© 2010 Nature America, Inc. All rights reserved. Biomedical Biomedical Research, Basel, should Switzerland. Correspondence be addressed to M.W. ([email protected]). France. 1 DNA methylation situation the formed in cultured cellular models that do not necessarily recapitulate genes germline as such genes developmental targeting by identity cellular maintaining in participate also may methylation reprogramming of rate the improves with methylation interfering DNA that and state pluripotent a to cells somatic gram vations that demethylation of pluripotency genes is required to repro by obser the is supported This of control pluripotency. to the linked methylation DNA moter in methylated cells is somatic islands CpG of fraction small a however, islands; unknown. remain embryogenesis early during methylation DNA dynamic undergoing sequences However, by immunostaining as visualized of DNA levels methylation, lower have cells extraembryonic whereas cells, embryonic in mostly tation ylation meth of by maintenance lack demethylated is progressively genome maternal the whereas fertilization, after shortly demethylated being genome paternal the with development, early during reprogrammed development embryonic for tial essen is and dinucleotides, CpG of context the in mostly cytosines, sequence-specific by memories cellular heritable establish regulated is that modifications epigenetic but entails it also factors, transcription development Mammalian suggesting activation De novo lineage-specific primarily implantation methylation process DNA Forné Thierry Julie Borgel during early mouse development Targets and dynamics of promoter DNA methylation Nature Ge Nature Received 8 April; accepted 14 September; published online 7 November 2010; Institute Institute of Molecular Genetics, Centre National de la Recherche Scientifique (CNRS) UMR 5535, Université Montpellier 2, Université Montpellier 1, Montpellier, Cytosine methylation is rare at regulatory regions containing CpG containing regions is rare at methylation regulatory Cytosine

methylation 2 4–6 Department Department of Molecular Genetics, Medical Institute of Kyushu Bioregulation, University, Higashi-ku, Fukuoka, Japan.

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to VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME 12,17

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© 2010 Nature America, Inc. All rights reserved. embryos, whereas group II genes are already Group I genes are with a methylated promoter in E9.5 embryos. the dynamics of DNA methylation at 691 genes the position of the CpGs. ( site is shown as a gray arrow. Red bars represent graphs as a gray box, and the transcription start shown for validation. The gene is shown below the with unamplified pooled MeDIPs at E9.5 are also in all figures, the MeDIP profiles we obtained ratios of individual oligonucleotides. Here and The graphs show smoothed MeDIP over input the presence of a germline methylation mark profiles at the imprinted gene function of the distance to the TSS. ( of tiles with a right axis). The bottom graph shows the fraction kilobase along the tiles is shown (red dotted line, For comparison, the average CpG count per function of the distance to the TSS (black arrow). fraction of tiles with a methylated region as a mouse promoters. ( NimbleGen HD2 arrays covering 11 kb of all epiblasts (EPB) and total E9.5 embryos on MeDIP samples from E3.5 blastocysts, E6.5 +400 bp relative to all TSS. ( compares average log2 ratios in −400 bp to DNA ( unamplified MeDIPs on 2 MeDIP-WGA on 150 ng of DNA and of pooled early mouse embryogenesis. ( Figure 1 arrays HD2 NimbleGen to epiblasts embryos E9.5 and blastocysts, embryos E3.5 E6.5 from from samples MeDIP hybridized We DNA low amounts of cells. from methylation DNAof profiling accurate allows protocol MeDIP DNA( of ng 150 on age log2 ratios measured with pooled MeDIPs and averwith MeDIP-WGA between correlation good a observed we genes, all comparing are also enriched with MeDIP-WGA ( MeDIPs pooled with enriched promoters gene most that showed 14 chromosome on comparison A sites. start transcription all from bp we averaged oligonucleotide log2 ratios in the region −400 bp to +400 toMeDIPs pooled ( regions that showed inaccurate profiles with MeDIP-WGA as comparedMeDIPs ( MeDIP-WGA both with similar areratios log2 very otide and pooled oligonucle that indicated profiles resulting The promoters. gene all prepared from 2 onic day (E) 9.5, as well as fromunamplified MeDIPs,20 pooled each MeDIP-WGAofDNAatng embry 150 samplesfrom embryos from hybridized we wide, genome protocol optimized our of accuracy the MeDIP on 2 enrichments with MeDIP-WGA on 150 ng of DNA and with standard To procedure, validate this we show by quantitative that (qPCR) PCR (WGA). amplification whole-genome by fractions MeDIP and input amplified We subsequently Methods). (Online DNA of ng 150 from DNA methylated immunoprecipitate to procedure MeDIP-on-ChiP Tomap DNA the we optimized methylation early embryos, mouse in An RESULTS s e l c i t r A 1094 hypermethylated in preimplantation blastocysts.

optimized

S

methylation upplementary Fig. 1

Profiling of DNA methylation during Supplementary Fig. 1b μ de novo g of DNA are similar (

MeDIP de novo μ c g g of same the DNA, on NimbleGen arrays covering ) The top graph shows the

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Average occurrence of a c

de novo MeDIP peaks lineage Average occurrence of log2 ratio (MeDIP-WGA of E3.5 →E9.5 cytosine methylation from 10,000 cells)

DNA 0.05 0.10 0.15 0.20 0.25 0.02 0.04 0.06 0.08 0.10 –2 –1 ). To assess 0 1 2 0 0 –8 –8 –2 ). ). When log2 ratio(pooledMeDIPs) r =0.81 –7 –7 –6 –6 –1 E9.5 E6.5 EPB E3.5 –5 –5 kb relativetoTSS kb relativetoTSS - - - –4 –4 0 –3 Snrpn ( DMRs germline carrying genes imprinted several at tocysts cells mammalian ( hypomethylated mostly remain HCPs epiblasts whereas E6.5 embryos, in E9.5 and hypermethylated are ICPs and LCPs of fraction but HCPs of ( ICPs hallmark or LCPs of a not is TSS the around hypomethylation that observed we Methods), Online islands; CpG strong (HCPs, moters low (LCPs), (ICPs, intermediate weak CpG islands) or high CpG pro ( regions to intergenic and as sites in (TSS) start was compared gene higher bodies scription tran low around was DNA methylation embryos, and E9.5 epiblasts of promoters low enriched ( fraction ( promoters the at content CpG and signals MeDIP between correlation no showed blastocysts E3.5 2a Fig. ( bodies Supplementary gene in ratio the of increase slight a noted we random reflect although regions, tiled along mostly distributed and are equally background ratios MeDIP hypomethylated, globally is the mouse genome ( of coverage 10% represents which 11 kb of promoters, gene ­covering ondary DMRs that are established postimplantation ( postimplantation established are that DMRs ondary in the absence of sufficient 5mC targets. 5mC sufficient of absence the in of antibody binding the nonspecific represents that this we speculate and shown), not (data regions intergenic several at blastocysts E3.5 in sequencing bisulfite by (5mC) 5-methylcytosine of presence the (see promoters gene It not we validate could regions. is noteworthy and intergenic below) rare covering enrichments MeDIP detected 3 Fig. Supplementary –3 Supplementary Fig. 2b Fig. Supplementary 1 –2 Importantly, we detected high MeDIP enrichments in E3.5 blas E3.5 in enrichments MeDIP high detected we Importantly, –2 –1 –1 VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME , , 2 Peg1 1 0 1 0 b blastocyst , , 2 2 E3.5 Peg3 Anti-5m 3 3 0 10 20 30 40 50 60

