Supporting Information

Rugg-Gunn et al. 10.1073/pnas.0914507107 SI Methods Chromatin Immunoprecipitation (ChIP). Different ChIP procedures Embryo Handling and Dissections. All animal work was performed are necessary for different applications/analyses. For example, in accordance with guidelines established by the Canadian where possible we used native ChIP to examine histone mod- Council on Animal Care. Mice used in this study included wild ifications, whereas fixed ChIP is needed to examine Polycomb type (strain ICR) and B5/EGFP, in which enhanced green binding and for sequential ChIP. Similarly, cChIP is used when ~ fluorescent protein is ubiquitously expressed (1). examining 1,000 cells. Importantly, the same assay was always Embryos were collected at appropriate time points from timed used within a figure or experiment, or when comparing cell lines. natural matings. Embryos on E3.5 were collected by flushing In addition, all key findings were replicated using alternative ChIP fi dissected uteri with M2 media (Specialty Media, Millipore). approaches where possible, con rming that the different meth- Embryos on E4.5, E5.5, and E6.5 were dissected from decidua in odologies do not affect the outcome of the experiment. In par- PBS with Ca2+ and Mg2+. Reichert’s membranes were removed ticular, the low prevalence of H3K27me3 sequence reads in TS fi from E5.5 and E6.5 embryos using 30-gauge needles before and XEN cells was con rmed for a subset of developmentally transfer to CO -calibrated DMEM/F12 supplemented with 10% important genes by quantitative PCR analysis of ChIP DNA. We 2 fi fi FBS. Isolation of epiblast (EPI), extraembryonic ectoderm also veri ed that our ndings were not unique to our methodology by obtaining highly similar results using an alternative H3K27me3 (ExE), and visceral endoderm (VE) was carried out as described fi (2). Briefly, E5.5 embryos were staged so that the proamniotic antibody and by performing ChIP on xed chromatin. Native (unfixed) ChIP was performed as described (5). Briefly, cavity was restricted to the embryonic portion of embryo, washed – four times in ice-cold PBS without Ca2+ and Mg2+, followed by 10 50 million cells were lysed in 0.2% (vol/vol) IGEPAL CA-630 (Sigma) for 10 min on ice, nuclei were collected by centrifugation incubation for 15–20 min in Hank’s based cell dissociation buffer at 10,000 × g for 20 min at 4 °C and suspended in digestion buffer (Invitrogen) on ice. Embryos were transferred to ice-cold drops at 1 mg/mL. Chromatin aliquots (0.5 mg in 500 μL) were digested of Ca2+- and Mg2+-free flushing and handling media (Specialty with 10 U micrococcal nuclease (GE Healthcare) for 6–9 min Media, Millipore) where the VE was reflected off the embryo at 37 °C and soluble chromatin recovered by overnight dialysis using 30-gauge needles. The remainder of the embryo was bi- followed by centrifugation. Successful chromatin fractionation sected along the embryonic/extraembryonic axis, and, if distin- (sample contains predominantly mononucleosomes to tetranu- guishable, the EPC was removed from the ExE and discarded. cleosomes) was verified by agarose gel electrophoresis. Frag- E6.5 embryos were processed similarly, except that EPCs were mented chromatin (35 μg per ChIP) was immunoprecipitated retained and all maternal tissue was removed. Isolated EPI, ExE, overnight at 4 °C with 2–10 μg of one of the following antibodies: VE, and EPC were pooled from multiple embryos and litters. – μ H3K4me3 (Abcam, ab8580), H3K9me2 (Millipore, 07 212), For cChIP analysis, tissues were snap-frozen in 50 L PBS sup- H3K9me3 (Abcam, ab8898), 1 μL H3K9me3 (6), H3K27me3 plemented with protease inhibitors (mini complete-EDTA free, (Millipore, 07–449), 1 μL H3K27me3 (7), H3K79me3 (Abcam, − Roche) and 5 mM sodium butyrate (Sigma) and stored at 80 °C ab2621), H4K20me3 (Millipore, 07–463), H3K9 acetylation until use. For mRNA expression analysis, tissues were homoge- (Millipore, 07–352), or rabbit anti-mouse IgG (Jackson, 315–005- μ nized in 800 L TRIzol (Invitrogen) and processed immediately 003). Protein A-sepharose (100 μL of 50% (vol/vol) slurry; GE ’ following manufacturer s instructions for small cell samples. Healthcare) was added to each sample and rotated for 4 h at 4 °C. Unbound material was removed, sepharose beads washed in salt Cell Culture. Mouse ES cell lines, R1 (passages 12–15) and – buffer (increasing sodium chloride concentration from 75 to E14TG2a (passages 19 21), were cultured on gelatin-coated 175 mM) and chromatin eluted from the beads with two 15-min surfaces in standard ES cell media (DMEM supplemented with μ incubations in 1% SDS at room temperature. DNA from bound, 15% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 g/mL unbound, and input samples was extracted by two rounds of μ streptomycin, 50 M 2-mercaptoethanol, 0.1 mM nonessential phenol/chloroform and precipitated with isopropanol overnight amino acids (NEAA), 2 mM glutamax (all from Invitrogen), and at −20 °C with 30 μg glycogen (Roche) as carrier. Air-dried DNA 1,000 U/mL LIF. pellets were reconstituted in TE buffer. Throughout the pro- – – TS cell lines, A4 (passages 6 15) and G3 (passages 7 14), were cedure, all solutions were ice-cold and freshly supplemented with × tg/tg derived from E3.5 blastocysts obtained from ICR B5/EGFP protease inhibitors (mini complete-EDTA free, Roche) and matings as previously described (3). TS cells were maintained in 5 mM sodium butyrate. RPMI (Sigma) supplemented with 20% FBS, 1 mM sodium Formaldehyde crosslinked and sonicated chromatin was ana- pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, 50 μM2- lyzed using the ChIP assay kit (Millipore) following manufacturer’s mercaptoethanol, 2 mM glutamax (all from Invitrogen), 25 ng/ protocol. Chromatin from 1 million cells was immunoprecipitated mL FGF4 (R&D Systems), and 1 μg/mL heparin (Sigma), with using the following antibodies: 3 μg Rnf2 (Abnova, H00006045- 70% of the media preconditioned by mitotically inactivated M01), 3 μg Ezh2 (Active Motif, 39103), 5 μg Ezh2 (Abnova, 5 embryonic fibroblasts. To induce TS cell differentiation, 1 × 10 PAB0648), 5 μg H3K27me3 (Millipore, 07–449), 5 μg Eed (Mil- cells were plated onto a 6-cm plate in standard TS cell conditions lipore, 09–774), and 5 μg anti-mouse IgG (Jackson 315–005-003). for 24 h, rinsed twice with PBS, and cultured for 6 days in un- Sequential ChIP was performed as described (8) with modi- conditioned TS cell media without FGF4 and heparin. Media fication to the first elution. Briefly, formaldehyde crosslinked and was changed every 2 days. sonicated chromatin (50 μg) was immunoprecipitated for 2 h at XEN cell lines, F3 (passages 7–12) and F4 (passages 7–14), were 4 °C using 2.4 μg H3K4me3 (Abcam, ab8580) that was prebound derived from E3.5 blastocysts obtained from ICR × B5/EGFPtg/tg to protein A Dynabeads (Invitrogen). Immune complexes were matings as previously described (4). XEN cells were maintained washed three times with RIPA and once with TE. The first on gelatin-coated surfaces in TS cell media (70% preconditioned) elution was carried out for 30 min at 37 °C with 30 μL20mM without FGF4 or heparin. Protocols can be found at http://www. Tris–HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, and sickkids.ca/research/rossant/custom/stemCells.asp. 20 mM DTT. Eluted chromatin was diluted 50-fold with RIPA and

