The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes

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1 LETTER doi:1.138/nture1443 The role of Tet3 DNA dioxygense in epigenetic reprogrmming y oocytes Tin-Peng Gu 1 *, Fn Guo 1 *, Hui Yng 2 *, Hi-Ping Wu 1 {, Gui-Fng Xu 1, Wei Liu 1, Zhi-Guo Xie 1, Linyu Shi 2, Xinyi He 3, Seung-gi Jin 4, Khursheed Iql 5, Yujing Geno Shi 6, Zixin Deng 3, Pirosk E. Szó 5, Gerd P. Pfeifer 4, Jinsong Li 2 & Guo-Ling Xu 1 Sperm nd eggs crry distinctive epigenetic modifictions tht re djusted y reprogrmming fter fertiliztion 1. The pternl genome in zygote undergoes ctive DNA demethyltion efore the first mitosis 2,3. The iologicl significnce nd mechnisms of this pternl epigenome remodelling hve remined uncler 4. Here we report tht, within mouse zygotes, oxidtion of 5-methylcytosine (5mC) occurs on the pternl genome, chnging 5mC into 5- hydroxymethylcytosine (5hmC). Furthermore, we demonstrte tht the dioxygense Tet3 (ref. 5) is enriched specificlly in the mle pronucleus. In Tet3-deficient zygotes from conditionl knockout mice, pternl-genome conversion of 5mC into 5hmC fils to occur nd the level of 5mC remins constnt. Deficiency of Tet3 lso impedes the demethyltion process of the pternl Oct4 nd Nnog genes nd delys the susequent ctivtion of pternlly derived Oct4 trnsgene in erly emryos. Femle mice depleted of Tet3 in the germ line show severely reduced fecundity nd their heterozygous mutnt offspring lcking mternl Tet3 suffer n incresed incidence of developmentl filure. Oocytes lcking Tet3 lso seem to hve reduced ility to reprogrm the injected nuclei from somtic cells. Therefore, Tet3-medited DNA hydroxyltion is involved in epigenetic reprogrmming of the zygotic pternl DNA following nturl fertiliztion nd my lso contriute to somtic cell nucler reprogrmming during niml cloning. To investigte whether loss of DNA methyltion in the mle pronucleus coincides with oxidtion of 5mC to 5hmC, recently reported type of modifiction of mmmlin DNA 5,6, we performed immunostining of mouse zygotes using n ntiody specificlly recognizing 5hmC (Supplementry Fig. 1). We found tht the 5hmC signl incresed mrkedly in the pternl pronucleus round the pronucler stge PN3 when the pternl pronucleus ecme lrger thn the mternl pronucleus (Fig. 1, ). By contrst, the 5mC signl ecme mrkedly weker in the mle pronucleus from the PN3 stge wheres there ws no cler chnge in the femle pronucleus (Supplementry Fig. 2), s reported previously 2. The inverse correltion etween the 5hmC nd 5mC signls in the two prentl genomes seemed to persist eyond the zygotic stge (Supplementry Fig. 3). Therefore, 5mC oxidtion in the mle pronucleus coincides with the loss of methyltion in the erly mouse emryo. A similr oservtion hs recently een reported in two independent studies 7,8. Next, we exmined the expression of Tet enzymes tht cn ctlyse the oxidtion of 5mC in DNA 9. The Tet3 mrna ws specificlly detected in oocytes nd zygotes (Supplementry Fig. 4). At the zygotic stge, the Tet3 protein ws concentrted in the mle pronucleus, ut loclized to the cytoplsm t other pre-implnttion stges (Fig. 1c nd Supplementry Fig. 5). The unique expression pttern of Tet3 suggests its possile role in modifying the zygotic pternl genome. To study the iologicl function of Tet3 in mouse, we generted conditionl knockout llele olishing its ctlytic ctivity (Supplementry Fig. 6). Becuse homozygous muttion led to neontl lethlity, we chieved germ-line-specific deletion of Tet3 from primordil germ cells (PGCs) in [Tet3 f/2, TNAP-Cre] conditionl knockout (CKO) mice. Femle CKO mice were norml in growth nd morphology. Although they displyed much reduced fecundity (see elow), they gve irth to heterozygous offspring when crossed with wild-type mles (Supplementry Fig. 6). The deletion of Tet3 in oocytes nd zygotes ws confirmed y immunostining nd PCR with reverse trnscription ssys (Fig. 2 nd Supplementry Fig. 6c). Strikingly, no 5hmC signl could e detected nd the 5mC signl intensity did not decline in the lte mle pronuclei of zygotes collected from the CKO femles mted with wild-type mles (Fig. 2 nd Supplementry Fig. 7). In contrst, deletion of Tet3 from the mle germ cells did not seem to ffect the chnge in 5hmC nd 5mC. Therefore, the loss of 5mC in the pternl genome in developing zygotes is cused y its conversion to 5hmC nd the mternl Tet3 is required for this conversion. We then ssessed the role of Tet3 in demethyltion of specific sequences in the mle pronucleus. Line1 trnsposons re known to e ctively demethylted in zygotes 1,11. Comprison of the methyltion level of mle pronucler DNA from Tet3-deficient zygotes t the PN3 4 stges with tht of wild-type zygotes showed tht the process of ctive DNA demethyltion ws impeded y Tet3 deletion (Fig. 2c, upper pnel). This finding lso indictes tht 5hmC serves s n intermedite etween 5mC nd unmethylted C, lthough isulphite nlysis cnnot distinguish 5hmC from 5mC 12,13. To confirm this, we ssyed for 5mC nd 5hmC on Line1 elements in the pternl DNA y MeDIP nd hmedip (methylted nd hydroxymethylted DNA immunoprecipittion). 