9,12,13

, , Average CpG/kb Average Peg10 C Fig. Fig. 1 E6.5 EPB Supplementary Fig. 2a Fig. Supplementary Fig. 1 Fig. Group II . Group e ), which validates our approach. We also also We approach. our validates which ), ). Consistent with global hypomethylation, hypomethylation, global with Consistent ). , and and b c ), which recapitulates previous results in in results previous recapitulates which ), ). In E3.5 blastocysts, because the genome ). because In E3.5 blastocysts, c I ) E9.5 22,25,26 Nnat Supplementary Fig. 2b Fig. Supplementary E3.5 ) but not in those carrying sec carrying those in not but ) d Supplementary Fig. 2c Supplementary 5mC (log2 ratio MeDIP/input) as promoters . classifying When –1 –1 –1 –1 E6.5 EPB 0 1 0 1 0 1 0 1 0 1 ). Consistently, a large large a Consistently, ). E9.5

2 kb Imprinted gene Nature Ge Nature Plagl1 Fig. 1 Fig. ) and a very very a and ) MeDIP/input –1 Fig. 1 Fig. log2 ratio ). ). In E6.5 E6.5 EPB 0 n MeDIPs Plagl1 pooled d etics c and and E9.5 E9.5 E3.5 and and 1 - - - - , © 2010 Nature America, Inc. All rights reserved. Circles represent CpG dinucleotides either either dinucleotides CpG represent Circles in shown are sequencing bisulfite by validations Other cells. epiblast in implantation during methylation of promoters ( digestion. the of products end representing fragments restriction mark asterisks and parenthesis, in indicated is of number the figures, all in and Here a control. as used novo de lineages extraembryonic in Oct4 of promoter The (EE). ectoderm extraembryonic novo de are genes germline-specific tested five All COBRA. by methylation DNA promoter S in given are examples More cells. epiblast novo de ( CpGs. of position the represent bars Red oligonucleotides. individual of ratios input over 4 Figure in given are genes pluripotency of examples Other cells. gene pluripotency the ( cells. epiblast in 2 Figure and of ers novo de development during methylation pro moter for targets pluripo primary are they that several confirms includes which I genes, tency Group cells. epiblast in implantation promoter ( of step embryos major E9.5 the in it gain few very epiblast and E6.5 in methylation promoter gain I group in genes Most novo De that are they bynot discriminated promoter CpG content. distribution of promoter classes ( ( development early throughout ( genes II group whereas cysts and gain promoter DNA methylation during early development, into two groups: group I genes ( DNAmethylation for targets primary are they that observation 10 ( genes these among represented 1 Table LCPs, 352 ICPs and 59 HCPs ( ated promoter in E9.5 embryos, including 280 methyl a with genes validated 691 identified ( events ylation tive stages of development, with rare demeth number of methylated promoters at consecu promoters ( gene in occurs also but regions ylation is frequent in intergenic and intragenic tion during early development. Nature Ge Nature (closed). methylated or (open) unmethylated b i. 1 Fig. upplementary Figure 5 Figure upplementary ) Examples of germline-specific genes genes germline-specific of ) Examples −16 Next we identified sites of Gdf3 , which has been shown to be methylated methylated be to shown been has , which , Wilcoxon test), which confirms our our confirms which test), Wilcoxon , Zfp42 methylated in E9.5 embryos E9.5 in methylated the and EPB the in E6.5 at methylated in implantation during methylated c . The graphs show smoothed MeDIP MeDIP smoothed show graphs . The methylated in the epiblast ( epiblast the in methylated . Cs n IP ae ihy over highly are ICPs and LCPs ). ). Indeed, there are an increasing increasing an are there Indeed, ). De novo De

are are CpG Taq n Dazl S (also known as as known (also etics α upplementary Figure 7 Figure upplementary de novo de I sites in the amplified fragment fragment amplified the in I sites

Supplementary Fig. 2c Fig. Supplementary island CpG island methylation methylation island CpG and and d a ) Bisulfite sequencing in the the in sequencing ) Bisulfite ) ) 9,13

De novo De VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME Tcl1 Sycp1 . We sorted these genes genes these We . sorted methylated in E9.5 embryos ( embryos E9.5 in methylated

. . ( de novo de methylation c in epiblast (EPB) (EPB) epiblast in 4 n ) Validation of ) Validation 8 methylation of methylation = 215) show promoter DNA methylation methylation DNA promoter show 215) = and partially partially and confirms confirms de novo Supplementary S n methylated methylated Rex1 De novo upplementary upplementary = 476) are unmethylated in E3.5 blasto e novo de Fig. 1 Fig. Table 3 . P 8 ), ), methyla

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marks Fig. 2 Fig. Dppa3 e meth 1 methylation occurs during during occurs methylation ). Both groups show a similar similar a show groups Both ).

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Group I embryos T Group II n calculated usingtheDAVID tool. table showsthepreferentialtissuesofexpressionandenrichedontologytermswithassociatedFisherexact in groupIandIIwithallgenespresentonthearray. ForgroupI,onlyICPandHCPgeneswereconsidered.The low orintermediateCpGcontent.To revealfunctionalannotations,theDAVID tool II ( The tableshowspromoterclassesandfunctionalannotationsassociatedwithgenesingroupI( epiblast =476 a c able able 1 e n ), indicating that that indicating ), χ Papolb 5mC (log2 ratio MeDIP/input) Spo11 Taq Sycp1 =215).BothgroupsshowedasimilardistributionofpromoterCpGcontentandareenrichedforpromoterswith Tex12 –1 –1 –1 –1 Tcl1 2 Asz1 Oct4 Supplementary Supplementary 0 1 0 1 0 1 0 1 test), indicating (3) (8) (4) (4) (6) (2) α 1 n

promoter is is promoter =215 Functional Functional annotation of genes with promoters hypermethylated in early mouse E3.5

– – stage + E6.5 EPB Stella Tcl1

+ LCP 196 E6.5 Promoter classes 84 – – EE - - - - )

+ + 0 E6.5 EPB cells ( cells dif restrict to cells embryonic proposed of been ferentiation has methylation promoter whose Elf5 ( analysis) tion Zfp42 Fig. Examples of genes specific to oocytes are are oocytes to specific genes of Examples ( components complex emal Spo11 Testisin ( germline activating female and male by the both in expressed genes for enrichment strong bypassed be a revealed that can analysis an ontology WeICPs and HCPs). performed and constitutive ­signals is LCPs at E9.5 MeDIPs pooled 244 108 ICP eas icesn eiec sget ht DNA that suggest evidence increasing Because 1 E9.5 E9.5 E3.5 , which encodes a trophoblast-specific transcription factor factor transcription trophoblast-specific a encodes which , * * * * * * * * * * * * *

