LETTER. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2

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1 doi:.8/nture98 Impired hydroxyltion of -methylytosine in myeloid ners with mutnt TET Myunggon Ko {, Yun Hung {, Ann M. Jnkowsk, Utz J. Ppe,, Mmt Thilini, Hozef S. Bndukwl, Jungeun An {, Edwrd D. Lmperti, Kin Peng Koh, Ree Gnetzky, X. Shirley Liu, L. Arvind, Suneet Agrwl, Jroslw P. Miejewski & Anjn Ro { TET is lose reltive of TET, n enzyme tht onverts -methylytosine (mc) to -hydroxymethylytosine () in DNA,. The gene enoding TET resides t hromosome q, in region showing reurrent mirodeletions nd opy-neutrl loss of heterozygosity (CN-LOH) in ptients with diverse myeloid mlignnies. Somti TET muttions re frequently oserved in myelodysplsti syndromes (MDS), myeloprolifertive neoplsms (MPN), MDS/ MPN overlp syndromes inluding hroni myelomonoyti leukemi (CMML), ute myeloid leukemis (AML) nd seondry AML (saml). We show here tht TET muttions ssoited with myeloid mlignnies ompromise tlyti tivity. Bone mrrow smples from ptients with TET muttions displyed uniformly low levels of in genomi DNA ompred to one mrrow smples from helthy ontrols. Moreover, smll hirpin RNA (shrna)-medited depletion of Tet in mouse hemtopoieti preursors skewed their differentition towrds monoyte/ mrophge lineges in ulture. There ws no signifint differene in DNA methyltion etween one mrrow smples from ptients with high versus helthy ontrols, ut smples from ptients with low showed hypomethyltion reltive to ontrols t the mjority of differentilly methylted CpG sites. Our results demonstrte tht Tet is importnt for norml myelopoiesis, nd suggest tht disruption of TET enzymti tivity fvours myeloid tumorigenesis. Mesurement of levels in myeloid mlignnies my prove vlule s dignosti nd prognosti tool, to tilor therpies nd ssess responses to ntiner drugs. We trnsiently trnsfeted HEK9T ells with My-tgged murine Tet nd ssessed mc nd levels y immunoytohemistry (Fig. nd Supplementry Figs ). My Tet-expressing ells displyed strong inrese in stining nd onomitnt derese in mc stining in the nuleus (Fig.,, quntified in Supplementry Fig. ). In ontrst, ws undetetle or rely deteted in nulei of ells expressing mutnt Tet with HY, DA sustitutions in the signture HxD motif,,7 involved in oordinting Fe, nd there ws no ovious derese in nuler mc stining (Fig., nd Supplementry Fig. ). These studies onfirm tht Tet is tlytilly tive enzyme tht onverts mc to in genomi DNA. Muttions in TET residues H88 nd R89, predited to ind Fe nd -oxoglutrte (OG), respetively, hve een identified repetedly in ptients with myeloid mlignnies,,7,.hek9tells expressing Tet mutnts H8R nd H8Q (Fig. nd Supplementry Fig. ) showed gretly diminished stining nd no loss of mc stining, onsistent with prtiiption of this residue in tlysis (Fig., nd Supplementry Fig., ). We nlysed missense muttions identified in TET in our own (Supplementry Tle ) nd other studies, (P7S, W9R, G9D, E8G nd I87T). HEK9T ells expressing Tet mutnts P87S, WR or C8D (Supplementry Figs nd ) displyed low stining nd strong mc stining (Supplementry Figs, nd, d), inditing role for these residues in the integrity of the tlyti or DNA inding domins. Cells expressing Tet(/M) (Fig., Supplementry Figs nd ) were positive for stining ut hnges in mc stining ould not e ssessed relily (Fig.,, Supplementry Figs, nd ). To