5hmC ws indeed present t Line1 sequences in wild-type zygotic mle pronuclei t significntly higher level compred to sperm, wheres the 5mC level ws mrkedly lower thn tht of sperm (Supplementry Fig. 8). In the nlysis of mle pronucler DNA from zygotes lcking Tet3, enrichment of 5hmC-contining Line1 elements ws significntly decresed wheres enrichment of 5mCcontining elements ws comprle with sperm. These results strengthen the conclusion tht 5mC oxidtion does occur t Line1 sequences in mle pronuclei. Emryonic stem cell mrker genes, such s Oct4 nd Nnog, re methylted during mle germ cell development nd their demethyltion occurs in the erly emryo In norml zygotes, the Oct4 gene of mle pronuclei hd undergone sustntil demethyltion y the PN3 4 pronucler stges, ut this process ws mrkedly hmpered in Tet3-null zygotes (Fig. 2c nd Supplementry Fig. 9). Moreover, Tet3 deletion lmost completely locked demethyltion t two other 1 Group of DNA Metolism, The Stte Key Lortory of Moleculr Biology, Institute of Biochemistry nd Cell Biology, Shnghi Institutes for Biologicl Sciences, Chinese Acdemy of Sciences, Shnghi 231, Chin. 2 The Stte Key Lortory of Cell Biology, Institute of Biochemistry nd Cell Biology, Shnghi Institutes for Biologicl Sciences, Chinese Acdemy of Sciences, Shnghi 231, Chin. 3 The Stte Key Lortory of Microil Metolism, School of Life Science nd Biotechnology, Shnghi Jiotong University, Shnghi 23, Chin. 4 Deprtment of Cncer Biology, Beckmn Reserch Institute of the City of Hope, Durte, Cliforni 911, USA. 5 Deprtment of Moleculr nd Cellulr Biology, Beckmn Reserch Institute of the City of Hope, Durte, Cliforni 911, USA. 6 Division of Endocrinology, Dietes, nd Hypertension, Deprtment of Medicine nd BCMP, Brighm nd Women s Hospitl nd Hrvrd Medicl School, Boston, Msschusetts 2115, USA. {Present ddress: Novrtis Institutes for BioMedicl Reserch Co., Shnghi 2123, Chin. *These uthors contriuted eqully to this work. 66 NATURE VOL SEPTEMBER 211 Mcmilln Pulishers Limited. 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2 LETTER RESEARCH 5hmC 5mC Merge PN2 PN1 PN PN3 c 2.5 Mle pronucleus PN5 PN4 Tet3 Dnmt1 5hmC level Dnmt3 PN PN1 2.5 PN2 PN3 PN4 PN5 Mle pronucleus 5mC level 2. DAPI Merge.5 PN PN1 PN2 PN3 PN4 PN5 Figure 1 Specific oxidtion of methylcytosine nd Tet3 distriution in the zygotic mle pronucleus., Immunofluorescent imges of 5hmC (green) nd 5mC (red) stining, nd overlid phse contrst imges. The pronucler (PN) stges re indicted. Mle nd femle symols indicte mle nd femle pronucleus, respectively., polr ody. Scle r, 25 mm., Quntifiction of the reltive levels of 5hmC nd 5mC in mle pronuclei in zygotes. Ech dt point is sed on the level of the 5hmC or 5mC signl reltive to the DAPI stining intensity of the sme pronucleus. Error rs indicte s.e.m. Numer of zygotes nlysed for ech stge: PN, 19; PN1, 1; PN2, 8; PN3, 17; PN4, 17; PN5, 15. c, Preferentil stining of Tet3 protein in the mle pronucleus. DNA ws stined with DAPI. Control stining shows Dnmt1 in the corticl cytoplsm nd Dnmt3 (red) in oth pronuclei. The nucleolus hd no stining signl. pternlly methylted genes, Nnog nd Lemd1 (ref. 17), which retined hypermethyltion similr to tht oserved in sperm (Supplementry Fig. 9). To ssess the significnce of pternl demethyltion on gene expression, Tet3-null oocytes from the CKO femles were fertilized y intrcytoplsmic injection (ICSI) of wild-type sperm crrying the enhnced green fluorescent protein (EGFP) reporter gene under the control of the Oct4 promoter, nd expression of EGFP ws monitored in cultured emryos. Compred with wild-type emryos, the mutnt emryos derived from oocytes lcking Tet3 showed significntly weker EGFP expression t the 8-cell nd morul stges (Fig. 2d nd Supplementry Fig. 1). Bsed on these glol nd sequence-specific nlyses of 5mC nd 5hmC, long with the reporter gene ssy, we conclude tht Tet3-medited 5mC oxidtion contriutes to the demethyltion in the zygotic pternl genome nd gene ctivtion in the erly emryo. We next investigted whether removing Tet3 from oocytes might compromise emryonic development. We first confirmed tht erly deletion of Tet3 from the PGC stge did not ffect epigenetic reprogrmming in the emryonic germ cells, oocyte development, mturtion nd fertiliztion (Supplementry Figs nd Supplementry Tle 1). Mle germ cell development nd sperm DNA methyltion were not ffected either (Supplementry Fig. 14). However, fecundity of the femle CKO mice ws significntly lower in terms of the frequency of successful pregnncy per mting nd the litter size (Fig. 3 nd Supplementry Tle 2). Deletion of mternl Tet3 did not seem to ffect the pre-implnttion development s