Yuan_FigS1. Control shrna. Vector. Eset shrna1. Eset shrna2. Eset overexpression. kda. anti-eset. anti-actin

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1 Yuan_FigS1 kda Vector Control shrna Eset shrna1 Eset shrna2 Eset overexpression anti-eset 11 anti-actin

2 Yuan_FigS2 A Relative expression (%) Nanog Pou5f1 Cdx2 Hand1 Eset B Control shrna Eset shrna 1 Eset shrna 1 Rescue Relative expression (%) Nanog Pou5f1 Cdx2 Hand1 Eset Control shrna Eset shrna 2 Eset shrna 2 Rescue

3 Yuan_FigS3 Luci shrna Eset shrna Pou5f1 shrna

4 Yuan_FigS4 Eset Control knockdown (triplicate expression measurements) Eset knockdown (triplicate expression measurements) ES cell up-regulated gene Tcl1 Nanog Esrrb Tbx3 Sox2 Zfp42 Klf4 Bmp4 Pou5f1 Trophoblast lineage associated genes Cdx2 Tcfap2a Fgfr2 Gata2 Plf Mmp9 Id2 Fold change

5 Yuan_FigS5 B A 5X 1X Control shrna 5X 1X Eset shrna 1 5X 1X Eset shrna 2 5X 1X C No. of colony / microscope field Control Eset Eset shrna shrna1 shrna2 AP stained Differentiated

6 B N na t G tl2 M es H t 19 Ig f Eo 2r m N es es Tc tin fa p2 a C dx 2 D az N l e Te nf x N 1 eu 9 ro O g1 lfr 9 M 1 Sp yog a Tc ca1 fa p2 c Fg Pr f5 dm 1 C 4 dh 3 Fold enrichment Yuan_FigS6 A Control shrna 3 25 Control shrna Eset shrna Anti-Eset DAPI Anti-Eset DAPI Eset shrna 35 Eset ChIP

7 Yuan_FigS7 5 GSE GSE3766 Average Expression Average Expression Non-Eset bound genes Eset bound genes Non-Eset bound genes Eset bound genes Mean Mean SE Mean.95 Conf. Interval

8 Yuan_FigS8 Peg1 H19 Grb1 Peg3 Kcnq1 Rasgrf1 Impact Mest Snrpn Gtl2 Nespas Gnas Nnat Control knockdown (triplicate expression measurements) Eset knockdown (triplicate expression measurements)

9 Yuan_FigS9 Relative Expression (%) Day 2 Day 3 Day 4 Day 5 Tcfap2a Relative Expression (%) Day 2 Day 3 Day 4 Day 5 Cdx2

10 Yuan_FigS1 A B Relative expression (%) Tcfap2c Control shrna Eset shrna Tcfap2c Exon 1 C D Fold enrichment H3K9me3 ChIP Fold enrichment Eset ChIP GFP ChIP Fold enrichment H3K9me2 ChIP Control shrna Eset shrna

11 Yuan_FigS11 3 Cdx2 Chr5:147,6,-147,63, Eset 1 2 H3K9Me3-control 1 2 H3K9Me3-Eset RNAi 1

12 Yuan_FigS12 A B Fold enrichment above background Cell 2-Cell 4-Cell 8-Cell Morula E3.5 E4.5 Relative Gene Expression Ratio TE/ICM Pou5f1 Nanog Sall4 Eset Eomes Tcfap2a Gapdh Nanog Pou5f1 Sall4 Eset

13 Yuan_FigS13 Anti-Nanog DAPI Control shrna Anti-Nanog DAPI Eset shrna

14 A Untreated 2-cell-stage embryo 2-cell-stage embryo (transfected with construct with shrna targeting gene of interest) These cells are labeled green due to GFP. Yuan_FigS14 Aggregation of 4-cell-stage embryos Gene involved in repressing ICM formation Gene plays no role in ICM or TE formation Gene involved in repressing TE formation B Luc shrna Eset shrna C Experiment Tranduced shrna Total number of blastocysts Blastocysts with GFP in ICM and TE Blastocysts with GFP in TE only 1 Control shrna Eset shrna Control shrna Eset shrna Control shrna 1 1 Eset shrna 1 5 5