quntify these findings, we developed dot lot ssys to detet in genomi DNA (Supplementry Fig. ). In the first ssy formt, the lot ws developed with speifi ntiserum to (Supplementry Fig., left), whose ility to reognize depended strongly on the density of in DNA (Supplementry Fig., top). We therefore developed more sensitive nd quntittive ssy in whih DNA ws treted with isulphite to onvert to ytosine -methylenesulphonte (CMS) (Supplementry Fig. ), fter whih CMS ws mesured with speifi nti-cms ntiserum (Supplementry Fig., right). Unlike nti- whih reted effiiently only with DNA ontining high densities of, the nti- CMS ntiserum reognized DNA with n verge of only single per se pirs (Supplementry Fig., ottom). This lk of density dependene llowed us to plot the signl otined with twofold dilutions of stndrd oligonuleotide ontining known mount of ginst the mount of CMS otined fter isulphite onversion. We ssumed % onversion effiieny nd used the liner portion of the stndrd urve to ompute the mount of CMS, nd therefore, in the DNA smples (for exmple, see Fig., right). To ssess levels, we otined uniform popultions of Tet- expressing HEK9T ells y trnsfetion with Tet-IRES-CD plsmid followed y mgneti isoltion of CD-expressing ells. Wild-type nd mutnt Tet proteins were expressed t omprle levels (Fig. d nd Supplementry Fig. d). Anti-/CMS dot lots of genomi DNA reveled, s expeted, tht ws rely detetle in DNA from ells trnsfeted with empty vetor; DNA from ells expressing wild-type Tet showed sustntil inrese in nd orresponding derese in mc; nd DNA from ells expressing the HxD mutnt Tet protein hd very low (Fig. e, Supplementry Figs e nd ). DNA from ells expressing seven of the nine mutnt Tet proteins tested H8Q/R, /M, WR, P87S nd C8D ontined signifintly less thn DNA from ells expressing wild-type Tet (Fig. e, Supplementry Figs e nd ), onfirming our previous onlusion tht these muttions impir enzymti tivity. We mesured (CMS) levels in genomi DNA extrted from one mrrow or lood (with.% immture myeloid ells) of 88 Deprtment of Pthology, Hrvrd Medil Shool, Immune Disese Institute nd Progrm in Cellulr nd Moleulr Mediine, Children s Hospitl Boston, Boston, Msshusetts, USA. Deprtment of Trnsltionl Hemtology nd Onology Reserh, Tussig Cner Institute, nd Deprtment of Hemtologi Onology nd Blood Disorders, Clevelnd Clini, Clevelnd, Ohio 9, USA. Deprtment of Biosttistis nd Computtionl Biology, Dn-Frer Cner Institute nd Hrvrd Shool of Puli Helth, Boston, Msshusetts, USA. Ntionl Center for Biotehnology Informtion, Ntionl Lirry of Mediine, Ntionl Institutes of Helth, Bethesd, Mrylnd 89, USA. Division of Peditri Hemtology/Onology, Children s Hospitl Boston nd Dn-Frer Cner Institute, Hrvrd Stem Cell Institute, Boston, Msshusetts, USA. {Present ddress: L Joll Institute for Allergy nd Immunology, L Joll, Cliforni 97, USA (M.K., Y.H., J.A., A.R.). These uthors ontriuted eqully to this work. 9 DECEMBER VOL 8 NATURE 89 Mmilln Pulishers Limited. All rights reserved