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 1of15 immunoprecipitated overnight at 4 °C with 2.4 μg H3K27me3 Immunofluorescence. Embryos were processed as described pre- (Millipore, 07–449), 0.5 μL H3K9me3 (6), or 2.4 μg anti-mouse viously (12). Primary antibodies included: Cdx2 (1:200, Biogenex, IgG (Jackson 315–005-003). After washing as before, the second CDX-88), H3K4me3 (10 μg/mL, Abcam, ab8580), H3K27me3 elution was carried out for 2 h at 68 °C in 250 μL elution buffer (6 μg/mL) (7), and H3K9me3 (1:300) (6). Secondary antibodies (without DTT) supplemented with 50 μg/mL proteinase K. An included: anti-rabbit Alexa546 and anti-mouse Alexa488 (5 μg/mL; additional elution was carried out with a further 250 μL for 5 min Invitrogen). Nuclei were stained with Draq5 (10 μM; Invitrogen) or at 68 °C and DNA was recovered by phenol/chloroform extrac- Hoechst 33342 (1 μg/mL, Invitrogen). Images were collected by tion and ethanol precipitation. confocal microscopy as previously described (12). Quantification of immunoprecipitated DNA was performed by qPCR. For each sample, immunoprecipitated DNA was calcu- Western Blot. Protein extracts (20 μg per lane) were resolved by lated as a percentage of total input DNA and normalized to an SDS/PAGE and transferred on to PVDF membranes. Mem- intergenic region. Primers were designed to target the promoter branes were blocked with 3% milk and incubated with anti-Rnf2 region of each gene. Primer sequences are detailed in Table S1. (1 in 1,000; Abnova, H00006045-M01), anti-Eed (1 in 500; Topreparesamplesfor Illumima sequencing,the concentrationof Millipore, 09–774), anti-Ezh2 (1 in 2,000; Abnova, PAB0648), immunoprecipitated DNA was determined using Quant-iT Pico- anti-Ezh1 (1 in 500; Thermo, PA1-41114), anti-Suz12 [1 in 1,000 Green dsDNA reagent (Invitrogen) and 100 ng of each sample was (diluted in 5% BSA/TBST), Cell Signaling, 3737S], anti- sent to BC Cancer Agency Genome Sciences Centre, Vancouver, H3K4me3 (1 in 2,000, Abcam, ab8580), anti-H3K27me3 (1 in Canada, where the service was performed. Sample processing and 1,000, Millipore, 07–449), anti-H3K9me3 (1 in 1,000, Abcam, initial raw data processing was carried out as described (9). ab8898), anti-unmodified H3 (1 in 5,000, Abcam, ab1791), or anti-β-actin (1 in 10,000; Sigma, A5441). Secondary antibodies Carrier Chromatin Immunoprecipitation (cChIP). cChIP was used to were HRP conjugated anti-mouse (1 in 40,000, Biorad) and fi analyze histone modi cations in small sample sizes and per- HRP conjugated anti-rabbit (1 in 20,000, Jackson). formed as described (10), with minor modifications. Briefly, 50 million Drosophila melanogaster S2 cells were added to each Histone Methyltransferase Assay. Nuclear proteins were extracted sample of 500–2,000 target mouse cells and nuclei were isolated from ES, TS, and XEN cells using the EpiQuik nuclear extraction by incubation in 0.1% Tween 40 (vol/vol) for 1 h on ice followed kit I (Epigentek), quantified by Bradford assay (Biorad), and snap by homogenization in a Dounce. Nuclei were collected by cen- frozen in aliquots. The EpiQuik H3K27 and H3K4 histone trifugation, washed twice in 5% sucrose (wt/vol), and suspended methyltranferase activity kits (Epigentek) were used following the in digestion buffer to a chromatin DNA concentration of 0.5 mg/ manufacturer’s protocol with modification to specifically in- mL. Chromatin was digested with 10 U micrococcal nuclease per vestigate H3K4me3 and H3K27me3 methyltransferase activity. μ 50 g chromatin for 5 min at 28 °C and soluble chromatin was Briefly, 10 μg of nuclear protein was incubated with 0.25 μLS- recovered by overnight dialysis and centrifugation. Successful fi adenosyl-methionine and 50 ng biotinylated substrate for 2 h at chromatin fractionation was veri ed by agarose gel electropho- 37 °C. Each sample was incubated with 1 μg/mL primary anti- resis. Fragmented chromatin was immunoprecipitated overnight body [anti-pan-methylated H3K4 (included with kit), anti- at 4 °C with 2 μg H3K4me3 (Abcam, ab8580), 2 μg H3K27me3 – μ μ H3K4me3 (Abcam, ab8580), anti-pan-methylated-H3K27 (in- (Millipore, 07 449), 1 L H3K27me3 (7), 2 g H3K9me3 (Ab- cluded with kit) or anti-H3K27me3 (7)] for 2 h then incubated cam, ab8898), 1 μL H3K9me3 (6), or 2 μg anti-mouse IgG with 0.1 μg/mL HRP-conjugated anti-rabbit IgG for 30 min. (Jackson 315–005-003). Protein A-sepharose (200 μL of 50% Developing solution was added for 5 min, the reaction stopped (vol/vol) slurry) was added to each sample and rotated for 3 h at by addition of sodium hydroxide, and samples analyzed at 450 room temperature. Unbound material was removed and kept, nM using a Molecular Devices microplate reader. Raw absor- sepharose beads washed in salt buffer (increasing sodium chlo- bance readings collected from each technical replicate were ride concentration from 50 to 150 mM), and chromatin was eluted from the beads with 2 × 15-min incubations in 1% SDS at corrected by subtracting background (obtained from control room temperature. DNA from bound and unbound samples was wells with no protein extract) and these values were then aver- fi aged. Each cell line was assayed using three biological replicates, extracted using the DNA puri cation kit (Qiagen) and eluted in fi 50–125 μL buffer EB. Immunoprecipitated DNA was subjected with nal mean plus SD presented. to qPCR analysis. For each sample, immunoprecipitated DNA Gene Expression Analysis. RNA was processed from ES, TS, and was calculated as a ratio to antibody unbound DNA. Primers XEN cells using the RNeasy mini kit (Qiagen) with on-column were designed to target the promoter region of each gene. Primer sequences are detailed in Table S1. RNase-free DNase (Qiagen) digestion. RNA was extracted from isolated EPI, ExE, and VE samples using TRIzol (Invitogen) DNA Methylation Analysis by Pyrosequencing. DNA (2 μg) was bi- following the protocol for isolation of RNA from small quantities μ sulphite modified using the Epitect bisulphite kit (Qiagen) fol- of tissue with 0.2 g RNase-free LPA (Sigma) per sample. Each μ lowing the manufacturer’s protocol. Modified DNA (10 ng) was air-dried RNA pellet was reconstituted in 10 L RNase-free amplified with primers specific to bisulphite converted DNA water (Qiagen) and immediately reverse transcribed. Tissues using HotStartTaq (Qiagen) for 35 cycles, with annealing tem- from reference adult mouse tissues (detailed in next paragraph) peratures ranging from 53 °C to 56 °C. We used the universal were homogenized and processed using TRIzol (Invitrogen) biotinylated primer strategy, developed by Royo and colleagues, following the standard protocol. Total RNA (1 μg for bulk cells to biotin label the DNA fragment during PCR amplification (11). and tissues or the entire 10 μL sample for embryonic tissues) was Each PCR was optimized so that only one DNA fragment was reverse transcribed using the SuperScript II first-strand cDNA generated. Quantification of DNA methylation was performed synthesis system (Invitrogen) and cDNA subjected to qPCR. on a PSQ 96 Pyrosequencer following the manufacturer’s pro- Data were normalized to Hmbs. Primers were designed to target tocol (Pyrosequencing AB) using reagents purchased from Bi- multiple exons in the cDNA sequence. Primer sequences are otage. All primers were designed using PSQ Assay Design detailed in Table S1. software (Biotage) to target the promoter regions of each gene Reference tissues used for Fig. 5 were: ES cells (Pou5f1, Nanog, and are detailed in Table S1. Hypermethylated and hyomethy- and Sox2), TS cells (Cdx2, Eomes, and Esrrb), XEN cells (Gata6, lated genomic mouse DNA was used as template to confirm the Sox17, Sox7, and FoxA2), differentiated TS cells (Dlx3, Lhx2, absence of methylation bias within the assay (Fig. S3D). Prdm1, Pik3r3, Prtg, Prl3b1, and Kcnq1ot1), brain (Dlx5), kidney

