced, either y genetic knockout of DNA methyltrnsferses or tretment with 5-z- -deoxycytidine (DAC) 6,4. Second, in dult cncers, the repressive chromtin present for DNA-hypermethylted genes is initilly more enriched for dimethylted H3K9 (refs. 6,4) thn is seen in the EC cells. The rtio of trimethylted H3K9 to dimethylted H3K9 in EC for unmethylted genes is greter thn 5: in cells, compred with men of.: (ref. 6) for DNA-hypermethylted promoters in dult cncers (Fig. 4d). Notly, in EC cells, the fully methylted RASSF gene nd the minimlly methylted gene oth demonstrted n incresed presence of the dimethylted H3K9 mrk t their promoters (Fig. 4). Most importntly, when DNA-hypermethylted genes re demethylted in dult cncer cells, dimethylted H3K9 is the only repressive mrk uniformly reduced 6,4, nd the rtio of trimethylted H3K9 to dimethylted H3K9 increses to 4: (see the men for previously pulished results in Fig. 4d), vlue virtully identicl to tht for unmethylted genes in EC cells 6,4 (Fig. 4d). The chromtin findings for genes in EC cells present n opportunity to study how the vrious repressive histone modifictions nd the proteins tht mintin them re ltered with differentition of these cells. This is key question, s DNA-hypermethylted genes in dult cncers re very deeply, heritly repressed nd thus re difficult to rectivte. d Suz SIRT Bmi Suz Sfmt SirT Rtio of to (multiple of untreted cells) Figure 5 Chnges in histone modifictions nd locliztion of known Polycom group (PcG) proteins to the gene promoters in Ter- cells with ATRA-induced differentition. () ChIP ws performed using ntiodies ginst Suz, nd SIRT in Ter- cells. We studied genes hving low to medium sl expression in undifferentited Ter- cells tht re upregulted with differentition (,, nd ) nd genes with high sl expression in Ter- cells tht re downregulted with differentition (, nd ). Representtive results of two independent experiments re shown. A negtive control without ntiodies is included for comprison. () RT- PCR ws performed s descried in Figure for Bmi, Suz,, Sfmt nd SIRT during ATRA-induced differentition of Ter- cells. Chnge in expression s multiple of expression in untreted cells (log scle) is shown fter,, 3, 6, 9 nd d of differentition. (c) Reltime ChIP PCR shows reduction in PcG protein locliztion to the promoters of,, nd fter ATRA-induced differentition ( µm, d) of Ter- cells. Protein locliztion is shown s multiple of tht in untreted cells is shown. A representtive experiment from two or more PCRs is shown. (d) Quntittive ChIP PCR showing chnge in the / rtio fter d of ATRA tretment in genes tht re downregulted fter ATRA tretment ( nd ) nd genes tht re upregulted fter ATRA tretment (, nd ). A representtive experiment from two or more PCRs is shown. unless the DNA methyltion is removed. We first oserved tht the key PcG proteins SUZ, nd SirT were enriched t the promoters of the genes in EC cells (Fig. 5). The stedy-stte levels of these proteins fell (Fig. 5), s did their levels t the ove promoters, with ATRA tretment (Fig. 5c). Additionlly, severl PcG proteins, including Bmi, Suz nd Sfmt, showed trnsient increse in expression t vrious points during the differentition process, followed y lowering of expression s cells entered more differentited stte. These dt support the extensive work of others in discerning role for this fmily during norml differentition 6. In mouse ES cells, mny developmentl genes mrked y the ove ivlent chromtin sttes re mintined in low expression stte ut demonstrte plsticity of chromtin nd expression y incresing trnscription nd shifting to more monovlent ctive chromtin pttern when differentition cues re received. We see this for (lso known s p6) nd, which hve moderte to low sl expression stte in EC cells nd n equivlent initil rtio of dimethylted H3K4 to trimethylted H3K7 (Fig. 4,) ut dopt more ctive, monovlent stte s their expression is distinctly incresed y ATRA (Fig. 5d). A second suset of genes (, nd ) showing frequent DNA hypermethyltion in dult cncers nd with higher sl expression level in EC cells nd higher initil rtio of ctive to repressive mrks (Fig. 4,), generlly showed lower expression fter ATRA tretment (Fig. ) nd showed decrese in the rtio of dimethylted H3K4 to trimethylted H3K7 (Fig. 5d). An exception is (lso known s p5), which is expressed t n intermedite level in undifferentited cells nd demonstrtes significnt upregultion with differentition (Fig. ) ut lredy shows n ctive, monovlent chromtin stte in EC cells (Fig. 4,) tht is not significntly ltered with differentition (Fig. 5d). If stem cell gene promoter chromtin pttern, including PcGmedited repressive histone modifictions, might help render certin genes vulnerle to DNA hypermethyltion, cn one pertur the system in emryonic cells to further test this hypothesis? We tested this y forcing overexpression of Bmi, centrl component of PRC. PRC is involved in recognition of the H3K7 mrk estlished y 4 VOLUME 39 NUMBER FEBRUARY 7 NATURE GENETICS