2 RESEARCH LETTER 7 7,9,, My DAPI My mc DAPI My Tet HxD mut H8Q H8R My Tet HxDmut H8Q H8R Cys-rih Core-DSBH Humn TET d HxD Hxs Rx Fe + Fe + OG H8 D8 HY DA H88Q R89S H88R R89M H8Q H8R Key signture Intertion H88 R89 Residue numers My Tet HxD mut H8Q H8R H87S Muttions in leukemi Murine ounterprts H87M My Atin e P <. vs wild-type Tet ng ng CMS (pmol per μg DNA) Stndrd Wild-type Tet mhxd ptients with myeloid mlignnies nd 7 helthy ontrols (Supplementry Tle ). In linded experiments, DNA ws treted with isulphite nd CMS levels were mesured. TET muttions were strongly ssoited with low genomi (Fig. nd Supplementry Fig. 7). To onfirm these onlusions in sttistilly rigorous H8Q H8R TET-CD TET-CD-HxDmut Figure The tlyti tivity of Tet is ompromised y muttions in predited tlyti residues., Shemti representtion of TET. The tlyti ore region ontins the ysteine-rih (Cys-rih) nd doule-strnded et-helix (DSBH) domins. Three signture motifs onserved mong OGnd Fe -dependent dioxygenses re shown,. Sustitutions in the HxD signture tht impir the tlyti tivity of TET (ref. ), leukemi-ssoited muttions in the roxy-terminl signture motifs, nd orresponding sustitutions introdued into murine Tet re indited., Tet expression results in inresed y immunoytohemistry. HEK9T ells trnsfeted with My-tgged wild-type nd mutnt Tet were o-stined with ntiody speifi for the My epitope (red) nd ntiserum ginst (green). DAPI (lue) indites nuler stining., Tet expression results in loss of nuler mc stining. HEK9T ells trnsfeted with wild-type nd mutnt My-tgged Tet were o-stined with ntiody speifi for the My epitope (green) nd ntiserum ginst mc (red). d, Equivlent expression of wild-type nd mutnt My Tet. CD ells were isolted from HEK9T ells trnsfeted with iistroni Tet-IRES-humn CD plsmids, nd Tet expression in whole ell lystes ws deteted y immunolotting with nti-my. -tin serves s loding ontrol. e, Genomi DNA purified from CD HEK9T ells overexpressing wild-type or mutnt Tet ws treted with isulphite to onvert to CMS (Supplementry Fig. ). CMS ws quntified y dot lot ssy using nti-cms nd syntheti isulphite-treted oligonuleotide ontining known mount of CMS. As positive nd negtive ontrols, we inluded DNA from CD HEK9T ells trnsfeted with TET tlyti domin (TET- CD) or TET-CD with muttions in the HxD motif (TET-CD-HxDmut). Twofold seril dilution Twofold seril dilution CMS (pmol per μg DNA)..... Stndrd CCF-Hetero (SX) CCF-Hemi (S9X) CCF7-Homo (InsT) CCF-Wild-type TET CCF-Wild-type TET TET sttus CCF-Wild-type TET CCF-Hetero (SX) CCF-Hemi (S9X) CCF7-Homo (InsT) CCF-Wild-type TET CCF-Wild-type TET CCF-Wild-type TET Intensity CMS (pmol per μg DNA) donors n = 9 y = x R =.99 CCF-Wild-type TET CCF-Wild-type TET CCF-Wild-type TET CCF-Hetero (SX) CCF7-Homo (InsT) CCF-Hemi (S9X) CMS (pmol) P =. P =.7 P = Mutnt TET n = Wild-type TET n = 8 Ptient smples TET sttus Figure TET muttionl sttus orreltes with levels in ptients with myeloid mlignnies., Quntifition of y nti-cms dot lot. Left, representtive dot lot of genomi DNA isolted from one mrrow spirtes of ptients with MDS/MPN nd TET muttionl sttus s indited. A syntheti oligonuleotide with known mount of CMS ws used s stndrd. Right, the liner portion of the stndrd urve ws used to estimte the mount of in DNA from ptient smples., Br grph of dt from pnel. The three ptients with TET muttions show lower levels thn the three ptients with wild-type TET. Error rs indite s.d. (n )., Correltion of levels with TET muttionl sttus. CMS levels in one mrrow smples from helthy donors nd ptients with myeloid mlignnies (Supplementry Tle ) re shown s the medin of triplite mesurements (Supplementry Fig. 7). In the TET mutnt group, squres, tringles, dimonds nd the str indite homozygous, hemizygous, heterozygous