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 2of15 (HoxA7 and Irx1), pituitary (Msx1), hippocampus (Prdm8), blood from the University of California Santa Cruz (UCSC) genome (Gata1), hypothalamus (Phf21b), and liver (ApoC2). browser for genome build mm8. For microarray analysis, 100 ng of total RNA was amplified and hybridized to the Mouse Gene 1.0 ST array (Affymetrix). This Processing of Genomewide DNA Methylation Data for ES and TS service was performed by The Centre for Applied Genomics, The Cells. Raw data (14) was processed using ChipMonk (http:// fi Hospital for Sick Children, Toronto, Canada. Array data were www.bioinformatics.bbsrc.ac.uk/projects/chipmonk/). A nal list processed using Expression Console (Affymetrix) to normalize of probes was generated by considering each cell line separately. fi and calculate the signal intensities using the PLIER algorithm. First, by applying a lter of replicates for individual probes with a cut off of P < 0.1, we tested for probe sets that behaved con- Gene expression was estimated testing the signal intensity against fi the global median. Signals that exceeded two times the global sistently over all three replicates. Second, we ltered this list for blocks of probes that behaved consistently in a 500-bp window median were considered to be expressed. Microarray annotation with a cutoff of P < 0.1. Using these data, a table of genome files were obtained from Affymetrix via their NetAffx web site. coordinates for all passing probes and their ratio of 5′ meC Quantitative PCR Analysis. Real-time qPCR was performed with immunoprecipitated DNA to input DNA was generated. This the following reaction conditions: 1× SYBR Green PCR master table was converted into a BED format and processed with the LiftOver tool from UCSC genome browser to transfer the mix (Roche), 500 nM of each primer, and 5 μL of template in genomic coordinates from mm6 to mm8 to match our ChIP- a 12-μL reaction volume. The volume of template corresponds to sequencing and microarray data. DNA methylation marks with- 1/80th of ChIP DNA sample or to 150 ng of cDNA. The reaction in ±1.5 kb of the TSS were included for analysis. and analysis were performed on a Lightcycler 480 (Roche). Melting curve analysis was performed to verify that only one Dataset Integration. Datasets were integrated on the basis of ge- amplicon was generated for each reaction. nome coordinates. MySQL code was used to test and group data on the basis of overlap or distance of TSS and histone methylation Process Wig Files. Raw ChIP-sequencing data were processed using peak distances or probe distances. FindPeaks (version 2.1.4) (13) with a false discovery rate of 0.001 and an average fragment size of 174 bp. A Java program was Statistical Analysis. Statistical comparison of ChIP sequencing, written to read the output Wig files and generate a BED file that DNA methylation, and expression array datasets was performed contained information on the maximum peak height of all in R (http://www.r-project.org/) using the RKWard interface. We thresholded H3K4me3 or H3K27me3 regions. Significant ChIP- reported the Pearson correlation, the significance of which was sequencing peaks within ±1 kb of the TSS for H3K4me3 and ± calculated by a Fisher exact test, and scored into bins of P values. 5 kb of the TSS for H3K27me3 were included in the final dataset. Two-tailed Student’s t test was used to compare Q-PCR datasets, All these data were imported as tables into a MySQL database. with a P value <0.05 deemed significant. One-tailed χ2 test, with 1 df, was used to compare expected and observed number of Genome Annotation. Tables of gene annotations (gene symbols, genes with different epigenetic states that are induced upon TS chromosome, and transcription start and stop) were obtained cell differentiation.

1. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A (1998) Generating 9. Robertson G, et al. (2007) Genome-wide profiles of STAT1 DNA association using chro- green fluorescent mice by germline transmission of green fluorescent ES cells. Mech matin immunoprecipitation and massively parallel sequencing. Nat Methods 4:651–657. Dev 76:79–90. 10. O’Neill LP, VerMilyea MD, Turner BM (2006) Epigenetic characterization of the early 2. Brons IG, et al. (2007) Derivation of pluripotent epiblast stem cells from mammalian embryo with a chromatin immunoprecipitation protocol applicable to small cell embryos. Nature 448:191–195. populations. Nat Genet 38:835–841. 3. Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J (1998) Promotion of 11. Royo JL, Hidalgo M, Ruiz A (2007) Pyrosequencing protocol using a universal trophoblast stem cell proliferation by FGF4. Science 282:2072–2075. biotinylated primer for mutation detection and SNP genotyping. Nat Protoc 2: 4. Kunath T, et al. (2005) Imprinted X-inactivation in extra-embryonic endoderm cell 1734–1739. lines from mouse blastocysts. Development 132:1649–1661. 12. Ralston A, Rossant J (2008) Cdx2 acts downstream of cell polarization to cell- 5. Umlauf D, Goto Y, Feil R (2004) Site-specific analysis of histone methylation and autonomously promote trophectoderm fate in the early mouse embryo. Dev Biol 313: acetylation. Methods Mol Biol 287:99–120. 614–629. 6. Cowell IG, et al. (2002) Heterochromatin, HP1 and methylation at lysine 9 of histone 13. Fejes AP, et al. (2008) FindPeaks 3.1: A tool for identifying areas of enrichment H3 in animals. Chromosoma 111:22–36. from massively parallel short-read sequencing technology. Bioinformatics 24: 7. Peters AH, et al. (2003) Partitioning and plasticity of repressive histone methylation 1729–1730. states in mammalian chromatin. Mol Cell 12:1577–1589. 14. Farthing CR, et al. (2008) Global mapping of DNA methylation in mouse pro- 8. Noer A, Lindeman LC, Collas P (2009) Histone H3 modifications associated with differentia- moters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4: tion and long-term culture of mesenchymal adipose stem cells. Stem Cells Dev 18:725–736. e1000116.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 3of15 Fig. S1. Analysis of ChIP-Seq dataset. (A) Number of sequence reads per sample. (B) Direct comparison of our H3K4me3 and H3K27me3 ES cell dataset (Upper two tracks; nonthresholded) with a previously published study (Lower two tracks; nonthresholded) (1) revealed a high degree of similarity. Example shown is a 600-kb region of chromosome 17 displayed in the UCSC genome browser. (C) Pearson correlation and Fisher’s exact t test of gene expression, histone modification, and DNA methylation datasets. Each correlation plot shows that there is a significant interaction with the gene expression array signal (sig) and the histone modification (either H3K4me3 or H3K27me3) or DNA methylation status (ratio). The Lower Left panels show the graphical interaction with a trend in red, whereas the Upper Right panels give the Pearson correlation coefficient and the Fisher’s exact t test significance. ***, P < 0.001; **, P < 0.01. (D) Histone state is predictive of expression status in ES, TS, and XEN cells. Boxplots showing median, 25th and 75th percentile expression levels, with whiskers indicating the interquartile range for ES, TS cells, and XEN cells (log 2 scale). Classes based on histone methylation state are indicated and divided into gene promoters with or without a CpG island. Classes with less than 50 or 100 genes are indicated. H3K4me3-marked genes have a range of microarray levels with a statistically significant tendency toward genes being ex- pressed, which is different to genes marked by H3K27me3 (bivalent or neither) that tend to have levels in the nonexpressed range.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 4of15 Fig. S2. Analysis of ChIP-seq datasets. (A) Examination of the width of H3K27me3 and H3K4me3 modified regions in ES, TS, and XEN cells. This analysis shows that the median width of H3K27me3 in ES (R1) cells is 2.2 kb and ~10% of all blocks are larger than 5 kb. In contrast, the median block width in TS (A4) and XEN (F4) cells is ~1 kb and only ~1% of all blocks are larger than 5 kb. H3K4me3 blocks were similar in all three stem cell lines (median width of ~1.4 kb and <1% are larger than 5 kb). (B) Chromosome 19 (arbitrarily selected) was divided into 174-bp bins and the number of sequence reads per bin is shown for H3K27me3 and H3K4me3. Note that this is nonthresholded data. A similar number of bins have a low sequence count per bin in all three cell lines; however, in contrast toES cells, few bins contain >11 H3K27me3 sequence reads in TS and XEN cells. Importantly, the randomization model that we used to identify significant peaks defined <11 reads per bin as background (i.e., expected by chance). (C) Direct pairwise comparison of average sequence reads per bin between ES and TS cells across chromosome 19. Despite the lower prevalence of H3K27me3 in TS cells, 12% of the bins have a greater average number of sequence reads than the same bin in ES cells. This is the figure expected by chance if background levels are equivalent. (D) Boxplots showing 25th and 75th percentile average peak height with whiskers indicating 5th and 95th percentiles for H3K27me3 and H3K4me3. H3K27me3 peak height was comparable between ES, TS, and XEN cells (P = 0.13, Student’s t test), suggesting that there were no differences in the ability to detect H3K27me3 between the cell lines. Together, these data provide strong evidence that background levels and the ability to detect significant H3K27me3 sequence peaks are highly similar between ES, TS, and XEN cells, which would be expected only if the ChIP efficiency was the same.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 5of15 Fig. S3. DNA methylation levels in ES and TS cells. Boxplots showing median, 25th and 75th percentile DNA methylation levels, with whiskers showing the interquartile range. DNA methylation data for ES and TS cells were obtained from a publication by Farthing et al. and integrated with our dataset (2). (A)ES cells and (B) TS cells show highly similar profiles. Levels >0 indicate hypermethylated DNA; <0 indicate hypomethylated DNA. Classes based on histone state are indicated and divided into two categories: genes that are expressed or not expressed. Classes with less than 30 genes are indicated. (C) Bar graphs showing pyrosequencing-based analysis of DNA methylation levels in two lines of TS cells. Levels generally do not significantly change at the promoters of candidate genes after 6 days of differentiation, despite changes in transcription of those genes. Left panel: TS cell line A4; Right panel: TS cell line G3. Increases and decreases in gene expression after differentiation (indicated) were assessed by qRT-PCR analysis (actual values for TS A4 cells are shown in Fig. 4A). Gapdh, expressed in both undifferentiated and differentiated TS cells, therefore appears in a separate category. (D) Hypo- and hypermethylated DNA was prepared and used as templates for DNA methylation analysis to assay for methylation-specific bias. Each black square represents a different gene. No strong bias was detected, although Prdm1 tended to overestimate and Lhx2 underestimate DNA methylation levels.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 6of15 Fig. S4. Analysis of H3K4me3 and H3K9me3 in an alternative TS cell line and in XEN cells. (A) ChIP experiments show the ratio of immunoprecipitated DNA for H3K4me3 and H3K9me3 relative to starting input DNA. Differentiated TS cells (black bars) were obtained by culturing undifferentiated TS cells (line G3) (gray bars) for 6 days in the absence of FGF4 and heparin. The Lower panel shows sequential ChIP experiments and confirms the coexistence of H3K4me3 and H3K9me3 marks in undifferentiated TS (G3) cells. In general, the data are in close agreement with the first TS cell line (Fig. 4). Mean plus SD are shown for two independent experiments. Asterisks, P < 0.05 (Student’s t test). (B) Sequential ChIP in ES cells confirms the coexistence of H3K4me3 and H3K27me3 at known bivalent domains Cdx2, Gata6, and Foxa2.(C) ChIP experiments show the levels of H3K4me3 (Upper) and H3K9me3 (Lower) for a panel of genes in un- differentiated XEN (F4) cells. Dashed lines indicate 2-fold of mean background levels, as determined using a nonspecific control antibody. The expression status of each gene is shown underneath. (D) Sequential ChIP confirms the coexistence of H3K4me3 and H3K9me3 at Npas2, Twist1, and Vdr. Gata6 and Gapdh are known to be modified by H3K4me3 only in XEN cells and served as controls for the specificity of the H3K9me3 immunoprecipitation. Dashed lines indicate 2- fold of mean background levels, as determined using anti-H3K4me3 in the first immunoprecipitation and a nonspecific control antibody in the second im- munoprecipitation.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 7of15 Fig. S5. Microdissection of lineage progenitors from early mouse embryos. (A) Schematic diagram alongside phase-microscopy images showing microdis- section of an E5.5 embryo to isolate lineage progenitor tissues. (Bar, 40 μM.) (B) Schematic diagram alongside phase-microscopy images showing microdis- section of an E6.5 embryo to isolate embryonic and trophoblast tissue. Maternal cells were removed from each EPC. (Bar, 100 μM.) (C) cChIP experiments examine ES (E14) cells, TS (G3) cells, and XEN (F3) cells. In general, the results are highly similar to the alternative cell lines in Fig. 5, demonstrating that differences between in vitro and in vivo cells are not due to variability among TS and XEN cell lines. Antibodies used were against H3K4me3 (green) and H3K27me3 (red). Dashed lines indicate 2-fold of mean background levels, as determined using a nonspecific control antibody. Mean plus SD are shown from two biological replicates.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 8of15 Fig. S6. Immunocytochemical localization of H3K27me3, Ezh2, and H3K9me3 in early mouse embryos. (A) Stacked images from confocal z-series following immunofluorescent localization of H3K4me3 and H3K27me3 in E3.5 and E4.5 mouse embryos. H3K4me3 signal was present in all lineages of the blastocyst (E3.5, n = 8/8; E4.5, n = 5/6), as shown by the complete overlap between histone marks (red) and either DNA (blue) or the TE (Cdx2, green). In contrast, high levels of nuclear H3K27me3 signals were specific to ICM, with lower H3K27me3 levels detected in TE of E3.5 embryos (n = 2/4). Dashed lines indicated ICM position. Additionally, H3K27me3 levels were low in PE at E4.5 (indicated by arrow); n = 2/4). At E5.5, H3K27me3 (red) was detected in all lineages (single image section). (B) Ezh2 (red) was detected in all lineages from E3.5 to E5.5 (all single image sections). (C) H3K9me3 in E3.5 (Upper panels; single image section) and E5.5 (Lower panels; stacked image of z-series) mouse embryos. The VE has been removed from the E5.5 embryo to increase antibody penetration. H3K9me3 was present in embryonic and trophoblast lineages at E3.5 and E5.5, with the strongest signal observed at heterochromatin foci.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 9of15 Top 20 GO Biological Process terms for H3K4me3 & H3K27me3 marked genes in TS and XEN cells