4a 2 kb and Supplementary Fig. 4d Fig. Supplementary , and and , 9,12,28,29 , ), ), b HCP b 36 23 ). We validated the timing of of timing the validated We ). Blastocyst d 5mC (log2 ratio MeDIP/input) Asz1 Dppa3 Table Table –1 –1 –1 –1 Epiblast Sycp1 0 1 0 1 0 1 0 1 expression E9.5 E6.5 E3.5 Tissue of , we focused our analysis on CpG islands (present in in (present islands CpG on analysis our focused we , , , Testis Testis Supplementary Fig. 4c Fig. Supplementary Mei1 Egg 1 promoters by COBRA (combined bisulfite restric bisulfite (combined by COBRA promoters , , Sycp3 ). Examples include include Examples ). , ,

Papolb Dazl Dazl , , 4.0 ×10 5.6 ×10 2.0 ×10 Syce1 P (also known as as known (also Fig. 2 Fig. ). E6.5 EPB and and 2 MeDIPs –6 –5 –2 0 7 pooled , is is , E9.5 E9.5 E3.5 1 Gamete generation Defense response Sexual reproduction Enriched ontologyterms Sexual reproduction Gamete generation Tex12 b de novo de 2 kb and and Dazl 4 de novo de 9 ). We also confirmed that that confirmed also We ).

5mC (log2 ratio MeDIP/input) wasusedbycomparinggenes , which encode synapton encode which , –1 –1 –1 –1 Supplementary Fig. Supplementary 0 0 0 0 1 1 1 1 , , methylated in epiblast epiblast in methylated Nlrp5 Prss21 Tpap methylation at the the at methylation Sycp1 s e l c i t r A n =476)andgroup (also known as as known (also (also known as as known (also ), ), Prss21

Tcam1 methylation methylation 1.5 ×10 1.3 ×10 1.3 ×10 4.9 ×10 6.0 ×10 P E6.5 EPB values MeDIPs , , P pooled Brdt 1095 E9.5 E9.5 E3.5

5 –3 –3 –3 –3 –4 ). ). - - - , © 2010 Nature America, Inc. All rights reserved. methylation is specifically erased during during erased specifically is methylation gene B-cell–specific the For liver. as such tissues other in maintained is but T cells and B cells HSCs, marrow bone Cytip (Ad), adults in Subsequently embryos. E10.5 from isolated (HSCs) cells stem hematopoietic in methylation DNA promoter substantial show also genes All embryos. E9.5 in and EE the EPB, the in E6.5 at hypermethylated are and implantation during methylation DNA promoter gain genes hematopoietic tested ( CpGs. of position the represent bars Red oligonucleotides. individual of ratios input over MeDIP smoothed show graphs The cells. EPB Cytip genes hematopoietic ( differentiation. hematopoietic during erased is genes hematopoietic at 3 Figure novo de response’; ‘defense term ogy (ontol genes hematopoietic of promoters ( eye during way a in cell-type–specific is erased ylation meth promoter that showed and lens and retina the in methylation DNA measured we differentiation, somatic during reversible is tion methyla DNA promoter that possibility the To test eye. the in sion expres subsequent with incompatible and maintained stably be to believed is methylation cytosine of density elevated because pected in as well as ( ectoderm epiblast E6.5 extraembryonic in methylation DNA of gain confirms genes eye-specific the of ( promoter the at COBRA embryos E9.5 in starts gene alternative protocadherin the (at by loci provided are examples Striking promoters. alternative at methylation DNA gain or clusters family gene of bers and genes, ( eye and brain in expressed genes tis ICP include with promoters. These differentiated are associated which sues, mostly to specific genes contains also I group Notably, Reversible genes. germline signature methylation vivo in promoter distinct a acquires epiblast tion postimplanta the that We conclude implantation. after or during that indicating shown), not (data 7 Fig. ( epiblast E6.5 in occurred already has ( genes germline Sycp1 four on sequencing at ectoderm ( E6.5 extraembryonic the in methylation DNA substantial ( E6.5 in hypermethylated are but cysts blasto E3.5 in unmethylated are promoters CpG-island these that Fig. Nalp5 s e l c i t r A 1096 B cells. into HSCs adult of differentiation b Supplementary Fig. 8b Fig. Supplementary Fig. 2 Fig. ) Validation by COBRA confirms that all three three all that confirms COBRA by ) Validation Notably, we also observed that several several that observed also we Notably,

and and in methylation DNA promoter gain 5

), which confirmed that that confirmed which ), Fig. 2 Fig. ). These genes are mostly unmethylated in E4.5 blastocysts blastocysts E4.5 in unmethylated mostly are genes These ). ). Validations by COBRA and HpaII digestion confirmed confirmed digestion HpaII and COBRA by Validations ). and and c , in particular with the methylation of a large number of of number large a of methylation the with particular in ,

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includes includes imprinted genes with a DMR germline near the TSS ( ( embryos implantation pre in present already methylation promoter show genes II Group Inheritance moderate with CpG promoters at demethylation of examples which provides differentiation, terminal during reversed be can epiblast in cells B into cells stem ( hematopoietic adult of differentiation ing gene whereas lineage, B-cell–specific the hematopoietic the embryos in erased E10.5 from subsequently is and isolated progenitors hematopoietic in dant cells. T and cells B progenitors, hematopoietic in methylation promoter measured we differentiation, hematopoietic during demethylated are genes these ( ectoderm extraembryonic and epiblast E6.5 in of pr109a Tlr1 Pscdbp development, B-cell in involved coactivator including HpaII digestion confirmed partial promoter methylation of methylation promoter partial confirmed digestion HpaII ( blastocysts E3.5 in alleles unmethylated and methylated of mixture a show but embryos E9.5 and ectoderm extraembryonic E6.5 epiblast, E6.5 in methylated are promoters tested all that cated ( hemat in expressed 10a Fig. Supplementary Spaca4 as germline male the in ( expressed genes in revealed enriched analysis is II group ontology that The imprinted. as reported not genes and Fig. Table Table Fig. Miwi Pou2af1 Supplementary Fig. 3 Fig. Supplementary and and

VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME

richness during somatic development. somatic during richness 4 3 E6.5 EPB E6.5 EPB a b , gn epesd n ekcts te toll-like the leukocytes, in expressed gene a ), , , MeDIPs ) ( ) 1 MeDIPs and poole ), ), pooled ). We conclude that promoter DNA methylation acquired acquired methylation DNA promoter that Weconclude ). ). Selected ICP and HCP examples are are examples HCP and ICP Selected ). Tuba3a E9.5 E3.5 E9.5 Fig. 3 Fig. E9.5 E3.5 E9.5 Pou2af1 Tlr6 Csnka2ip , , d

Supplementary Fig. 10b Fig. Supplementary Cytip of Pou2af1 , , Taq

Cytip a promoter Cxcl9 (also known as as known (also b Tlr6

5mC (log2 ratio MeDIP/input) promoters the of COBRA shown). not data and (4) (2) (2) α –1 –1 –1 –1 and and (also known as as known (also 1 0 1 0 1 0 1 0 1 (also known as as known (also Tlr6 o E3.5 – oei cls ( cells poietic , , + + Gzmk Tlr6 and and Fig. 1 Fig. Pou2af1 E6.5 EPB – ). Group II also contains somatic genes genes somatic contains also II Group ).