heterozygous zygotes collected from CKO femles mted with wild-type mles developed to lstocysts in vitro normlly (Supplementry Tle 3). We then exmined the effect of mternl Tet3 deletion on prentl development y trnsplnttion of 2-cell emryos into oviducts of pseudo-pregnnt femles. Wheres the trnsferred emryos lcking mternl Tet3 implnted normlly, they showed much reduced rte of full-term development (Supplementry Tle 4). Dissection of trnsferred [Tet3 Mt2/Pt1] mutnt emryos in the pregnnt wild-type foster mothers reveled high frequency of degenertion nd the ppernce of morphologicl normlities, strting from midgesttion (Fig. 3 nd Supplementry Tle 4). Moreover, deletion of Tet3 lter from growing oocytes 2 9 S E P T E M B E R VO L N AT U R E Mcmilln Pulishers Limited. All rights reserved

3 RESEARCH LETTER Tet3 DAPI Merge 5hmC 5mC Merge +/+ Mt + /Pt c Line1 Sperm +/+ PN % 57.5% 83.2% Oct4-PE PN3-4 Sperm MII oocyte +/+ 89.3% 33.9% 21.4% 92.9% +/+ d EGFP intensity +/+ *** ** 2-cell 4-cell *** 8-cell Morul Blstocyst Figure 2 The role of Tet3 in 5mC oxidtion, demethyltion of pternl DNA, nd ctivtion of the pternl Oct4 llele., Loss of the Tet3 protein in heterozygous zygotes (Mt 2 /Pt 1 ) otined from CKO femles mted with wild-type mles. PN3 zygotes were stined with n nti-tet3 ntiody rised ginst the deleted region. Scle r, 25 mm., 5hmC (green) nd 5mC (red) immunostining in wild-type (1/1) nd mternlly (Mt 2 /Pt 1 ) or pternlly (Mt 1 / Pt 2 ) Tet3-deficient zygotes t the PN5 stge. c, Methyltion nlysis of Line1 nd Oct4 (PE, the proximl enhncer region) in mle pronuclei isolted from wild-type (1/1) nd Tet3-deficient (Mt 2 /Pt 1 ) zygotes. Open nd filled circles represent unmethylted nd methylted CpG sites, respectively. Percentge of methylted CpGs is indicted. d, Pternl Oct4 ctivtion in wild-type emryos (1/1) nd emryos lcking mternl Tet3 (Mt 2 /Pt 1 ). Emryos were derived from ICSI using sperm crrying the Oct4-EGFP trnsgene nd the EGFP signl ws quntified, reltive to the level in 2-cell lstomere. Numer of emryos nlysed t ech stge, 1/1: 2-cell, 13; 4-cell, 9; 8-cell, 9; morul, 12; lstocyst, 7. Mt 2 / Pt 1 : 2-cell, 11; 4-cell, 9; 8-cell, 8; morul, 8; lstocyst, 5. Error rs indicte s.e.m. **P,.1 nd ***P,.1. using Zp3-Cre lso led to filure in zygotic 5hmC genertion, retention of pternl 5mC, impired demethyltion t Line1 nd Oct4, nd compromised emryonic development (Supplementry Fig. 15). Therefore, lcking mternl Tet3 locks pternl genome reprogrmming nd cuses mrkedly incresed developmentl filure of the emryo. Somtic cell nuclei injected into eggs undergo profound epigenetic reprogrmming, including DNA demethyltion. The cytoplsm of germinl vesicle (GV) oocytes 18, metphse II (MII) oocytes 19, zygotes 2 nd 2-cell emryos 21 hs een shown to possess reprogrmming ctivity. The existence of the Tet3 protein cross these stges indicted tht Tet3 might e one of the cytoplsmic fctors contriuting to the reprogrmming ctivity. We tested therefore whether Tet3-medited hydroxyltion is prt of the reprogrmming process in somtic cell nucler trnsfer (SCNT). Remrkly, following ctivtion of nucler trnsfer (NT) oocytes reconstructed fter injection of somtic nucleus into oocyte with or without enucletion (intct oocyte), the Tet3 protein originting from the oocyte cytoplsm ecme concentrted in the pseudo-pronucleus () formed from the trnsferred somtic nucleus, ut not in the femle pronucleus derived from the spindle-chromosome-complex tht existed in oocytes (Fig. 4). Significntly, the formed in the NT emryos from wild-type intct or enucleted oocytes underwent 5mC oxidtion wheres this modifiction did not occur in emryos derived from Tet3-null oocytes (Fig. 4). Wheres sustntil demethyltion ws detected t the Oct4 promoter in the of Tet3-proficient NT emryos, the somtic hypermethyltion persisted when the oocyte Tet3 ws deleted (Fig. 4c). To investigte the role of Tet3-medited DNA oxidtion in the ctivtion of pluripotency genes, we used donor somtic cells crrying the Oct4-EGFP trnsgene. Compred with emryos from wild-type oocytes, the EGFP signl ws significntly lower in emryos derived from the Tet3-null oocytes t the Productive mtings (%) Averge litter size P <.1 f /+ f/ P <.1 f/+ f/ E8.5 E11.5 E14.5 E18.5 +/+ (7/24) (2/23) (12/27) (3/24) (21/45) Figure 3 Mternl Tet3 deficiency compromises emryonic development., Reduced efficiency of productive mting nd litter size in femles (f/2) with germline Tet3 deficiency. Error rs indicte s.e.m. [Tet3 f/1, TNAP-Cre] mice, n 5 9; [Tet3 f/2, TNAP-Cre] mice, n 5 7., Developmentl filure mong emryos lcking mternl Tet3. Pregnnt foster femles receiving wild-type (1/1) nd mutnt (Mt 2 /Pt 1 ) 2-cell emryos trnsferred were dissected t different stges of gesttion. Numers in rckets indicte proportion of emryonic dy 8.5 (E8.5) decidus nd E emryos showing smller size nd morphologicl normlities. The rrow indictes degenerted conceptuses otined. Detiled dt re presented in Supplementry Tle NATURE VOL SEPTEMBER 211 Mcmilln Pulishers Limited. All rights reserved 211