TS Cells

Term GO ID Level (Average) P value (Average) Gene Hits E value kidney development GO:0001822 5.86 0.000005 5 19.88 urogenital system development GO:0001655 5 0.000008 5 18.2 nucleosome assembly GO:0006334 6.8 0.000016 5 15.74 chromatin assembly GO:0031497 6 0.000030 5 13.86 protein-DNA complex assembly GO:0065004 5.5 0.000054 5 12.28 chromatin assembly or disassembly GO:0006333 7 0.000084 5 11.19 DNA packaging GO:0006323 4 0.000090 5 11.03 pattern specification process GO:0007389 3.67 0.000143 6 7.52 metanephros development GO:0001656 6.86 0.000504 3 19.29 establishment and/or maintenance of chromatin architecture GO:0006325 6 0.002111 5 5.51 macromolecular complex assembly GO:0065003 4.5 0.004330 5 4.66 regulation of cell differentiation GO:0045595 4.5 0.005146 4 5.76 negative regulation of cell differentiation GO:0045596 5.17 0.005644 3 8.32 organ development GO:0048513 4.75 0.005672 11 2.38 ureteric bud development GO:0001657 7.38 0.006313 2 16.79 organ morphogenesis GO:0009887 5.33 0.007938 6 3.43 cellular component assembly GO:0022607 4 0.008389 5 3.98 chromosome organization and biogenesis GO:0051276 5 0.008662 5 3.94 regulation of transcription GO:0045449 5.89 0.010387 13 2.01