nd illeli heterozygous muttions, respetively (for detiled definition, see Supplementry Methods). The horizontl r indites the medin for eh group. P-vlues for group omprisons were lulted y two-sided Wiloxon rnk sum test. Ptients ering TET muttions show uniformly low expression levels. fshion, we tested smples for whih suffiient mount of DNA ws ville to mke independent dilutions in triplite, so tht medin nd stndrd devition for (CMS) levels in eh ptient ould e derived (Supplementry Fig. 7). Anlysis of DNA from 9 helthy donors nd ptients (8 with wild-type TET nd with TET muttions, Supplementry Tle ) reveled strong, sttistilly signifint orreltion of TET muttions with low (Fig. ). In ontrst, smples from ptients with wild-type TET showed imodl distriution, with levels rnging from, to,.8 pmol per mg DNA (Fig., Supplementry Fig. 7, lso see Fig. ). We exmined Tet expression in hemtopoieti ell susets isolted from one mrrow nd thymus of C7BL/ mie (Supplementry Figs 8 nd 9). Tet mrna ws highly expressed in linege-negtive (Lin ) S- -Kit hi (S- is lso known s Ly) multipotent 8 N AT U R E V O L 8 9 D E C E M B E R Mmilln Pulishers Limited. All rights reserved

3 RESEARCH progenitors (LSK), t levels similr to those in emryoni stem ells (ESC). Expression ws mintined t high levels in myeloid progenitors (ommon myeloid progenitors, CMPs, nd grnuloytemonoyte progenitors, GMPs), ws low in mture grnuloytes (Gr- M-, lso known s Lyg nd Cd or Itgm, respetively) nd high in monoytes (Gr- M- ) (Supplementry Fig. 9, middle pnel). To test the role of Tet in myelopoiesis, we trnsdued one mrrow stem/progenitor ells with Tet shrna (Supplementry Fig. ), effetively downregulting Tet mrna nd protein reltive to ontrol ells trnsdued with empty vetor or srmled shrna (Fig., ) (refer to Supplementry Fig. for hoie of Tet shrna). Tet depletion promoted expnsion of M- F/8 (lso known s Emr) nd M- CD (lso known s Csfr or M-CSFR, mrophge olony stimulting ftor reeptor) monoyte/mrophge ells in the presene of G-CSF (grnuloyte olony-stimulting ftor) or (grnuloyte-mrophge olony-stimulting ftor), ytokines tht support grnuloyte nd grnuloyte/ monoyte development respetively, ut not in the presene of M-CSF (mrophge F/8 Tet expression reltive to Gpdh Kit + BM ells shtet Srmle My Tet Srmle + ++ shtet + ++ Srmle shtet Srmle shtet M-. 7. CD (M-CSFR) My Atin Cytokine(s) ( ng ml ) M- M-CSF + M-CSF G-CSF + G-CSF Figure Tet regultes myeloid differentition.,, Tet shrna represses Tet mrna nd protein expression., -Kit stem/progenitor ells from one mrrow of C7BL/ mie were trnsdued with retroviruses (Supplementry Fig. ). After seletion with puromyin for dys, Tet mrna expression ws ssessed y quntittive RT PCR (PCR with reverse trnsription). Error rs show the rnge of duplites., HEK9T ells were otrnsfeted with expression plsmids enoding My-tgged Tet nd retrovirl shrnas. Tet protein expression ws quntified 8 h lter y nti-my immunolotting of whole-ell extrts., Effet of Tet depletion on myeloid differentition. Lin ells purified from one mrrow of C7BL/ mie were trnsdued with ontrol (srmle) or shtet retroviruses, then grown in the presene of ng ml stem ell ftor (SCF), puromyin ( mgml ) nd ytokines ( ng ml )s indited (lso see Supplementry Fig. ). After dys, flow ytometri nlysis of M- versus F/8 (left pnel) or CD (right pnel) ws performed. All ells were GFP