4 LETTER RESEARCH Tet3 DAPI Merge WT 5hmC 5mC Merge KO WT d EGFP intensity WT KO ** ** KO c Oct4 promoter, 6 8 hp Cumulus WT KO 4-cell 8-cell Morul Blstocyst 78.6% 22.6% 69.% Figure 4 Tet3 contriutes to reprogrm the somtic nucleus trnsferred into oocytes., Tet3 enrichment in the pseudo-pronucleus () formed in emryos reconstructed y trnsplnttion of cumulus somtic nuclei into n intct (top row) or enucleted oocyte (middle row). The enrichment did not occur in the femle pronucleus in intct oocytes (top row) nd in the two pronuclei of prthenogenetic emryos (ottom row). Scle r, 25 mm., Impired 5mC oxidtion in of 1-cell NT emryos derived from injection of cumulus nuclei into intct or enucleted oocytes lcking Tet3. Emryos round 1 h post ctivtion (hp) were stined with nti-5hmc (green) nd nti-5mc (red) ntiodies. c, Impired Oct4 demethyltion in NT emryos derived from Tet3-null oocytes. More thn 2 NT emryos derived from enucleted wild-type (WT) or Tet3-null (KO) oocytes were collected 6 8 hp for DNA methyltion nlysis. d, Wekened ctivtion of the somtic Oct4 EGFP reporter in cultured NT emryos from Tet3-null oocytes. The EGFP intensities were reltive to the level in 4-cell lstomere. Error rs indicte s.e.m. Numer of emryos nlysed for ech stge, WT: 4-cell, 11; 8-cell, 13; morul, 9; lstocyst, 7. KO: 4-cell, 8; 8-cell, 7; morul, 6; lstocyst, 7. **P,.1. 8-cell nd morul stges (Fig. 4d nd Supplementry Fig. 16). Consistently, the Oct4 mrna level ws reltively low in 8-cell emryos from Tet3-null oocytes (Supplementry Fig. 16). Therefore, deficiency in oocyte Tet3 could cuse wekened or delyed ctivtion of the somtic Oct4 in NT emryos. We hve otined sustntil evidence tht oxidtion of 5mC in the pternl genome in fertilized eggs y Tet3 initites DNA demethyltion nd fcilittes the ctivtion of the pternl copy of erly emryonic genes, thus contriuting to the estlishment of iprentl totipotency in the erly emryo y countercting the silencing function of 5mC. Blocking oxidtion of 5mC y Tet3 deletion ffects pternl gene ctivtion, leding to reduced developmentl fitness nd fetl survivl. It remins to e determined whether the developmentl filures could e cused y hploinsufficiency 22 for the genes ffected in the erly emryo. The involvement of Tet3 in somtic Oct4 ctivtion indictes tht Tet-medited 5mC oxidtion contriutes to epigenetic reprogrmming of the donor nucler DNA in SCNT. Reprogrmming in SCNT might thus shre common mechnism with pternl genome remodelling in fertilized eggs. Further investigtions re needed to revel the signls regulting Tet3 nd the events susequent to 5hmC formtion in oth fertilized nd cloned emryos. METHODS SUMMARY Preprtion of nti-5hmc ntiody. To prepre nti-5hmc ntiody, 5-hydroxymethylcytidine 59-monophosphte (5hmCMP) hpten ws synthesized from cytidine 59-monophosphte (CMP) using MilA hydroxymethylse 23.The rionucleoside ws then conjugted to ovine serum lumin (BSA) s previously descried 24 nd used to immunize rits. The ntiody ws ffinity-purified from ntiserum with 5hmCMP BSA conjugte coupled to grose eds. Immunostining for 5hmC in fertilized oocytes. Immunofluorescence detection of 5hmC in zygotes followed the procedure for 5mC 11. Fluorescent imges were cquired t 2-mm Z-xis intervls using confocl microscope (LEICA TCS SP5 II) nd their signl intensity ws determined. Gene trgeting. A Tet3 trgeting vector ws electroported into 129Sv ES cells for homologous recomintion. The floxed region contins exons 8-9, which includes the coding region for the conserved Fe 21 -inding motif of dioxygenses. To inctivte Tet3 in the germ line, we crossed the mice crrying floxed llele with TNAP-Cre knock-in mice on 129 genetic ckground 25. The conditionl knockout mice were on C57BL/6J-129Sv genetic ckground. Zygote collection nd stging. Femle mice 4- to 8-week-old were injected with pregnnt mre s serum gondotropin nd humn chorionic gondotropin were mted with wild-type mles. Zygotes were hrvested nd the PN stges of individul zygotes were clssified ccording to ref. 11, y tking into ccount the pronucler morphology nd the presence of 5mC signl. Oservtion of Oct4-EGFP ctivtion. To exmine the role of Tet3 in pternl gene ctivtion, MII oocytes collected from wild-type nd Tet3 CKO femles were 29 SEPTEMBER 211 VOL 477 NATURE Mcmilln Pulishers Limited. All rights reserved