XEN Cells

Term GO ID Level (Average) P value (Average) Gene Hits E value antigen processing and presentation GO:0019882 3 0.000184 4 14.12 cell division GO:0051301 3 0.001327 6 4.98 cell cycle GO:0007049 3 0.001696 10 3.03 inner ear morphogenesis GO:0042472 7.32 0.002093 3 11.82 tRNA processing GO:0008033 6.78 0.002332 3 11.38 biopolymer metabolic process GO:0043283 4 0.00264 32 1.6 ear morphogenesis GO:0042471 6.47 0.003003 3 10.41 RNA metabolic process GO:0016070 5 0.003149 21 1.87 ubiquitin-dependent protein catabolic process GO:0006511 8.92 0.003992 4 6.21 modification-dependent protein catabolic process GO:0019941 7.92 0.004324 4 6.07 modification-dependent macromolecule catabolic process GO:0043632 6 0.004324 4 6.07 proteolysis involved in cellular protein catabolic process GO:0051603 7.15 0.004674 4 5.94 inner ear development GO:0048839 7 0.004674 3 8.9 cellular protein catabolic process GO:0044257 6.2 0.004918 4 5.85 nucleobase, nucleoside, nucleotide and nucleic acid metabolic process GO:0006139 4 0.00515 24 1.7 ear development GO:0043583 6.75 0.006345 3 7.98 ubiquitin cycle GO:0006512 8 0.006396 7 3.19 anti-apoptosis GO:0006916 8.29 0.007053 3 7.68 cellular metabolic process GO:0044237 3 0.008407 42 1.37

Fig. S7. List of top 20 GO-terms for H3K4me3/H3K27me3 marked genes in TS and XEN cells.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 10 of 15 Fig. S8. Control ChIP-qPCR data using a nonspecific antibody. In parallel with other ChIP experiments, chromatin was incubated with a nonspecific isotype control antibody and the ratio of immunoprecipitated DNA relative to starting input DNA is shown. Enrichment ratios were low or negligible. Mean plus SD are shown for two to three independent experiments. (A) Control for TS and XEN cells described in Fig. 3. (B) Control for undifferentiated and differentiated TS cells described in Fig. 4 B and C.(C) Control for TS cells described in Fig. 4D.(D) Control for ES cells described in Fig. 5A.

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 11 of 15 Table S1. Primers Primer Name Sequence (5′ to 3′) Reference

ChIP Primers ApoC2 F CCATGCGTAGGGCATTAGAAGA New ApoC2 R GGCCCATCCTGTAACAGAGCTT Cdx2 F CCAGGTTGGAAGGAGGAAGC New Cdx2 R ACCACCCCCAGAAACACGAT Dlx3 F ACAGCGCTCCTCAGCATGAC New Dlx3 R CTGCGAGCCCATTGAGATTG Dlx5 F GCTTCGCTGGCTAATCCAGACT New Dlx5 R CAGCCCTAGTGGTGTTTGCGTA Eomes F CCTCTGGGACCTGCCAAACT New Eomes R CTCTATGGCGCCGGAGAAAC Epas1 F CTCGGACCTGCGAGCACTAA New Epas1 R CGGAGCACCTGGGTTCCTTA Esrrb F CAGCCAGCCCAACCATGTAA New Esrrb R AGGAGGATGTGTCGGGAGGA FoxA2 F TCCTCCTGAAGTCATCCCACAA New FoxA2 R TAAATCCAAGGTGCCCAAAGC Gapdh F TCCTATCCTGGGAACCATCACC New Gapdh R TCTTTGGACCCGCCTCATTT Gata1 F TGCCCCAACTTCTTCCCATT New Gata1 R CAGGCCTGGGAGGATGAAGA Gata6 F CTGGGTGGCGGGTATGACTT New Gata6 R CGCCCAGCTAAAGGACACCA Gbx1 F CAAGCCCTTCTGAACTATCCCAAT New Gbx1 R AGCTCCCAGAGTTAGGAGACAGGA Hoxa7 F GAGAGGTGGGCAAAGAGTGG (3) Hoxa7 R CCGACAACCTCATACCTATTCCTG Hoxa9 F GGAGGGAGGGGAGTAACAAA (4) Hoxa9 R TCACCTCGCCTAGTTTCTGG Intergenic F GGAGAGAAGTGGAGTGGCCAAG New Intergenic R TTGCCAGCCTAATCATGAGGAA Irx1 F CGGTCACCTCGGTGCTAGG New Irx1 R ATAGGGCAAGAAGGCGCTGT Kcnq1ot1 F CAAAGCACACTGAGGATGGCTAGT New Kcnq1ot1 R GCCTCAGCATATTTGTCCACAGTT Lhx2 F GATGCACTGGGCCGGTTA New Lhx2 R GCCCGACAGACTGTGGAACA Major Satellite F GACGACTTGAAAAATGACGAAATC (5) Major Satellite R CATATTCCAGGTCCTTCAGTGTGC Msx1 F ACAGAAAGAAATAGCACAGACCATAAGA (3) Msx1 R TTCTACCAAGTTCCAGAGGGACTTT Nanog F CAGACTGGGAGGGAGGGAAA New Nanog R GAGGTGCAGCCGTGGTTAAA Nostrin F TGCTTGATGAGGTGCCAACA New Nostrin R GTGTGGAGGGGAGGCAAATC Npas2 F TTGTGTCACTACGTTCCTGGGTCT New Npas2 R GAGCGCAGAGCTGTCTAAGCAC Phf21b F GCCCCTCCTTACTTGTTTGTCG New Phf21b R CCCGCTCCTCTGTGTCTTCATA Pik3r3 F TTCCCTTTGTGGCGATTCCT New Pik3r3 R TGAGAGAAGCACGGAGTCTCAAA Pou5f1 F TGGCTGAGTGGGCTGTAAGG New Pou5f1 R CAAACCAGTTGCTCGGATGC Prdm1 F GGGTGGACATGAGAGAGGCTTA New Prdm1 R GGTTCCTTACCAAGGTCGTACCC Prdm8 F GGAGGATCTGCGAAGGAAGAGA New Prdm8 R CAGGACCCCGGGCTTTATAGTA Prl3b1 F GGAGGGCTTTCGTTACCACCT New Prl3b1 R GGTTCCATAGTGACGCAGACCA Prtg F GGCCGCACGTGGTTTTATTT New Prtg R GAGGAACCCCACTGCAAACC Sox17 F CACCAACCCGCTTGCTACAG New Sox17 R TAAGCCACATCCCCAAAGCA