on the dy of nlysis. olony-stimulting ftor), whih promotes growth of monoyti progenitors (Fig. nd Supplementry Fig. d). Simultneous tretment with nd M-CSF, or nd G-CSF, lso led to inresed numers of monoyte/mrophge ells (Fig. ). These results indite tht Tet hs n importnt role in norml myelopoiesis. However, Tet does not mrkedly influene short-term prolifertion of myeloid-linege ells: when shrna-trnsdued Lin ells were ultured in the presene of nd pulse-lelled with romodeoxyuridine (BrdU), Tet depletion promoted monoyte/ mrophge expnsion ut CD (M-CSFR ) ells from the two ultures showed no differene in ute BrdU inorportion (Supplementry Fig. ). We sked whether levels in tumour smples orrelted with DNA methyltion sttus. A histogrm of normlized vlues from 88 ptients nd 7 helthy individuls showed the expeted imodl distriution (see Supplementry Methods): helthy ontrols nd most ptient smples with wild-type TET hd high, wheres the mjority of ptient smples with mutnt TET hd low (Fig. ). The DNA methyltion sttus of smples ws interrogted t 7,78 CpG sites. As expeted, the resulting histogrms were strikingly imodl, with sites within nd outside CpG islnds showing low nd predominntly high methyltion, respetively (Fig. ). Comprison of 8 ontrol smples with high tumour smples ( wild-type TET, mutnt TET) showed no signifint differene in DNA methyltion; in ontrst, omprison of the ontrol smples with 9 low tumour smples (7 wild-type TET, mutnt TET) yielded, differentilly methylted sites, of whih the mjority (, sites) were hypomethylted ompred to ontrols (Fig. d nd Supplementry Tle ). Thus TET loss-of-funtion is predominntly ssoited with deresed methyltion t CpG sites. To summrize, our studies demonstrte strong orreltion etween myeloid mlignnies nd loss of TET tlyti tivity. The leukemi-ssoited missense muttions ssoited with diminished levels provide lues to the struture of the TET tlyti domin. The WR, P87S nd C8D muttions ffet positions tht re highly onserved within the tlyti domin of the TET sufmily of dioxygenses : W is loted t the eginning of the strnd just mino-terminl to the ore of the doule-strnded et-helix (DSBH), nd is predited to onstitute prt of the mouth of the tive site poket of the enzyme; P87 is predited to stilize the onformtion of the juntion etween the N-terminl helix nd the first ore strnd of the DSBH; nd G9/C8 is predited to e the N-terminl pping residue of helix tht lines the mouth of the DSBH nd potentilly interts with sustrte DNA. The E8G muttion hd no detetle effet on prodution in our overexpression ssys; however, the ptient with this muttion lso showed CN-LOH spnning q, feture tht likely ontriutes to the signifint redution in levels oserved in the one mrrow. levels were oserved in suset of ptients with pprently wild-type TET, whose linil phenotypes resemled those of ptients with mutnt TET. In severl of these ptients, TET mrna expression ws not signifintly different from ontrols; muttions in other TET proteins hve not een desried (Supplementry Text). Some ptients in the wild-type TET/low tegory my hrour muttions in regultory or prtner proteins for TET, or in is-regultory regions ontrolling TET mrna expression. Alterntively, the primry event in some of these ptients my e CpG hypomethyltion, resulting in deresed seondry to depletion of the sustrte, mc. There is little onsensus on whether