); however, we also identified many other other many identified we also however, ); methylation confirmed gain of DNA methylation methylation DNA of gain confirmed Cytip , , Tlr6 e E6.5 – EE Abcg3 ). As could be expected, this group group this expected, be could As ). + + is specifically demethylated dur demethylated specifically is Obf1 Tuba3 promoter methylation is abun is methylation promoter E9.5 – Cd4 Ckt2 ). ). Validations by indi COBRA and and ), encoding a transcriptional a transcriptional encoding ),

and and from ) and and ) E10. HSCs – ), ), 9130005N14Rik Niacr1 + + Dpep3 5

HSCs Cytip Fyb – parental Ad

Gykl1 Piwil1 Nature Ge Nature Fig. 3 Fig. o rtn ( retina or ) (also known as as known (also B cells , , (also known as as known (also – Ad Hdhd1a ( + + E6.5 EPB 0 (also known known (also MeDIPs b i. 4 Fig.

T cells pooled gametes – ). To test if if To test ).

1 Ad E9.5 E3.5 E9.5 receptors receptors Fig. 4 Fig. 2 kb Tuba3a , , n Fig. – Tssk2 a liver etics Ad and and Rrh +

b 1 * * * * * * * ). ). d - - - - ) ,

© 2010 Nature America, Inc. All rights reserved. ylation reprogramming during preimplantation development. preimplantation during reprogramming ylation that nonimprinted This sequences resist DNAidentifies global meth ( methylation DNA retained cysts showed that predominantly maternal maternal predominantly that blasto showed E3.5 cysts JF1 × allele BL6 in parental polymorphisms one this, on of support In perhaps only. fertilization, after methylation of and ( embryos in preimplantation but partially only gametes both maternal are ( blastocysts E3.5 JF1 × BL6 in alleles methylated that in found were polymorphisms (no the in mice JF1 and BL6 used we between polymorphisms hypothesis, this test To implantation. during are methylated alleles paternal whereas oocytes from methylation inherit they E9.5 and epiblast in ( methylated embryos fully are and blastocysts and las moru in methylated partially are spermatozoa, in not but oocytes embryos. Dpep3 of promoters the in sequencing bisulfite we performed Next, gametes. from ( alleles methylated inherit II group in seven) morulas genes most that of E2.5 out in (seven alleles genes methylated tested showed All morulas. E2.5 on COBRA performed we gametes, from methylation DNA inherit potentially are genes these and (closed). or (open) methylated unmethylated either CpG dinucleotides ( ( alleles. pat, paternal alleles; Mat, maternal carry DNA alleles methylation. of promoter ( and alleles. E2.5 show E3.5 E6.5 of morulas and a blastocysts pattern embryos. unmethylated mixed methylated consistent Red the of bars position represent CpGs. ( MeDIP over smoothed input oligonucleotides. of ratios individual are given in More examples early development. throughout methylation ( genes ( hematopoietic ( genes. from at oocytes nonimprinted 4 Figure Nature Ge Nature f Piwil1 Fig. c a ) Bisulfite sequencing in ) the sequencing Bisulfite 5mC (log2 ratio MeDIP/input) –1 –1 –1 –1 spermatozoa Supplementary Fig. 11c Supplementary Dpep3 0 1 0 1 0 1 0 1

4 and , , Piwil1 1 0 d Oocyte

Csnka2ip ). In contrast, contrast, In ). Inheritance of DNA promoter methylation Inheritance Piwil1 Fig. 4 Fig. Csnka2ip Piwil1 S n in E3.5 blastocysts ( blastocysts E3.5 in upplementary Figure 10 Figure upplementary 2 kb etics de novo de , , c in and gametes ( early embryos. , , and and Dpep3

Tssk2 morula ) ) with high of levels DNA promoter E2.5 Fyb VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME Tssk2 Fyb Supplementary Fig. 11a Fig. Supplementary ) and germline-specific genes ) and germline-specific , , and and methylated in blastocysts or whether they they whether or blastocysts in methylated Spaca4 Tssk2 , , E6.5 EPB – Spaca4 Csnka2ip Fig. 4 Fig. MeDIPs blastocyst e pooled ), suggesting ), incomplete suggesting maintenance E3.5 E9.5 E9.5 E3.5 , , promoter in promoter BL6 × represent Circles shows JF1 that carry DNA alleles only E3.5 maternal methylation. blastocysts Supplementary Fig. 6 Fig. Supplementary Rrh . . The show graphs a ) Examples ) of Examples f Dpep3 , , and and and and Rrh b promoters are methylated in in methylated are promoters ) ) Validation in by of EPB DNA All COBRA. promoter and methylation genes tested show EE hypermethylation in 5mC (log2 ratio MeDIP/input) –1 –1 –1 –1 and and Supplementary Fig. 11c Fig. Supplementary 0 1 0 1 0 1 0 1 or or epiblast Cd4 E6.5 Tssk2 Csnka2ip d Cd4

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ing that absence of DNA methylation is not sufficient to promote promote to sufficient not is methylation DNA of absence that ing in promoter hypomethylation genes hematopoietic ( repression gene stable ( gene a 13–50-fold reactivation at most germline genes and one pluripotency embryos, hypomethylation in all genes and were Although in wildtype barely detectable embryos. mutant E9.5 and by RT-qPCR wildtype genes in target eral sev of expression the measured we methylation, DNA promoter of with Dnmt3b to methylate these targets. To further assess the function and and Notably, DNA methylation at genes certain such as promoter for required ( embryos E9.5 in reduced markedly also is genes eye and genes hematopoietic genes, pluripotency at methylation DNA ( Dnmt3a of not but Dnmt3b line germ that showed sequencing bisulfite and COBRA embryos. E9.5 in DNA methylation measured we ment, which enzyme is responsible for specificities target and patterns expression ferent The De novo 5mC (log2 ratio MeDIP/input) –1 –1 –1 –1 Piwil1 0 1 0 1 0 1 0 1 - Crygd Supplementary Fig. 12c Fig. Supplementary specific genes are severely hypomethylated in the absence of of absence the in hypomethylated severely are genes specific de novo de morula d Zfp42 E2.5 Ma Pat promoter in promoter BL6 × shows JF1 that only E3.5 maternal blastocysts

t DNA is only partially reduced in reduced is only partially E3.5 blastocyst ), showing that somatic DNA methylation is required for is required DNA somatic that methylation ), showing BL6 xJF1 methyltransferases Dnmt3a and Dnmt3b display dif display Dnmt3b and Dnmt3a methyltransferases Csnka2ip Piwil1