5 c d Expression (multiple of uninfected control) NL Pool A p5 IVD U M U M -Bmi B p -Bmi clone 5 p Pool B p5 -Bmi A p5 U M U M Pool B Pool B p p5 -Bmi B p5 U M U -Bmi B p M UM U -Bmi pool B p5 -Bmi M U M U M U M in the PRC complex nd hs role in susequent mintennce of PcG-medited long-term gene silencing 3,4. Bmi is endogenously expressed in the wild-type cells nd shows trnsient increse nd susequent decline during ATRA-induced differentition (Fig. 5). Forced, stle overexpression of Bmi in these cells resulted in n initil increse in BMI mrna tht ws sustined for more thn ten pssges nd then returned to seline (Fig. 6). This ws ccompnied in pooled cells, nd multiple cloned popultions studied, y n overll increse in cell prolifertion, cell numer nd loss of contct inhiition of susets of cells in vitro tht ws seen only infrequently in wild-type cells (Fig. 6). The overexpression of Bmi did not cutely induce methyltion of most unmethylted tumor-suppressor genes exmined, including (lso known s p6), E-cdherin, or. However, in the pooled cells nd in two of five seprte clones studied, incresed DNA methyltion of the Wnt ntgonist gene occurred over time (Fig. 6c), result never oserved in wild-type cells over chronic pssges. The histone modifictions t the promoter remined unchnged in the pooled Bmi-overexpressing cells through pssge (Fig. 6d). However, s DNA methyltion incresed t pssge for one clone exmined, the rtio of dimethylted H3K9 to trimethylted H3K9 ecme pproximtely equl (Fig. 6d) nd thus ws similr to tht for DNA-hypermethylted genes in dult cncers 6 (Fig. 4c). In summry, our studies demonstrte tht genes showing frequent DNA hypermethyltion nd deep trnscriptionl silencing in dult -Bmi B p5 -Bmi clone p5 -Bmi clone p -Bmi clone 5 p5 -Bmi clone 5 p Figure 6 Overexpression of Bmi cn cuse progressive promoter DNA methyltion of the gene in EC cells. () cells were stly infected with Bmi-expressing lentivirus, nd quntittive RT-PCR for Bmi trnscript is shown in Bmi-infected pools of cells t pssges 5 (p5), nd 5 post-infection. A representtive experiment from two PCRs is shown. () Wild-type (top) nd Bmi-overexpressing cells (ottom) in culture ( mgnifiction). Arrow indictes smll, infrequent cluster of cells in showing incresed prolifertion nd loss of contct inhiition. (c) Methyltion nlysis y methyltion-specific PCR (MSP) for,, nd for nd Bmi-overexpressing pools t pssges 5, nd 5 nd five individul clones t pssges 5 nd. For clones, representtive results for two of five clones re shown. For MSP, representtive results of two independent experiments re shown. M = methylted signl; U = unmethylted. Norml lymphocytes (NL) nd in vitro methylted DNA (IVD) re included s positive nd negtive controls for methylted DNA. (d) ChIP ws performed t the promoter in cells, in pooled Bmi infected cells t pssge post-infection nd in single clone of Bmi-infected cells t pssge. ChIP ws performed using ntiodies to,, nd s descried in Figure 4. cncers usully lck such DNA methyltion in norml nd neoplstic emryonic cells. However, the chromtin of these genes is virtully identicl in emryonic cncer cells to tht of the genes in dult cncers, especilly when the DNA methyltion in the ltter cells is reduced nd the involved genes re re-expressed. The repressive pttern for the EC cells my, then, represent trnsition stte somewhere etween tht for genes in ES cells nd tht for fully DNA-hypermethylted nd tightly silenced genes in dult cncers tht fcilitte neoplstic progression. When DNA demethyltion is trnsiently induced y drugs in DNA-hypermethylted genes in dult cncers, they retin promoter chromtin pttern virtully identicl to wht we now show in EC cells; this my e importnt in explining why these sme genes recquire the DNA methyltion nd silencing once the drug is removed 6,5. In terms of humn cncer iology, our findings suggest tht stem cell like promoter ground stte involving the key PcG mrk, trimethylted H3K7, my e indictive of the contriution of stem cell nd/ or progenitor cells to the derivtion of dult cncers (Supplementry Fig. online). We nd others hve suggested tht stem nd progenitor cells re especilly t risk for cncer initition owing to their continued expnsion during sttes such s chronic wound heling nd inflmmtion 9,6. If further repressive mrks for H3K9 re dded to the stem cell chromtin stte descried here, the comintion of H3K7 nd H3K9 methyltion my provide progrm for mking key tumor suppressor genes vulnerle for imposition of promoter CpG islnd DNA methyltion during such expnsion (Supplementry Fig. ). These chnges my enhnce the