TET muttions orrelte with linil outome. One study reported n ssoition with deresed survivl in AML, wheres others report little prognosti vlue in MPN diseses 7,,. Assys for my inrese our options for the moleulr lssifition of myeloid mlignnies, mking it possile to sk whether ptients with high or low levels of genomi show differenes in disese progression or therpeuti response. Notly, histone deetylse nd DNA methyltrnsferse inhiitors show linil effiy 9 DECEMBER VOL 8 NATURE 8 Mmilln Pulishers Limited. All rights reserved

4 RESEARCH LETTER vlue d... ontrols n = 7 8 ontrols Wild-type TET Mutnt TET...8. vlue Frequeny ontrols P =.8 P =. 8 P =.8 9 Mutnt TET n = SP Wild-type TET n = Ptient smples.8. Frequeny... All sites ontrols smples smples...8. Sites outside CpG islnds...8. Sites inside CpG islnds...8. density AIM SURF C9orf RABIP..... TMEM FSDNL PSMD TMEM ZFHXB LRRC.... ontrols ontrols Figure Reltion of levels to DNA methyltion sttus., Normlized (CMS) levels in DNA from three different groups: helthy ontrols (lk dimonds), ptients with mutnt TET (red symols) nd ptients with wild-type TET (lue irles). Among TET mutnts, we distinguish homozygous (squres), hemizygous (tringles), heterozygous (smll dimonds) nd illeli heterozygous (str) muttions (for definitions see Supplementry Methods). The horizontl r indites the medin for eh group. The numer of smples in eh group is indited., Histogrm of normlized (CMS) levels in DNA from helthy donors (lk dimonds), ptients with mutnt TET (red retngles) nd ptients with wild-type TET (lue irles). The frequeny ws lulted sed on Gussin kernel estimtor. The lol minimum etween oth modes ws used s threshold (vertil dotted line) etween low nd high vlues., Density of methyltion vlues for helthy ontrols (lk), high smples (lue) nd low smples (red) of ll sites (top pnel), sites outside CpG islnds (middle pnel) nd sites inside CpG islnds (lower pnel). d, Box plot for group-speifi methyltion for the only two hypermethylted sites (SP, AIM; top pnel) nd the top nine hypomethylted sites (lower pnels) etween helthy ontrols nd low smples (totl numer of differentilly methylted sites ws,). in ptients with CMML nd AML 7 ; nd genomi levels ould potentilly e useful prognosti inditor or preditor of ptient responses or refrtoriness to epigeneti therpy with demethylting gents. DNA methyltion is highly errnt in ner 8. Beuse TET opertes on mc, we were surprised to find tht TET loss-of-funtion in myeloid tumours ws ssoited with widespred hypomethyltion rther thn the expeted hypermethyltion t differentilly-methylted CpG sites. Tumour smples with low my hve expnded ells with lolized hypomethyltion t these sites, or TET my ontrol DNA methyltion indiretly, for instne y regulting the expression or reruitment of one or more DNA methyltrnsferses, perhps vi -inding proteins. Alterntively, if TET nd re required for ells to exit the stem ell stte, loss of TET funtion in myeloid neoplsms my retivte stem-like stte hrterized y generlized hypomethyltion nd onsequent genomi instility,. Indeed, hypomorphi DNMT muttions ssoited with genome-wide DNA hypomethyltion skew hemtopoieti differentition towrds myeloerythroid lineges, nd promote the development of ggressive T-ell lymphoms due to tivtion nd insertion of endogenous retroviruses,. Further studies of the role of TET in hemtopoieti differentition should