Fig. 5 Fig. methylation blastocyst E3.5 b Pou2af1 ). This shows that Dnmt3b is the main enzyme ). shows is that This Dnmt3b main the enzyme E6.5 EPB e novo de Fig. 5 Fig. MeDIPs pooled E9.5 E9.5 E3.5

by , Dnmt3b and and d Dnmt3b Fig. 5 Fig. epiblast c

), suggesting that Dnmt3a cooperates cooperates Dnmt3a that suggesting ), E6.5 Dnmt3B ). Other tested genes, including the the including genes, tested Other ). de novo Csnka2ip methylation during implantation. implantation. during methylation Hdhd1a Spaca4 Dpep3 Taq b Tlr6 Tssk2 Piwil1 Tssk2 Cd4 a Fyb Rrh α (1) (3) (2) (3) (4) (2) (2) (3) (1) −/− and and Dnmt3b Dnmt3b 1 −/− , were not reactivated despite despite reactivated not were , methylation in early develop in and gametes early embryos. embryos was associated with

– maintains E2.5 Mor. embryos ( embryos Dnmt3a c ) Bisulfite sequencing in ) the sequencing Bisulfite Supplementary Fig. 12 Fig. Supplementary + E9.5 Blast. E3.5 −/− −/− – + embryos ( embryos but not 30,31 −/− s e l c i t r A Brdt

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© 2010 Nature America, Inc. All rights reserved. (71 out of 675) methylated in embryonic stem cells are never meth never are cells stem embryonic in methylated 675) of out (71 Finally, cells. stem ating a groupembryonic minor of promotersgene in this study identified could not in be studies of identified differenti many that highlights also comparison ( embryos E9.5 in methylated also cells are also methylated in E6.5 epiblasts and 85% (572 out of 675) are (463 out of 675) of the promoters hypermethylated in embryonic stem as genes the hematopoietic genes germline-specific the is, the ones that also gain methylation in postimplantation embryos (that and development (including early throughout DNA of methylation levels high show that II group blastocysts ( E3.5 in enriched also were 675) outof (255 38% only cells, stem onic embryonic stem cells. Out of 675 gene promoters methylated in embry Dppa3 ( genes pluripotency several include embryos that gain promoter methylation in both neuronal progenitors and total methylation specific to cells committed to the neuronal lineage. Targets ated in ( total embryos most promoters methylated in neuronal progenitors are hypomethyl embryonic stem cells differentiating into neurons methylation in early embryos compares with promoter methylation in related most are they population what to unclear is it however, blastocysts; plantation preim of mass cell inner the from derived are cells stem Embryonic early Comparison embryos developing in silencing gene maintains and Dnmt3b by mediated primarily is methylation DNA this developmental stage. Altogether, this demonstrates that promoter the absence of suitable transcription factors at reflects probably which transcription, gene experiments. independent three or two from deviations standard represent ( genes housekeeping three to normalization after units arbitrary are Values embryos. E9.5 (WT), wildtype in qPCR real-time by measured was genes indicated of Expression reactivation. gene with promoter ( (right). genes eye and (middle) genes hematopoietic (left), genes pluripotency and wildtype ( in shown are sequencing bisulfite by validations Additional in unaffected were but in methylation DNA promoter of reduction severe showed genes for homozygous or heterozygous embryos E9.5 mutant and (WT) wildtype in COBRA by analyzed was genes germline-specific indicated the of vivo repression gene maintains Dnmt3b by 5 Figure s e l c i t r A 1098 ( embryos in ylated b ) Promoter DNA methylation by COBRA in in COBRA by methylation DNA Promoter ) Gpr109a . ( . Cd4

a embryos ). Next, we focused on gene promoters hypermethylated in in hypermethylated promoters gene on focused we Next, ). ) DNA methylation in the promoter promoter the in methylation DNA ) Gapdh

). Many genes hypermethylated in embryonic stem cells are cells stem embryonic in hypermethylated genes Many ). Promoter DNA methylation mediated mediated methylation DNA Promoter de novo de ) and the eye genes genes eye the and ) Supplementary Fig. 13b S Dnmt3 , ,

upplementary Figure 12 Figure upplementary of Rpl13A Dnmt3a Dnmt3

methylation is associated associated is methylation DNA mutant E9.5 embryos at at embryos E9.5 mutant Dnmt3b Supplementary Fig. 13b Fig. Supplementary and and deletions. Most tested tested Most deletions. −/− Dnmt3a Supplementary Fig. 13a Supplementary

methylation Dpep3 and and Pou2af1 Actb −/− Dnmt3b embryos embryos −/− , c ). Error bars bars Error ). Cplx4 Dazl Piwil1 ) Absence of of Absence ) embryos. embryos. , , Cytip 3 2 Supplementary Fig. 13b Fig. Supplementary , , . Thus, we asked how promoter promoter how asked we Thus, . ,

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embryonic de novo de Tlr1 Cryaa , , and Sycp1 , . Tssk2 ), which might reflect reflect might which ), Tcl1 ), which reflects DNA ), reflects which ). Consequently, ). 69% 1 methylation targets targets methylation 3 Niacr1 b a . First, we show that , , A P , , Dpep3 S Spo1 , Spo11 Ta

dam2 apolb Te Gdf3 ycp1 Tuba3a stem Dazl Dppa3 q Ta x1 Zfp42 in vivo in (2) (1) (4) (8) (4) (4) (6) α Oct4 q 2 1 1 (3) (1) (3) α (also known (also 1 , , and and + –