likelihood of tumor initition nd progression from cell trnsformtion s it renders trnsient trnscription-redy stte to one of heritle, permnent gene silencing. METHODS Cell culture. Ter-, Ter- nd HCT-6 (ATCC) cells were mintined in McCoy s 5A medium supplemented with % FBS nd grown t 37 C under 5% CO, s were HCT 6 cells in which oth DNA methyltrnsferses nd 3 re geneticlly deleted (DKO cells) 7. DNA for methyltion-specific PCR (MSP) ws isolted in the lortory of S.J.S. (Johns Hopkins University) from the humn ES cell line WA (WiCell Reserch Institute) ccording to estlished protocols. Cross-linking for humn ES cell chromtin immunoprecipittion (ChIP) nd susequent DNA isoltion ws performed in the lortory of L. Cheng (Johns Hopkins University) using the humn ES cell lines WA nd WA9 (WiCell Reserch Institute). Bisulfite tretment nd MSP. Bisulfite tretment nd MSP were performed s descried previously 8. DNA ws extrcted following stndrd phenol-chloroform NATURE GENETICS VOLUME 39 NUMBER FEBRUARY 7 5

6 extrction method. Primer sequences nd cycling conditions re included in Supplementry Tle online. Any informtion not included is ville y request from the uthors. RNA purifiction nd rel-time RT-PCR nlysis. RNA ws isolted with TRIzol Regent (Invitrogen) ccording to the mnufcturer s instructions. For RT-PCR, µg of totl RNA ws reverse trnscried using the Superscript First-Strnd Synthesis System (Invitrogen). Quntittive rel-time RT-PCR ws performed s descried previously 9. PCR primers nd mplifiction conditions re included in Supplementry Tle. Any informtion not included is ville y request from the uthors. ChIP. ChIP ssys were performed s descried previously 6, with the modifiction tht immunoprecipittion ws performed using Dynl Mgnetic eds purchsed from Invitrogen (Protein A eds (-D) nd Protein G eds (-4D)). Antiodies to trimethylted H3K7, dimethylted H3K9 nd trimethylted H3K9 were produced s previously descried 6. Antiodies to Suz (Acm);, (Upstte) nd SIRT (Delt Biols) were purchsed from commercil sources. Primers nd mplifiction conditions re included in Supplementry Tle. Any informtion not included is ville y request from the uthors. Lentivirl vector preprtion, virl production nd infection. An untgged Bmi lentivirl expression clone ws generted using the full-length cdna from pbabe-puro retrovirl construct (see Acknowledgments) nd using the VirPower Promoterless Lentivirl Gtewy System (Invitrogen). Briefly, Bmi entry clone with n intct stop codon ws incorported long with second UC promoter contining entry clone otined with this kit (pentr5 -UCp) into the plenti6/ R4R/V5-DEST vector from Invitrogen. The expression clone ws trnsformed into Stl3-competent cells (Invitrogen), nd Bmi-contining recominnts were selected using mpicillin nd lsticidin resistnce nd were confirmed y restriction digest. Plsmid DNA ws purified using GenElute Plsmid Mxiprep Kit (Sigm- Aldrich), nd lentivirus ws pckged in 93FT cells nd infected in cells using the mnufcturer s recommended protocols. Blsticidin (.5 µg/ml) ws dded to complete medium 48 h post-infection, nd stle expressing pools nd clones were mintined in.5 µg/ml lsticidin for the durtion of the experiments. Note: Supplementry informtion is ville on the Nture Genetics wesite. ACKNOWLEDGMENTS Specil thnks to L. Meszler (Cell Imging Core Fcility, The Sidney Kimmel Comprehensive Cncer Center), P. Argni (Pthology Deprtment, Johns Hopkins University) nd G. Dimri (Northwestern University) for providing the BmicDNA. The pbabe-puro retrovirl construct ws provided y G. Dimri (Northwestern University). This work ws supported y US Ntionl Institutes of Helth grnts CA66 to S.B.B. nd HL7378 to L.C. nd Ntionl Cncer Institute grnt CA4338 to S.B.B. AUTHOR CONTRIBUTIONS J.E.O. nd S.B.B. designed this study. J.O., K.M.M., X.Y., K.E.S., H.P.M.,W.C.,V.C.D. nd K.P. contriuted collortive experimentl results. L.C. nd S.G.S. mnged the growth nd mnipultion of ES cells. D.M.B. ssisted with chrcteriztion of the tertocrcinom cell explnts. D.N.W. nd J.G.H. ssisted J.E.O. nd S.B.B. with dt nlysis nd preprtion of the mnuscript. L.C. nd W.Y. helped with performnce nd interprettion of microrry results tht contriuted to the dt. T.J. provided ntiodies generted in his lortory for most of the histone modifictions studied y ChIP nlysis. COMPETING INTERESTS STATEMENT The uthors declre competing finncil interests (see the Nture Genetics wesite for detils). Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions. Al-Hjj, M., Wich, M.S., Benito-Hernndez, A., Morrison, S.J. & Clrke, M.F. Prospective identifiction of tumorigenic rest cncer cells. Proc. Ntl. Acd. Sci. USA, (3).. Clrke, M.F. & Fuller, M. Stem cells nd cncer: two fces of eve. Cell 4, 5 (6). 3. 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