5 RESEARCH LETTER fertilized y ICSI with sperm from trnsgenic mice (tg/tg) crrying the Oct4-EGFP trnsgene 26 nd the resulting emryos were cultured to oserve EGFP expression. To exmine the role of Tet3 in somtic gene ctivtion in SCNT, we monitored the Oct4-EGFP trnsgene expression from the donor cell DNA in NT emryos, which were derived fter injection of cumulus nucleus into wild-type or Tet3-null oocyte without enucletion. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 21 Jnury; ccepted 16 August 211. Pulished online 4 Septemer Surni, M. A., Hyshi, K. & Hjkov, P. Genetic nd epigenetic regultors of pluripotency. Cell 128, (27). 2. Myer, W., Niveleu, A., Wlter, J., Fundele, R. & Hf, T. Demethyltion of the zygotic pternl genome. Nture 43, (2). 3. Oswld, J. et l. Active demethyltion of the pternl genome in the mouse zygote. Curr. Biol. 1, (2). 4. Ooi, S. K. & Bestor, T. H. The colorful history of ctive DNA demethyltion. Cell 133, (28). 5. Thilini, M. et l. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mmmlin DNA y MLL prtner TET1. Science 324, (29). 6. Kriucionis, S. & Heintz, N. The nucler DNA se 5-hydroxymethylcytosine is present in Purkinje neurons nd the rin. Science 324, (29). 7. Iql, K., Jin, S. G., Pfeifer, G. P. & Szo, P. E. Reprogrmming of the pternl genome upon fertiliztion involves genome-wide oxidtion of 5-methylcytosine. Proc. Ntl Acd. Sci. USA 18, (211). 8. Wossidlo, M. et l. 5-Hydroxymethylcytosine in the mmmlin zygote is linked with epigenetic reprogrmming. Nture Commun. 2, 241 (211). 9. Wu, S. C. & Zhng, Y. Active DNA demethyltion: mny rods led to Rome. Nture Rev. Mol. Cell Biol. 11, (21). 1. Jmil, A. Z., Iql, K., Fwd Ur, R. & Mirz, K. A. Effect of phcoemulsifiction on introculr pressure. J. Coll. Physicins Surg. Pk. 21, (211). 11. Wossidlo, M. et l. Dynmic link of DNA demethyltion, DNA strnd reks nd repir in mouse zygotes. EMBO J. 29, (21). 12. Jin, S. G., Kdm, S. & Pfeifer, G. P. Exmintion of the specificity of DNA methyltion profiling techniques towrds 5-methylcytosine nd 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (21). 13. Hung, Y. et l. The ehviour of 5-hydroxymethylcytosine in isulfite sequencing. PLoS ONE 5, e8888 (21). 14. Httori, N. et l. Epigenetic control of mouse Oct-4 gene expression in emryonic stem cells nd tropholst stem cells. J. Biol. Chem. 279, (24). 15. Immur, M. et l. Trnscriptionl repression nd DNA hypermethyltion of smll set of ES cell mrker genes in mle germline stem cells. BMC Dev. Biol. 6, 34 (26). 16. Frthing, C. R. et l. Glol mpping of DNA methyltion in mouse promoters revels epigenetic reprogrmming of pluripotency genes. PLoS Genet. 4, e1116 (28). 17. Iql, K. et l. Sucutneous pnniculitis-like T-cell lymphom in ssocition with srcoidosis. Clin. Exp. Dermtol. 36, (211). 18. Bui, H. T. et l. The cytoplsm of mouse germinl vesicle stge oocytes cn enhnce somtic cell nucler reprogrmming. Development 135, (28). 19. Yng, H. et l. High-efficiency somtic reprogrmming induced y intct MII oocytes. Cell Res. 2, (21). 2. Egli, D., Rosins, J., Birkhoff, G. & Eggn, K. Developmentl reprogrmming fter chromosome trnsfer into mitotic mouse zygotes. Nture 447, (27). 21. Egli, D., Sndler, V. M., Shinohr, M. L., Cntor, H. & Eggn, K. Reprogrmming fter chromosome trnsfer into mouse lstomeres. Curr. Biol. 19, (29). 22. Seidmn, J. G. & Seidmn, C. Trnscription fctor hploinsufficiency: when hlf lof is not enough. J. Clin. Invest. 19, (22). 23. Li, L. et l. The mildiomycin iosynthesis: initil steps for sequentil genertion of 5-hydroxymethylcytidine 59-monophosphte nd 5-hydroxymethylcytosine in Streptoverticillium rimofciens ZJU5119. ChemBioChem 9, (28). 24. Erlnger, B. F. & Beiser, S. M. Antiodies specific for rionucleosides nd rionucleotides nd their rection with DNA. Proc. Ntl Acd. Sci. USA 52, (1964). 25. de Vries, W. N. et l. Expression of Cre recominse in mouse oocytes: mens to study mternl effect genes. Genesis 26, (2). 26. Oho, K. et l. Identifiction nd chrcteriztion of stem cells in prepuertl spermtogenesis in mice. Dev. Biol. 258, (23). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk C. Wlsh nd M. Rots for criticl reding of the mnuscript, J. Wlter for discussions, H. Qi for providing cdna of mouse oocytes, R. Zhng & Q. Cui for Tet3 cdna, L. Li for help with 5hmCMP synthesis, Shnghi Reserch Center for Model Orgnisms for lstocyst injection, nd J. Go for mouse work. This study ws supported y grnts from the Ministry of Science nd Technology Chin (27CB94753 to G.-L.X., 27CB94711 to J.L., nd 29CB94111 to G.-L.X. nd J.L.), Ntionl Science Foundtion of Chin (37359 to G.-L.X. nd to J.L.), the Chinese Acdemy of Sciences (XDA1131 to G.-L.X.; XDA1143 nd KSCX2-YW-R-11 to J.L.) nd the NIH (GM78458 to Y.G.S.). Author Contriutions G.-L.X. nd J.L. conceived the projects. Y.G.S., H.-P.W. nd G.-L.X. contriuted tothe knockoutdesign. F.G., T.-P.G., H.-P.W., G.-F.X., nd W.L. performed the experiments on erly emryos. X.H. nd Z.D. contriuted to the synthesis of the 5hmC hpten. H.Y. nd L.S. performed the nucler trnsfer nd emryo trnsfer experiments. S.-g.J., K.I., P.E.S., G.P.P. nd Z.-G.X. chrcterized Tet3 expression in PGCs nd ovries. G.-L.X. wrote nd G.P.P. revised the mnuscript. Author Informtion Reprints nd permissions informtion is ville t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of this rticle t Correspondence nd requests for mterils should e ddressed to J.L. (jsli@sis.c.cn) or G.-L.X. (glxu@sis.c.cn). 61 NATURE VOL SEPTEMBER 211 Mcmilln Pulishers Limited. All rights reserved 211