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+ – 3a + – –/ WT is reduced in extraembryonic as compared to embryonic lineages embryonic to compared as extraembryonic in reduced is potential. development their impact not does it why explain might which genes, pluripotency target not does cells stem embryonic in DNA methylation to EpiSCs, contrast in that note also an in back placed are cells when reversed cells stem embryonic between geneity blastocysts mature and with naïve share properties epiblast from cells similar blastocysts into reintroduced when layers germ all to contribute to able cells still are stem embryonic because question into put be can differences methylation H3K27 and H3K4 in data showing that derivation of embryonic stem cells induces changes the from blastocysts which they are derived. This is in line with recent methylation signature that promoter resembles thata of postimplantationadopt embryos rather than cells stem embryonic that show we epiblast in not but EpiSCs in methylated are that genes pluripotency the by exemplified as culture, and derivation during methylation DNA abnormal by explained be also could EpiSCs of potency reduced The cells. epiblast of potency the limits that boundary chimeras an epigenetic constitutes DNA methylation to contribute rarely (EpiSCs) cells stem epiblast postimplantation that fact the by highlighted potency, fluorescence cells, with epiblast coincides in which implantation during occurs methylation gene of step major the that reveal to protocol MeDIP optimized Wean used DISCUSSION that embryos. of resembles postimplantation embryonic stem cells bear a promoter DNA methylation signature that implantation after and during occurs ture, embryonic stem cells accumulate promoter DNA methylation that cul and derivation during that indicates Altogether, this differences. strain or conditions culture by induced methylation DNA abnormal + Immunostaining experiments have shown that DNA methylation DNA methylation that shown have experiments Immunostaining + – 3a * * * * * * VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME –/ – + – WT P ou2af1 Ta C 7 q Tlr6 ytip . Notably, this correlates with a reduction in cellular cellular in reduction a with correlates this Notably, . (4) (2) (2) α * * * * * * * * * * * * * * * 1 c + – 3b Relative RNA quantity (AU) 3 0.5 1.0 1.5 2.0 2.5 3.0 –/ 2 . It is possible that this reflects a strong hetero strong a reflects this that possible is It . – 0 de novo de + – 3a Brdt –/ –

Dazl + – WT methylation observed by immuno by observed methylation

Sycp1 3 * * * * * * * 6 . The impact of these epigenetic epigenetic these of impact The . in vivo

3 Tex12 7 or that DNA methylation is is DNAmethylation that or Taq Crygd Cryaa Cplx4 Tuba3a in vivo in . Consequently, cultured Consequently,. cultured (3) (3) (1) α 1 33,34

Dppa3 3b + – in vivo in Nature Ge Nature environment. We environment. –/ Dppa3 , suggesting that that suggesting , – Zfp42 + – 3a WT Dnmt3a Dnmt3 3 –/– 5

and and Pou2af1 . Similarly, Similarly, . b + – –/ n –/ WT – – Zfp42 etics Tlr6 * * * * * * * * 7 - - ­ ,

© 2010 Nature America, Inc. All rights reserved. are necessary to clarify this issue. this clarify to necessary are stages developmental and targets other at studies further however, in genes hematopoietic of reactivation demonstrate to able been not have we far, So development. during activation gene to early leads of DNA methylation absence that dicts to in cells H3K27 similar pluripotent polycomb-mediated function a genes, differentiation key of expression early the restrains DNA that reversible methylation it In suggests contrast, development is associated with increased promoter DNA methylation. contradicts the model whereby loss of differentiation potential during and are what gene activation mechanisms involved. This observation ness, and studies further are required to test if this is rich a consequence of CpG higher with promoters at demethylation report we Here genes hormone-response the for recently shown as activation, gene of consequence a possibly is vivo in described been have demethylation promoter of examples few very methylation promoter acquired in epiblast is Toduring differentiation. erased terminal Second, date, promoters. duplicated regulate to mechanism a as evolved have might DNA methylation that suggests the (the clusters family of gene are members targets several First, observations. unexpected two made we targets, these studying By and lineages. in neuronal the hematopoietic opment, in particular devel during later expressed be to programmed genes are embryos integrity cellular somatic impacts negatively reactivation ectopic their that suggests these which cancer, in of overexpressed many are genes Second, silent. already are they when implantation during support methylation DNA acquire arguments genes Two germline First, model. cells. this somatic in reactivation ectopic by caused effects deleterious prevent to system locking a as acts rather but silencing gene initiate not does methylation this that speculate genes germline to methylation DNA recruit specifically that mechanisms of existence the suggests strongly This loci. imprinted in DMRs germline at except genome, the in associated hypomethylation otherwise with is that feature a bp), 100 per CpGs four than (more promoters their in CpGs of concentrations high show genes silencing gene maintain to required is and organogenesis before implantation during occurs methylation that showing by observations these extend and targets methylation in differentiated cells island CpG of targets as genes germline-specific identified have ies embryos developing in program expression germline the repress to is methylation DNA of function important Nanog expression spatiotemporal pathways. different of control the under are they that indicate might which patterns, methylation DNA global follow stem cells trophoblast stem and embryonic cultured between promoter methylation similar Cryaa at ( genes some ectoderm in tion extraembryonic the methyla reduced a slightly note we although at E6.5, ectoderm onic gain promoter DNA in equally epiblast and methylation extraembry evident at the level of promoters. Our data show that most genes tested compared to proper the embryo hypomethylatedis placenta the persistentbecause is thatdifference a Nature Ge Nature for Our results show that Dnmt3b is the main enzyme responsible responsible enzyme main the is Dnmt3b that show results Our early in methylation promoter of targets other Surprisingly, Few genes have been shown to require DNA methylation for correct de novo de Cxcl and (ref. , mostly at very CpG-poor promoters CpG-poor very at mostly , cluster, cluster, Cd4 n 38), as 38), well as the methylation at tested genes in early embryos. This is is This embryos. early in genes tested at methylation etics ). ). This is in line with a previous study’s results showing protocadherin

VOLUME 42 | NUMBER 12 | DECEMBER 2010 DECEMBER | 12 NUMBER | 42 VOLUME 1 1 . Therefore, CpG island methylation does . not Therefore, does CpG methylation island in vivo in in vivo in Rhox TFF1 loci and the the and loci 2 . It is remarkable that some of these these of some that remarkable is It . 3 9,10,39,40 . . Interestingly, is not this difference . Rare examples include include examples Rare . genes (ref. (ref. 1 . . Here we identify many new 29) and and 29) Dnmt3b 6 . Our data reveals that . one data Our reveals in vivo in 4 Tlr1–6 2 . This demethylation demethylation This . 43,44 CYP27B1 −/− . This model . preThis model . Previous stud Previous . cluster), which which cluster), E9.5 embryos; embryos; E9.5 Pou2af1 Cryg 4 1 . (ref. (ref. Oct4 cluster, , , Cplx4 4 0