unover the reltion etween TET loss-offuntion, DNA methyltion hnges nd myeloid neoplsi. METHODS SUMMARY Ptient smples. Genomi DNA ws extrted from one mrrow/ peripherl lood smples from helthy donors nd ptients with MDS, MDS/MPN, primry nd seondry AMLs. Clinil fetures nd other detiled informtion pertining to the ptient smples re summrized in Supplementry Tle. Quntittive nlysis of nd CMS levels using dot lot. For CMS detetion, genomi DNA ws treted with sodium isulphite using the EpiTet Bisulfite kit (Qigen). DNA smples were dentured nd twofold seril dilutions were spotted on nitroellulose memrne in n ssemled Bio-Dot pprtus (Bio-Rd). The lotted memrne ws wshed, ir-dried, vuum-ked, loked nd inuted with nti- or nti-cms ntiody (:,) nd horserdish peroxidseonjugted nti-rit IgG seondry ntiody. To ensure equl spotting of totl DNA on the memrne, the sme lot ws stined with.% methylene lue in. M sodium ette (ph.). To ompre results otined in different experiments, we used the normliztion proedure desried in Supplementry Methods (see Fig.,,whihinorportedtfrom Fig. nd Supplementry Fig. ). nlysis. The DNA methyltion sttus of isulphite-treted genomi DNA ws proed t 7,78 CpG dinuleotides using the Illumin Infinium 7k rry (Illumin). sttus ws lulted from the rtio of methyltionspeifi nd demethyltion-speifi fluorophores (-vlue) using BedStudio Module (Illumin). We removed sites on the Y nd X hromosomes from the nlysis euse of inonsistent methyltion sttus with respet to gender ( known prolem sed on ommunition with Illumin). Clultions re sed on vlues, whih orrespond to the methyltion sttus of site rnging from to, returned y Illumin s BedStudio softwre. We tested sites for differentil methyltion using n empiril Byes pproh employing modified t-test (LIMMA). The flse disovery rte (FDR) is ontrolled t level of % y the Benjmini Hoherg orretion. Reeived 9 Mrh; epted 9 Otoer. Pulished online 7 Novemer; orreted 9 Deemer (see full-text HTML version for detils).. Thilini, M. et l. Conversion of -methylytosine to -hydroxymethylytosine in mmmlin DNA y MLL prtner TET. Siene, 9 9 (9).. Iyer, L. M., Thilini, M., Ro, A. & Arvind, L. Predition of novel fmilies of enzymes involved in oxidtive nd other omplex modifitions of ses in nulei ids. Cell Cyle 8, 98 7 (9).. Viguié,F.et l. Common q deletion in four ses of hemtopoieti mlignny: erly stem ell involvement? Leukemi 9, ().. Adel-Wh, O. et l. Geneti hrteriztion of TET, TET, nd TET ltertions in myeloid mlignnies. Blood, 7 (9).. Delhommeu, F. et l. Muttion in TET in myeloid ners. N. Engl. J. Med., 89 (9).. Jnkowsk, A. M. et l. Loss of heterozygosity q nd TET muttions ssoited with myelodysplsti/myeloprolifertive neoplsms. Blood, (9). 7. Lngemeijer, S. M. et l. Aquired muttions in TET re ommon in myelodysplsti syndromes. Nture Genet., 88 8 (9). 8. Levine, R. L. & Crroll, M. A ommon geneti mehnism in mlignnt one mrrow diseses. N. Engl. J. Med., 7 (9). 9. Mullighn, C. G. TET muttions in myelodysplsi nd myeloid mlignnies. Nture Genet., 7 77 (9).. Tefferi, A. et l. Frequent TET muttions in systemi mstoytosis: linil, KITD8V nd FIPL-PDGFRA orreltes. Leukemi, 9 9 (9).. Tefferi, A. et l. Detetion of mutnt TET in myeloid mlignnies other thn myeloprolifertive neoplsms: CMML, MDS, MDS/MPN nd AML. Leukemi, (9).. Tefferi, A. et l. TET muttions nd their linil orreltes in polyythemi ver, essentil thromoythemi nd myelofirosis. Leukemi, 9 9 (9). 8 N AT U R E VO L 8 9 D E C E M B E R Mmilln Pulishers Limited. All rights reserved