6 LETTER RESEARCH METHODS Preprtion of nti-5hmc nd nti-tet3 ntiodies. To prepre 5hmC ntiody, 5-hydroxymethylcytidine 59-monophosphte (5hmCMP) hpten ws synthesized from cytidine 59-monophosphte (CMP) in rection contining formldehyde nd tetrhydrofolte ctlysed y recominnt MilA hydroxymethylse 23. The rionucleoside hpten ws then conjugted to BSA s previously descried 24 for immuniztion of rits (Supplementry Fig. 1). Mss spectrl nlysis confirmed tht the 5hmCMP se moiety ws unltered under the conjugtion condition. The ntiody ws ffinity-purified from ntiserum with the 5hmCMP BSA conjugte coupled to grose eds. To ensure specificity, crossrectivity ws removed y incution with grose eds crosslinked with CMP BSA conjugte. For the detection of Tet3, two rit polyclonl ntiodies were rised ginst C-terminl region (mino cids , GenBnk ccession numer NP_898961) nd the trgeted region (mino cids ) respectively, ffinitypurified nd evluted s descried previously 27. Immunostining for 5hmC nd Tet3 in fertilized oocytes. Immunofluorescence detection of 5hmC in zygotes derived from nturl mtings followed the procedure for 5mC 11. The signl ws detected y Alex Fluor-conjugted got nti-rit or nti-mouse IgG (see Supplementry Tle 4 for detiled ntiody informtion). Fluorescent imges were cquired t 2-mm Z-xis intervls using confocl microscope (Leic TCS SP5 II) nd their signl intensity ws determined using the Leic Appliction Suite-Advnced Fluorescence softwre. The distriution of the Tet3 protein in emryonic cells ws determined on emryos fixed with 4% prformldehyde (PFA) nd permeilized with.5% Triton. Gene trgeting. A trgeting vector for Tet3 ws prepred using the recomineering technique 28 nd electroported into 129Sv ES cells for selection of trgeted clones. The floxed region contins exons 8-9, which code for the region (76 mino cids from EEVLR to NGCTV, GenBnk ccession numer NP_898961) contining the conserved Fe 21 -inding motif of the ctlytic domin. Deletion of the floxed region leds to the loss of 76 mino cids with in-frme fusion etween exons 7 nd 1. Neomycin-resistnt emryonic stem clones were screened y PCR using pir of primers crossing the shorter right homologous rm. Positive clones were further chrcterized y Southern lotting to confirm homologous recomintion on the left side of the trgeted genomic region (refer to Supplementry Fig. 7). Eemryonic stem cells crrying correctly trgeted llele (with neo) were injected into lstocysts to generte germline chimers. Mice with floxed llele were otined y reeding with C57BL/6J mice. The neo selection mrker ws removed in mice y crossing with ACTFLPe mice 29. To inctivte Tet3 in germ cells from the PGC stge onwrds, we generted conditionl knockout mice y crossing floxed mice with TNAP-Cre knock-in mice. TNAP-Cre is expressed in primordil germ cells from emryonic dy 9.5 to lte gesttion 3. To inctivte Tet3 in femle germ cells from the growing oocyte stge, we generted conditionl knockout mice y crossing floxed mice with Zp3-Cre trnsgenic mice which express Cre exclusively in growing oocytes 31. Mice were genotyped y PCR (primer sequences re presented in Supplementry Tle 6). The conditionl knockout mice were on mixed C57BL/6J-129Sv genetic ckground. Zygote collection. Wild-type BDF1 (from C57BL/6 R 3 DBA2 =) femle mice 4- to 8-week-old injected with pregnnt mre s serum gondotropin nd humn chorionic gondotropin were mted with BDF1 mle. Zygotes were hrvested t different time points fter humn chorionic gondotropin injection. The PN stge of ech individul zygote ws clssified ccording to ref. 11, y tking into ccount the pronucler morphology nd the presence of 5mC signl. We stined the DNA with DAPI (for fixed zygotes) or Hoechst (for live zygotes), nd used Nomrski differentil interference contrst microscope for etter oservtion of zygotic pronuclei, when necessry. Tet3-deficient zygotes were otined from [Tet3 f/2,tnap2cre] femle mice crossed with wild-type mles. Fertility test. [Tet3 f/2,tnap2cre] femle mice t 6 8 weeks of ge were housed with 8- to 12-week-old wild-type mles of proven fertility. Rte of fertiliztion ws judged y the presence of two pronuclei in zygotes collected from femles fter pregnnt mre s serum gondotropin nd humn chorionic gondotropin tretment nd mting with wild-type mle mice of proven fertility. Numer of completed pregnncies per plug seen (litters per plug) nd numer of vile pups orn per litter (litter size) were clculted from the dt included in Supplementry Tle 2. [Tet3 f/1,tnap2cre] femle mice were used s control group for comprison. Student s t-test ws performed to compre verges in the two different experimentl