. We . and and 28). 28). ------,

fraction of the genome. It will be important to test whether similar similar whether test to important be will It genome. the of fraction is transgenerational transmission of DNA methylation at a substantial demethylated transiently at a not specific developmentalare stage, this genes suggests that therethese that condition the At implantation. after methylation allele-specific maintains genes these of none and gametes, both in methylated are some because genes imprintedfrom genes requiredfromdiffer These spermatozoa. to testoccurs itif also however, from oocytes; methylationoccurs validations are additional far, all tested genes indicate that inheritance post-fertilization of DNASo gametes. parental from methylation DNA promoter inherit and fertilization after demethylation global resist genes, somatic also but targets. their to enzymes Dnmt3 recruit that mechanisms the understanding on concentrated ferent specificities for Dnmt3 enzymes. In the future, efforts should be embryos action of Dnmt3a and Dnmt3b at synergistic a showing studies with line in is This targets. these at ate absence of Dnmt3b or Dnmt3a, suggesting that both enzymes cooper and embryos late preva in more lent is Dnmt3a whereas epiblast, and embryos plantation preim in detected is Dnmt3b that showing studies with ­compatible supervised the study,supervised participated in data analysis and wrote the manuscript. participated in the study design and writing of the manuscript. M.W. anddesigned Y.L., H.C. and H.S. prepared samples from of the manuscript. S.G. developed R scripts and participated in data analysis. J.B. performed all experiments and data analysis and contributed to the writing (LRI-EMSG49-CNRS-08). Initiative Research Long (CEFIC) Council Industry Chemical European and 4868) contract (ARC Cancer le sur Recherche la pour Association 02), (ANR-07-BLAN-0052- Recherche la de Nationale Agence (CNRS), Scientifique Recherche la de National Centre Foundation, Research Novartis 2006-037415), by Epigenome NoE the (LSHG-CT- supported was research This dissection. on embryo for advice Hirasawa Posfai and E. and R. R programming, with T. cytometry, We flow the for with help Vicente R. Gostan for assistance thank Note: Supplementary information is available on the GSE22831. code accession under database codes. Accession online the at http://www.nature.com/naturegenetics/. paper the of in version available are references associated any and Methods M software, Annotation, Genome DAVID functional annotation tool, URLs. methylation that is linked to environmental factors mammals, our results justify the search for transmission of altered DNA mission of DNA methylation can atoccur nonimprinted sequences in of development. In addition, by showing that transgenerational trans epigenetic information that might be important to guide the early steps embryo the to gametes the from transmitted are modifications histone that showing work recent with Together reprogramming. epigenetic to resistance confer elements sequence specific whether and genes, line mechanisms maintain DNA methylation at imprinted genes and germ AUTH A c et Finally, we reveal that certain genes, in particular germline genes genes germline particular in genes, certain that reveal we Finally, kno Crygd h O R software for statistical computing, ods wl R R C 20,21 16,38 http://www.ur ) only show minor or partial reduction of methylation in in methylation of reduction partial or minor show only ) e ON , this indicates that fertilization involves the transmission of dgm . This indicates that different genomic regions show regions genomic dif that . different indicates This TRIBUTI en Microarray data are accessible from the GEO GEO the from accessible are data Microarray ts 31,45 ON h ogene.org/methprimer ttp://www.genome.ucs . However, rare genes ( genes rare However, . S Oct4 http://david Dnmt , , Nanog mutant embryos. D.S. and T.F. N http://www.r-proje a t .abcc.ncifcrf.gov u and r /index1.htm e c.edu

G Brdt e s e l c i t r A 4 Rhox n 6 e . t i / c , , ; MethPrimer MethPrimer ; s website. Dpep3 in E8.5–E9.5 l .

/ ; UCSC ct.org , , Cytip 1099 / ------;

© 2010 Nature America, Inc. All rights reserved. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturegenetics/. The authors declare no competing interests.financial s e l c i t r A 1100 C O

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© 2010 Nature America, Inc. All rights reserved. log2 ratios to have identical normal distributions using the Limma package Limma the using distributions normal haveto ratios identical log2 To compare microarray results from different samples, we average normalized r = 0.48 with E3.5 blastocysts, r = 0.78 and r = 0.79 with pooled MeDIPs at E9.5). (r = 0.77 andreproducibility r = good 0.67 showed with E9.5which embryos, stage, r =embryonic 0.60 same and the r = of 0.58 replicates with E6.5 epiblasts, as a total signal reference. We log2 ratios averaged from normalized biological package Limma from the method loess the using normalized were datasets All NimbleGen. Roche provided by values MeDIP/Input fluorescence raw from calculated oligonucleotide we each for First, ratios log2 URLs). (see software computing R the analysis. Microarray arrays. Promoter RefSeq 385K NimbleGen DNA Roche to 2 from generated MeDIPs (each unamplified at ( files E9.5 to standard procedure by Roche NimbleGen. Control unamplified MeDIP pro start sites. Sample and labeling were hybridization microarray done according in regions covering −8,200 bp to +3000 bp from 23,517 potential transcription Deluxe Promoter arrays, which contain 50–75 monomer oligonucleotides tiled blastocysts of number high the required. of Amplified samples were hybridized to Roche NimbleGen HD2 2.1M because duplicates in done was E3.5 at manufacturer’s the MeDIP whereas MeDIP at and was done E6.5 E9.5 in triplicates, instructions. following (Sigma-Aldrich), kit Amplification Genome Whole the with product MeDIP entire the DNA input and ng 5 amplified we Subsequently, methylation. global reduced the for account to 1/30 and 1/10 1 used we blastocysts, and epiblasts buffer. For E6.5 DNA, 2 of 1 amounts used and low DNA of ng on 150 MeDIP sonicated we For (Invitrogen). beads magnetic IgG 30 and mg/ml) 1 33D3, clone Serotec, (AbD described 2 on MeDIP unamplified as performed was MeDIP Bioruptor. MeDIP-on-ChiP. Zp3 and one mRNA in the regions 200 bp upstream or downstream of the poten the of downstream or upstream bp 200 regions the in mRNA one and UCSC Genome annotations to identify promoters with at least one RefSeq gene The promoterNimbleGen). onwith (Roche array software set the filtered was SignalMap the with visualized files GFF created and package Ringo the using For data representation, we smoothed average log2 ratios over 400-bp windows delete crossing by enzymes Weproduced respectively. CD4 as nodes lymph adult from Lin– cells by flow cytometry. T cells and c-Kit B cells were isolated isolated by we flow cytometry then and beads, magnetic IgG anti-rat by followed CD4) and GR-1 Mac-1, B220, (Ter119, markers lineage by cells against negative with (Lin–) rat directed incubation mouse antibodies lineage We isolated tibias. first and femurs from cells marrow bone collected To BD Biosciences). we adult HSCs, isolate (FacsAria, by cells cytometry flow aorta-gonad- pooled we embryos, mesonephros (AGMs) from dissected ten and embryos isolated CD34 E10.5 from (HSCs) cells stem opoietic hemat To isolate shown). not (data hypomethylated appeared which DMR, the in sequencing bisulfite performing by eliminated was oocytes the in contamination of somatic presence The mm in diameter. 60–80 were oocytes of majority The females. 5-week-old of ovaries dissected from medium M2 in collected were oocytes Grown medium. M2 with oviducts the flushing by at E2.5 stage cell 16–32 at the collected were Morulas blastocysts. pooled 300 on performed then was MeDIP Each medium. M2 with uteri the flushing by performed on DNA from 30 pooled epiblasts. Blastocysts were collected at E3.5 using proper dissection for controlled and ectoderm extraembryonic the from epiblast the separated we At E6.5, (Sigma-Aldrich). medium M2 in E9.5 and E6.5 at dissected were C57BL/6 mice. The morning of the vaginal plug was designated E0.5. Embryos Isolation of cells and embryos. ONLINE doi:10.1038/ng.708 promoters. validated 18,577 To identified which TSS, tial promoter determine μ l M280 magnetic beads in a final volume of 150 150 of volume final a in beads magnetic M280 l -Cre females with with females -Cre Dnmt3a