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 12 of 15 Table S1. Cont. Primer Name Sequence (5′ to 3′) Reference

Sox2 F CCATCCACCCTTATGTATCCAAG (3) Sox2 R CGAAGGAAGTGGGTAAACAGCAC Sox7 F TGCCAGTTTAGGGAAGTCAGT New Sox7 R GTCATCTCGCCCCAGTAAAC Tbx2 F CTTACTGCTGAGGCTTCCGACAC New Tbx2 R TTTGGACCAATTGTGGGTCTCC Tcfap2a F ACAGGGGAGACGCTGGAGAT New Tcfap2a R GGGGAAAGAGTGGAACACGA Twist1 F GGGAATCCCTTGGGACTAGAGGTT New Twist1 R AAAGTTTCAACAACCGAGTCCATC Vdr F CTCCCTTCTTACTCCTCCACTCCA New Vdr R AGTCCTTAGCTAGGAGGGTGCTCA Wnt5b F GATGTCTGTCACAGCCGCTCAT New Wnt5b R TCATAAGATGTTGAAGGGCAGGTG

Expression Primers ApoC2 F AGACATACCCGATCAGCATGGA New ApoC2 R GCCTGCGTAAGTGCTCATGG Ash1 F CCAGCTGGGACAGAGCTTACCTAT New Ash1 R TCCAATGATTCCTCGACACTTCTC Cdx2 F CGAAACCTGTGCGAGTGGAT New Cdx2 R AGCCGCTGATGGTCTGTGTA Dlx3 F GCTGGGCCTCACACAAACAC New Dlx3 R TGTTGTTGGGGCTGTGTTCC Dlx5 F AGCCAGCCAGAGAAAGAAGTGG New Dlx5 R GTCCTGGGTTTACGAACTTTCTTTG Eed F AGACCCAAACCTTCTCCTGTCAGT New Eed R ACCTCCGAATATTGCCACAAGAGT Eomes F ACTGGCTCCCACTGGATGAG New Eomes R GCAGCCTCGGTTGGTATTTG Epas1 F TCCTTGCGACCATGAGGAGA New Epas1 R GCTCGGTGGACACGTCTTTG Esrrb F AATTGGCAGATCGGGAGCTT New Esrrb R GCTCATCTGGTCCCCAAGTG Ezh1 F CGAGTCTTCCACGGCACCTA New Ezh1 R GCAAACTGAAAGACCTGCTTGC Ezh2 F CCTTCCATGCAACACCCAAC New Ezh2 R GCTCCCTCCAGATGCTGGTAA FoxA2 F CTCCCTACTCGTACATCTCGCTCA New FoxA2 R GGTAGAAAGGGAAGAGGTCCATGA Gapdh F GCCAAGGTCATCCATGACAACTT New Gapdh R ACAGTCTTCTGGGTGGCAGTGAT Gata1 F GCAGCATCAGCACTGGCCTA New Gata1 R CATAAGGTGAGCCCCCAGGA Gata6 F CGGTCTCTACAGCAAGATGAATGG New Gata6 R TAGTGGTTGTGGTGTGACAGTTGG Hmbs F CGTGGGAACCAGCTCTCTGA New Hmbs R GAGGCGGGTGTTGAGGTTTC Hoxa7 F GAAGCCAGTTTCCGCATCTACC New Hoxa7 R ATGGAATTCCTTCTCCAGTTCCAG Hoxa9 F AAAACAATGCCGAGAATGAGAGC New Hoxa9 R GTTTTGTGTAGGGGCATCGCTTC Irx1 F ACCTCAGCCTCTTCTCGCAGAT New Irx1 R AGGGATAATAAGCAGGCGTTGTGT Kcnq1ot1 F AGTTAGCTGCCTGTCTCCTCCACT New Kcnq1ot1 R AATCAATGAAGCCACTGACCAGAA Lhx2 F TTCAGCAAGGATGGCAGCAT New Lhx2 R GCGCATCACCATCTCTGAGG Major Satellite F GACGACTTGAAAAATGACGAAATC (6) Major Satellite R CATATTCCAGGTCCTTCAGTGTGC Mll1 F TTCTCGGACTACCAGTCACTTGCT New Mll1 R ATGCTTTCCTCAGTCTCAGGGTTT

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 13 of 15 Table S1. Cont. Primer Name Sequence (5′ to 3′) Reference