groups nd P,.5 ws considered to e significnt. Isoltion of primordil germ cells (PGCs). Femle CF1 (Chrles River Lortories) mice were mted with mle OG2 (ref. 32) mice. This trnsgenic mouse line expresses the enhnced green fluorescent protein (EGFP) from the Oct4 promoter nd thus enles the selective purifiction of emryonic nd fetl germ cells. Emryo prts enriched in PGCs nd genitl ridges were dissected t 9.5 nd 11.5 dys post coitum (dpc), respectively. At 9.5 dpc nd 11.5 dpc, the sex of the emryo ws determined y rel-time PCR mplifiction of two genes in single rection. The Sry mplicon indicted the presence of Y chromosome nd mle sex, wheres mplifiction of the Snrpn gene served s positive control for DNA. Gonds were dissected from mle nd femle fetuses t 13.5, 15.5 nd 17.5 dpc. Mle nd femle gonds were distinguished y their distinct morphology t these stges. Gonds were incuted t 37 uc for 15 min in trypsin-edta nd triturted to chieve single cell suspension contining germ cells nd somtic cells. Dulecco s Modified Egle s Medium (DMEM) (Invitrogen) supplemented with 2% FBS ws dded to inctivte trypsin. Cell suspensions were nlysed nd sorted on MoFlo flow cytometer (Beckmn Coulter). Dt were cquired using 488 nm excittion from n Innov-36 Argon lser (Coherrent) t 5 mw. EGFP emission ws mesured through 53DF3 filter (Omeg Opticl). Quntittive reverse trnscription PCR. Poly(A1) mrnas were isolted from zygotes (n 5 2) nd femle PGCs (emryonic dys 9.5, 11.5, 13.5, 15.5 nd 17.5) y using the Dyneds mrna DIRECT Micro Kit (Invitrogen). Oligo (dt)25- coupled Dyneds nd mrna complexes were immeditely used for reverse trnscription using SuperScript III reverse trnscriptse (Invitrogen), ccording to the mnufcturer s instructions. Rel-time quntittive PCR rections were performed t 5 uc for 2 min nd 95 uc for 1 min followed y 5 cycles t 95 uc for 15 s nd 6 uc for 1 min using TqMn Gene Expression Mster Mix (Applied Biosystems) on n iq5 rel-time PCR cycler (Bio-Rd). PCR ws performed with TqMn MGB primers with 6FAM-sed proes (Applied Biosystems) using the following ssy ID numers: Tet1 (Mm116988_m1), Tet2 (Mm131297_m1), Tet3 (Mm85754_m1) nd Stell/Dpp3 (Mm _g1). The cdna levels of trget genes were nlysed using comprtive C t method nd normlized to the internl stndrd gene Gpdh. Collection of oocytes nd production of prthenogenetic emryos. For collection of GV oocytes, the ovries were removed from the femle mice h fter pregnnt mre s serum gondotropin injection. Antrl follicles were punctured y 3G needles, nd cumulus-enclosed GV oocytes were relesed into HEPESuffered CZB medium (HCZB) contining.2 mm 3-isoutyl-1-methylxnthine (IBMX) to inhiit germinl vesicle rekdown. Cumulus cells were removed y pipetting. For collection of mture oocytes, oviducts were removed from the femle mice h fter humn chorionic gondotropin injection. Cumulus oocyte complexes were relesed into HCZB contining.1% ovine testiculr hyluronidse (3 USP units per mg; ICN Biomedicls Inc.). MII oocytes were ctivted for 6 h in ctivtion medium (clcium-free CZB medium contining 1 mm Sr 21 nd 5 mgml 21 cytochlsin B) to generte prthenogenetic emryos, which were cultured in KSOM medium with mino cids nd hrvested t 8 h post ctivtion. Intrcytoplsmic sperm injection (ICSI). ICSI ws performed ccording to the method of ref. 33 except for eing performed t room temperture (out 25 uc). Briefly, sperm were collected from dult mice (Oct-delt PE-GFP #18) crrying Oct4-EGFP trnsgene (tg/tg) 26 nd the hed ws seprted from the til y pplying pulses to the hed-til junction y mens of Piezo-driven pipette (Piezoelectric ctutor; PrimeTech). Only the sperm hed ws injected into ech oocyte. Injected oocytes were cultured in KSOM medium for 96 h to exmine their development in vitro. Imges of resulting emryos were cquired with n IX51 inverted microscope (Olympus) under the sme exposure prmeters nd the EGFP intensity of ech emryo ws quntified with ImgeJ softwre 34. Emryo trnsfer nd Cesren section. Fertilized eggs derived from nturl mtings were cultured in KSOM medium until the 2-cell stge. Two-cell emryos were then trnsferred into oviducts of surrogte femles t dy 1 of pseudopregnncy. For strict comprison, eight mutnt nd control 2-cell emryos (Supplementry Tle 4) were trnsferred into the left nd right oviducts of recipients, respectively. Recipient mothers were euthnized t 8.5, 11.5 nd 14.5 dys of gesttion nd emryos were dissected. For emryos developed to term, cesren section ws performed on dy 19 nd living pups were nursed y lctting ICR femles. Isoltion of mle pronuclei. Mle pronuclei, which were distinguished from femle pronuclei on the sis of their size nd distnce from polr odies were hrvested from zygotes of PN3-4 stges y reking the zon using Piezo drive (Prime Tech) nd spirting using micromnipultor. At lest 4 mle pronuclei from control or Tet3-deficient zygotes were collected nd sujected to isulphite sequencing nlysis. Bisulphite sequencing. For DNA methyltion nlysis in oocytes, pronuclei from zygotes nd pseudo-pronuclei from NT emryos with limited numers, isulphite conversion ws performed in grose eds s descried 35. Unised mplifiction for methylted nd unmethylted sequences ws ensured y testing isulphite PCR primers using 1:1 mixture of unmethylted nd in vitro methylted DNA frgments. The PCR products were cloned into pmd19-t vectors (Tkr Inc.) nd individul clones were sequenced y BGI Ltd, Shnghi. Bisulphite primer 211 Mcmilln Pulishers Limited. All rights reserved