MET Supplementary Fig. 1 Supplementary in growing oocytes, with H Sonication of DNA was performed with a Diagenode Diagenode a with performed was DNA of Sonication ODS Data processing and calculations were performed with with performed were calculations and processing Data Dnmt3a Dnmt3b Oct4 5 0 μ , which was modified to use input signal instead of instead input to use signal modified was which , Dnmt3 g sonicated DNA, we used 2 2 used we DNA, sonicated g DNAas a methylation marker. MeDIP Each was 2lox/2lox +/− Embryos were obtained by natural breeding of + males /CD8 mutant embryos without oocyte-derived oocyte-derived without embryos mutant ) were generated by hybridizing 20 pooled 20 ) pooled by were generated hybridizing ; ; Zp3 Dnmt3a + 4 /CD3 5 . -Cre females, which conditionally conditionally which females, -Cre μ l 5mC antibody diluted 1/5 and and 1/5 diluted antibody 5mC l μ + μ g of DNA) together with input g with of DNA) together cells and B220 and cells +/− l of M280 sheep anti-mouse anti-mouse sheep M280 of l + males, and /Sca-1 2 4 μ , with adaptations. For For adaptations. with , μ l 5mC antibody diluted diluted antibody l 5mC l immunoprecipitation immunoprecipitation l + μ /B220 l of 5mC antibody antibody 5mC of l Dnmt3b + /CD19 − HSCs from from HSCs + 2lox/2lox /c-Kit + cells, cells, H19 5 0 + - - - ; .

was performed with the DAVID functional annotation tool annotation DAVIDfunctional the with performed was analysis ontology The embryos. E9.5 and E3.5 in >0.3 ratio log2 average an <0.3 in E3.5 and an average log2 ratio >0.3 in E9.5 embryos; group II genes had ratio anaverageGroup had log2 package. genes gplots I the using created was located within in the methylation E9.5 identified The embryos. peak Heatmap atTo E9.5. generate the X chromosome and genes showing inconsistent profiles with pooled MeDIPs (411 ICPs and HCPs) after removal of duplicates, receptors, olfactory genes on promoters validated 691 identified this upstream Inof TSS. embryos, the E9.5 overlapping bp 300 MeDIP promoters than a or less peak ylated with genes as smoothed log2 ratio >0.5 using the Ringo package. Wea with oligonucleotides defined consecutive genessix than more with with methregions identified we tion of the distance to the using TSS the Ringo package. To MeDIPfind peaks, func a as blastocysts) E3.5 in >0.25 ratio MeDIP smoothed a with oligos tive with a MeDIPsmoothed ratio >0.5 in E9.5 and embryos less than six consecu ratio and >0.25) a MeDIP smoothed a with oligos consecutive six than more as (defined region along tiled regions ( regions covering to −400 bp+400 relative To to TSS. all represent methylation were onperformed different array designs ( that E9.5 atMeDIPs ToMeDIP-WGA pooled compare and criteria. previous the meet not do ICPs >55%; content GC and >0.65 ratio CpG a with window 500-bp one least at HCPs contain >0.45; ratio CpG a with window 500-bp no contain LCPs follows: as defined were classes Promoter TSS. the to relative values in sliding 500-bp windows with a 5-bp offset in regions classes, −900we measured bp the toGC content+400 and bp the CpG ratio of observed to expected 50. ( genes housekeeping three PCR and real-time to the were values the normalized mean expression level of as enzyme a using scription were negative control. performed Quantifications 1 treated we parallel, In primers. random using Healthcare) (GE Kit cDNA Synthesis First-Strand the with transcribed reverse RNA was total of microgram One instructions. manufacturer’s the to analysis. Expression in given are Primers experiments. qPCR quantification. Values given are the average of two or three independent 2 used we and h, 2 for °C 37 at incubated were Samples 1 containing in tubes three 20U Xba with DNA of ng 200 mixed we experiments, HpaII For conversion. of with the QIAGEN PCR cloning kit and removed clones with patterns identical for the control.undigested For we bisulfite sequencing, cloned PCR fragments U 10 with by 30 of cycles with annealing at 52 °C. For consisted COBRA, we digested the PCR programs product PCR DNA. 20 cycles of bisulfite-treated touchdown on PCR (62–52 °C, primers with a 0.5 design °C decrease to per cycle) followed tion of DNA oocyte with was the performed EpiTect kit. We used MethPrimer pH 5.0, 1.5 of 200 at for °C 55 addition 4 h after and converted 1.1 of addition after min 15 for °C 50 at denatured glycogen, 32.5 in °C 37 at min 90 for tion and phenol-chloroform extraction. For we oocytes, incubated 600 oocytes with the EpiTect kit (Qiagen) following DNA byextraction proteinase K diges HpaII and sequencing digestion. bisulfite COBRA, by analysis methylation DNA annotations.on RefSeq based matched be could that promoters all for region same the in ratios log2 oligonucleotide averaged we data, our with comparison a For >0.4. ratio log2 TSS the average MeDIP ratios in regions covering −700 bp to +200 bp relative to the array.the data measuring Forwe used stem cells, embryonic comparison with on present classes promoter same the of genes all versus genes II and I group

mt, .. Sed T Nraiain f DA irary data. microarray cDNA of Normalization T. Speed, & G.K. Smyth, 265–273 (2003). 265–273 1 I, 20U HindIII, restriction buffer, and split the mixture in equal amounts amounts equal in mixture the buffer, split and restriction HindIII, 20U I, 3 . . We promotershypermethylated defined stem in with a embryonic cells For all samples except oocytes, we performed bisulfite conversion conversion bisulfite performed we oocytes, except samples For all Taq μ l l 75 mM hydroquinone and 5 α 1 for 1 h at 65 °C and used an equal amount of PCR product product PCR of amount equal an used and °C 65 at h 1 for de de novo Fig. 1 Figure 1 Figure Total RNA was treated with the RQ1 DNase according according DNase RQ1 the with Total treated RNA was c methylation peak (more methylation peak than oligos six consecutive ), we calculated the fraction of tiles with a methylated μ Gapdh μ e l l H l 1 mM SDS, 280 280 SDS, mM 1 l , , we averagedfor ratios oligonucleotides log2 all 2 O, 1 , , Supplementary Table 2 Supplementary Rpl13A μ l l of HpaII 10 U/ μ and and Fig. Fig. 1 g g glycogen. Subsequent desulfona μ g of RNA without reverse tran reverse without RNA of g μ Actb g/ml proteinase K and 10 10 and K proteinase g/ml a ), ), we averaged log2 ratios in μ ). l 4 M sodium bisulfite at bisulfite l 4 M sodium μ l l or 1 μ Nature Ge Nature l of the samples for for samples the of l . 4 μ 9 l l μ by comparing comparing by Msp l NaOH 10 N 10 NaOH l Methods I I 10 U/ n etics

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