5 RESEARCH. Ito, S. et l. Role of Tet proteins in mc to onversion, ES-ell self-renewl nd inner ell mss speifition. Nture, 9 ().. Hytsu, H. & Shirgmi, M. Retion of isulfite with the -hydroxymethyl group in pyrimidines nd in phge DNAs. Biohemistry 8, 7 (979).. Hung, Y. et l. The ehviour of -hydroxymethylytosine in isulfite sequening. PLoS ONE, e8888 ().. Lister, R. et l. Humn DNA methylomes t se resolution show widespred epigenomi differenes. Nture, (9). 7. Tefferi, A. Epigenetiltertions ndnti-epigeneti therpy inmyelofirosis. Leuk. Lymphom 9, (8). 8. Smith, L. T., Otterson, G. A. & Plss, C. Unrveling the epigeneti ode of ner for therpy. Trends Genet., 9 (7). 9. Esteller, M. Epigenetis in ner. N. Engl. J. Med. 8, 8 9 (8).. Gl-Ym, E. N., Sito, Y., Egger, G. & Jones, P. A. Cner epigenetis: modifitions, sreening, nd therpy. Annu. Rev. Med. 9, 7 8 (8).. Ehrlih, M. DNA hypomethyltion in ner ells. Epigenomis, 9 9 (9).. Lenguer, C. Cner. An unstle liison. Siene, ().. Bröske, A.-M. et l. DNA methyltionprotets hemtopoieti stemellmultipoteny from myeloerythroid restrition. Nture Genet., 7 (9).. Gudet, F. et l. Indution of tumors in mie y genomi hypomethyltion. Siene, 89 9 ().. Wlsh, C. P., Chillet, J. R. & Bestor, T. H. Trnsription of IAP endogenous retroviruses is onstrined y ytosine methyltion. Nture Genet., 7 (998).. Jing, Y. et l. Aerrnt DNA methyltion is dominnt mehnism in MDS progression to AML. Blood, (9). Supplementry Informtion is linked to the online version of the pper t Aknowledgements This work ws supported y NIH grnts R AI nd RC DA8 (to A.R.), NIH grnts K HL77 nd R HL98, n Estlished Investigtor wrd from the Aplsti Anemi & MDS Foundtion, nd n wrd from the Bo Duggn Memoril Reserh Fund (to J.P.M), NIH grnt R HG9 (to X.S.L.) nd pilot grnt from Hrvrd Ctlyst, The Hrvrd Clinil nd Trnsltionl Siene Center (NIH Grnt # UL RR 78-, to S.A.). Y.H. ws supported y postdotorl fellowshipsfromthe GlxoSmithKline-ImmuneDisese Institute (GSK-IDI) Alline nd the Leukemind LymphomSoietyof Ameri. H.S.B. is supported y postdotorl fellowship from the GSK-IDI Alline. Author Contriutions M.K. nlysed the iohemil effets of ptient-ssoited TET muttions nd performed the in vitro differentition studies; Y.H. generted nd hrterized the nti-cms ntiserum, developed the quntittive dot-lot ssy nd quntified in DNA smples from ptients nd helthy ontrols. A.M.J., R.G. nd J.P.M. provided ptient nd ontrol DNA for quntifition, performed DNA methyltion rrys nd nlysed TET muttionl sttus in ptients. U.J.P. nd X.S.L. rried out the sttistil nlysis of levels ndmethyltion dt; M.T., H.S.B. nd K.P.K. provided ritil regents; J.A. nd E.D.L. ontriuted to moleulr loning nd mouse mintenne respetively; nd L.A. nd S.A. provided essentil intelletul input. A.R. set overll gols, oordinted ollortions nd wrote the mnusript. Author Informtion Dt hve een deposited t GEO under ession numer GSE7 (methyltion sttus of eh CpG site (et vlue) n e found in Supplementry Tle ). Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of this rtile t Correspondene nd requests for mterils should e ddressed to A.R. (ro@idi.hrvrd.edu nd ro@lii.org) or J.P.M. (miejj@f.org). 9 DECEMBER VOL 8 NATURE 8 Mmilln Pulishers Limited. All rights reserved