7 RESEARCH LETTER informtion is presented in Supplementry Tle 6. For the determintion of the methyltion stte of ech sequence, the experiment ws performed t lest twice strting from the isoltion of cells, pronuclei nd emryos. DNA immunoprecipittion with nti-5mc nd nti-5hmc ntiodies. To detect the existence of 5hmC t Line1 repets in the pternl genome in mouse zygotes,.1 mle pronuclei were hrvested from zygotes t PN3-4 stges, digested with proteinse K nd RNse A, nd the genomic DNA ws purified y phenol-chloroform extrction. The genomic DNA ws mixed with 25 ng of crrier lmd DNA (dm 2, dcm 2 ) nd frgmented y AluI digestion, hetdentured (1 min, 95 uc), nd immunoprecipitted s descried previously 36 using 1 mg of nti-5hmc or nti-5mc ntiodies (Eurogentec, BI-MECY-1) nd 1 ml Dyneds (coupled with M-28 sheep nti-rit IgG for the 5hmC ntiody or nti-mouse IgG for the 5mC ntiody). qpcr ws performed on BioRd CFX96 Rel-Time PCR Detection System for the input nd immunoprecipitted DNA. Mouse genomic DNA (1 ng) from Dnmt TKO emryonic stem cells lcking DNA methyltion 37 nd thus contining no 5hmC, ws used s negtive control. Mouse sperm DNA ws used for comprisons. Nucler trnsfer with intct nd enucleted oocytes. NT ws performed s descried 38 with modifictions 19. Briefly, metphse II-rrested oocytes were collected from superovulted B6D2F1 or CKO femles, nd cumulus cells were removed using hyluronidse. In the stndrd NT procedure, the oocytes were collected from wild-type nd Tet3 CKO femles, nd enucleted in droplet of HEPES-CZB medium contining 5 mgml 21 CB using lunt Piezo-driven pipette. After enucletion, the spindle-free oocytes were wshed extensively nd mintined in CZB medium up to 2 h efore nucleus injection. The cumulus cells collected from superovulted Oct4-EGFP trnsgenic mice were spirted in nd out of the injection pipette to remove the cytoplsmic mteril nd then injected into enucleted oocytes. The reconstructed oocytes were cultured in CZB medium for 1 h nd then ctivted for 5 6 h in ctivtion medium. For NT with intct oocytes, oocytes were ctivted for 2 min nd then directly injected with cumulus cells. The reconstructed oocytes were ctivted for 5 6 h in ctivtion medium. Following ctivtion, the reconstructed emryos were cultured in KSOM medium with mino cids t 37 uc under 5% CO 2 in ir. Emryo imging nd EGFP quntifiction followed the sme procedure s in the ICSI experiment descried ove. The EGFP levels were determined from n. 6 emryos t ech stge. 27. Ge, Y. Z. et l. Chromtin trgeting of de novo DNA methyltrnsferses y the PWWP domin. J. Biol. Chem. 279, (24). 28. Liu, P., Jenkins, N. A. & Copelnd, N. G. A highly efficient recomineering-sed method for generting conditionl knockout muttions. Genome Res. 13, (23). 29. Rodriguez, C. I. et l. High-efficiency deleter miceshowthtflpe is nlterntive to Cre-loxP. Nture Genet. 25, (2). 3. Lomelí, H., Rmos-Meji, V., Gertsenstein, M., Loe, C. G. & Ngy, A. Trgeted insertion of Cre recominse into the TNAP gene: excision in primordil germ cells. Genesis 26, (2). 31. Lewndoski, M., Wssrmn, K. M. & Mrtin, G. R. Zp3 cre, trnsgenic mouse line for the ctivtion or inctivtion of loxp-flnked trget genes specificlly in the femle germ line. Curr. Biol. 7, (1997). 32. Szó, P. E., Huner, K., Scholer, H. & Mnn, J. R. Allele-specific expression of imprinted genes in mouse migrtory primordil germ cells. Mech. Dev. 115, (22). 33. Kimur, Y. & Yngimchi, R. Intrcytoplsmic sperm injection in the mouse. Biol. Reprod. 52, (1995). 34. Aràmoff, M. D., Mglhães, P. J. & Rm, S. J. Imge processing with ImgeJ. Biophotonics Int. 11, (24). 35. Hjkov, P. et l. DNA-methyltion nlysis y the isulfite-ssisted genomic sequencing method. Methods Mol. Biol. 2, (22). 36. Weer, M. et l. Chromosome-widend promoter-specific nlyses identifysites of differentil DNA methyltion in norml nd trnsformed humn cells. Nture Genet. 37, (25). 37. Tsumur, A. et l. Mintennce of self-renewl ility of mouse emryonic stem cells in the sence of DNA methyltrnsferses Dnmt1, Dnmt3 nd Dnmt3. Genes Cells 11, (26). 38. Wkym, T., Perry, A. C., Zuccotti, M., Johnson, K. R. & Yngimchi, R. Full-term development of mice from enucleted oocytes injected with cumulus cell nuclei. Nture 394, (1998). 211 Mcmilln Pulishers Limited. All rights reserved