Msx1 F GATGCAGAGTCCCCGCTTCT New Msx1 R GGTTGGTCTTGTGCTTGCGTAG Nanog F ATGCCTGCAGTTTTTCATCC New Nanog R GAGGCAGGTCTTCAGAGGAA Nostrin F GAGTTGGAAGCGATAAAGCCAAC New Nostrin R TCCACTTCATTATCAAGCGACTTTCTC Npas2 F GGAAATGTGTGTAGCTGACGAACC New Npas2 R CCTATGATTGGAGGAGCTCTGTGA Phf21b F TCAAGAAGCAGCTCCACGAAAG New Phf21b R GAGCTGACCTGAGGACCTGTGA Pik3r3 F TGCAGAGTGGTACTGGGGAGA New Pik3r3 R ATCCCCCTGCATTTTCGTTG Pou5f1 F AGCTGCTGAAGCAGAAGAGG New Pou5f1 R AGATGGTGGTCTGGCTGAAC Prdm1 F AGCCGAGGCATCCTTACCAA New Prdm1 R ATGAGGGGTCCAAAGCGTGT Prdm8 F GCGACTCGGTCCTGACCTTATT New Prdm8 R AGACACTGTTGGACAGCCTTGG Prl3b1 F ACAACGCCCATGATCTTGCT New Prl3b1 R CAGGCCATAGGTCCAAGCTG Prtg F GGAAAAGCATGGAGGCTTCG New Prtg R GCGGTTACATTCTGTGGACTGG Rnf2 F GCTGAAGATACAGGCCATGAACAG New Rnf2 R CAGTGGGAGCTGTCACCATTATCT Sox2 F GGACCGTTACAAACAAGGAAGGAG New Sox2 R AACGGTCTTGCCAGTACTTGCTCT Sox7 F GAGCATGGTCACCCCCATC New Sox7 R AGGGCTAAAGAACCTAGAGGG Sox17 F CAGAACCCAGATCTGCACAA New Sox17 R GCTTCTCTGCCAAGGTCAAC Suz12 F CCTGGTTCTGTTAAACCTGCACAA New Suz12 R CTGGCGACTTTCATTTGAATTATCC Tcfap2a F TGCCAGCAGGGAGACGTAAA New Tcfap2a R CGCACACGTACCCAAAGTCC Twist1 F ATGTCCGCGTCCCACTAGCAG New Twist1 R TGTCCATTTTCTCCTTCTCTGGAA Vdr F AGCTGAACCTCCATGAGGAAGAAC New Vdr R TCAACCAGCTTAGCATCCTGTACC

Pyrosequencing Primers Cdx2 F ATTATGTTGTTTGGGGATAAA New Cdx2 R CGCCAGGGTTTTCCCAGTCACGACATACAACCAACTACCTTTATCTCT Cdx2 Seq TTATGTTGTTTGGGGAT Dlx3 F GTTTGGGAATAGGAGTTGAAATT New Dlx3 R CGCCAGGGTTTTCCCAGTCACGACACAACATTAAAAACCCCCATAATC Dlx3 Seq GATTTTTAGAGGGGTGAA Eomes F TGAGTGTAGGGTGGAGGGATTTTA New Eomes R CGCCAGGGTTTTCCCAGTCACGACAATTCATTCCCAAATTTCCATCT Eomes Seq GGGTGGAGGGATTTT Epas1 F CGCCAGGGTTTTCCCAGTCACGACGGGGTTAAGGAATTTAGGTG New Epas1 R CTTACTCAAAACCCCTCAACAA Epas1 Seq CTCCTTATCAACTATCATTA Esrrb F CGCCAGGGTTTTCCCAGTCACGACTTTGTTTTTTGATTTGGTAGGG New Esrrb R CAAAACCTATACAATCAACAATCT Esrrb Seq ACAATCTAACTTAAAAACAT Gapdh F GGAAGTTTAAGTTTGGGAGATGA New Gapdh R CGCCAGGGTTTTCCCAGTCACGACACCCCCAACTACTACACCTCTAAT Gapdh Seq TTGTTTTTAATATTTAAGAT Lhx2 F CGCCAGGGTTTTCCCAGTCACGACGGGGTTGAGAGTTGGGATTT New Lhx2 R CCTTACACCCTATCCAAAAATTCC Lhx2 Seq ATTCCCCAACCTTAAT Nanog F GGATTTTGTAGGTGGGATTAATTG New

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 14 of 15 Table S1. Cont. Primer Name Sequence (5′ to 3′) Reference

Nanog R CGCCAGGGTTTTCCCAGTCACGACCTACCCTACCCACCCCCTATTCT Nanog Seq TGAATTTATAGGGTTGGTG Pik3r3 F GGTGTAAAGGAGTGGAAAGGATAG New Pik3r3 R CGCCAGGGTTTTCCCAGTCACGACCCCTAACTCCAACCACTAAAATTT Pik3r3 Seq AAAGGAGTGGAAAGGAT Pou5f1 F TTAAGGTTAGAGGGTGGGATTG New Pou5f1 R CGCCAGGGTTTTCCCAGTCACGACTCTAAAACCAAATATCCAACCATA Pou5f1 Seq GGAGGGAGAGGTGAAAT Prdm1 F CGCCAGGGTTTTCCCAGTCACGACGGAAAAAGTTTTTTTTAAAGAGGA New Prdm1 R CAAATAAACATCTTTCCCCTTC Prdm1 Seq AAACATCTTTCCCCTTC Prtg F CGCCAGGGTTTTCCCAGTCACGACGGGTTTGTAGTGGGGTTTTTTTAG New Prtg R CATCCTCCCATCTTCTTACTCAAT Prtg Seq TCCCATCTTCTTACTCAAT Tcfap2a F CGCCAGGGTTTTCCCAGTCACGACTTAATAAGGGAAGAATGTTTGGAA New Tcfap2a R TTCACTTTTACAAACCCTACAACC Tcfap2a Seq CTCCCTCTCCCCACC B-M13-S BIOTIN-CGCCAGGGTTTTCCCAGTCACGAC (7)

Dataset S1. Table shows all expression, histone methylation and DNA methylation data used in this study. H3K4me3 and H3K27me3 values represent the maximum ChIP-sequencing peak height in the final thresholded dataset; “0” means that the peak height was below threshold. DNA methylation for ES and TS cells was obtained from a publication by Farthing et al. and integrated into our Dataset S1 (2). For each gene, the P value, ratio of 5′ mCpG immunoprecipitated DNA to input DNA (where >0 indicates hypermethylated and <0 indicates hypomethylated) and the number of array probes used in the analysis are given.

1. Mikkelsen TS, et al. (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448:553–560. 2. Farthing CR, et al. (2008) Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4:e1000116. 3. Stock JK, et al. (2007) Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol 9:1428–1435. 4. Terranova R, et al. (2008) Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell 15:668–679. 5. Jorgensen HF, et al. (2007) The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells. Genome Biol 8:R169. 6. Lehnertz B, et al. (2003) Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol 13:1192–1200. 7. Royo JL, Hidalgo M, Ruiz A (2007) Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping. Nat Protocols 2:1734–1739.

Dataset S1 (XLS)

Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 15 of 15