15 Yuan_FigS15 A Control shrna Eset shrna B Relative expression (%) Pl1 Pl2 Plf Control shrna Eset shrna

16 Control shrna Eset shrna Yuan_FigS16

17 Yuan_FigS17 A Relative expression (%) Eomes Cdx2 Tpbpa Pl1 Pl2 Plf Control shrna Eset shrna TS markers Intermediate diploid trophoblast marker Trophoblast giant cell markers B GFP Anti-Kip2 DAPI Merge Control shrna GFP Anti-Kip2 DAPI Merge Eset shrna

18 Yuan_FigS18 A B Control shrna Control shrna Eset shrna Eset shrna L S D L: Labyrinth; S: Spongiotrophoblast; D: Decidua

19 Yuan_FigS19 Transcription Number of Number of TF-Eset % Eset Number of TF-control p-value (Fisher's exact test) factor (TF) sites * overlap overlap library overlap Sox Nanog Oct E-8 Suz E-25 * Chen et al (28). Cell. 133(6):

20 Yuan_FigS2 A Relative expression (%) ES cell Eset TS cell B Fold enrichment Eset ChIP Cdx2 Tcfap2a Fold enrichment Eset ChIP Nnat C Fold enrichment H3K9me3 ChIP Cdx2 Tcfap2a Fold enrichment H3K9me2 ChIP Cdx2 Tcfap2a ES cell TS cell

21 Supplemental Figure Legends Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells Ping Yuan, Jianyong Han, Guoji Guo, Yuriy L. Orlov, Mikael Huss, Yuin-Han Loh, Lai-Ping Yaw, Paul Robson, Bing Lim and Huck-Hui Ng Supplemental Figure S1. Characterization of antibodies Characterization of antibodies raised against Eset by Western blotting. ES cell were transfected with control vector (psuper plasmid), control shrna (Luc shrna targeting luciferase), Eset shrna1, Eset shrna 2 or Eset overexpression vector. The ES cell lysates were harvested 72 hrs after transfection and used to test the specificity of the antibody. Western blot analyses of ES cell lysates were carried out using anti-eset antibody. Supplemental Figure S2. Analysis of marker gene expression after Eset rescue. (A) The ES cells were transfected with plasmids expressing control Luc shrna, Eset shrna1 or Eset shrna1 and Eset RNAi-immune cdna with silent mutation at Eset shrna1 region. The RNA samples were harvested 5 days later. Data are presented as the mean ± s.d., n = 3. *, p<.5; **, p<.5. (B) The ES cells were transfected with plasmids expressing control Luc shrna, Eset shrna2 or Eset shrna2 and Eset RNAi-immune cdna with silent mutation at Eset shrna2 region. The RNA samples were harvested 5 days later. Data are presented as the mean ± s.d., n = 3. *, p<.5; **, p<.5. 1

22 Supplemental Figure S3. Eset depleted cells show trophoblast giant cell-like morphology Five days after transfection of Eset shrna plasmid, some cells show enlarged nuclei (labeled with white arrow) and are morphologically similar to trophoblast giant cells. A similar phenotype was observed for Pou5f1 knockdown cells (marked with white arrow). Supplemental Figure S4. Global gene expression changes after depletion of Eset. Microarray analysis was performed to measure gene expression changes at 5 days after Eset knockdown. Microarray heatmaps depicting expression changes of selected ES cellassociated and trophoblast lineage associated genes are shown. Red indicates increased expression compared to control samples, whereas green means decreased expression. The genes expression levels were mean centered to show their relative change. *, p<.5. Supplemental Figure S5. Colony formation assay of Eset depleted cells. (A) Bright-field microscopy of re-plated ES cells. The ES colonies were stained for alkaline phosphatase activity. (B) Eset knockdown ES cells formed less colonies in secondary re-plating assay compared to control knockdown ES cells. (C) Counting of the alkaline phosphatase-positive and differentiated colonies of replating cells. The average of 5 different microscopy fields from two independent experiments was shown. Supplemental Figure S6. Specificity of Eset antibody. (A) Specificity of the Eset antibody was confirmed by immunofluorescence staining of control shrna and Eset shrna knockdown cells. 2

23 (B) To validate the Eset ChIP-seq dataset, we performed ChIP assays using chromatin extract from ES cells expressing Eset shrna. The chromatin extract transfected with control RNAi construct was used as control. 2 loci were tested and they showed reduced ChIP signal upon Eset depletion. Supplemental Figure S7. Expression analysis of Eset-bound genes versus non-eset bound genes. Comparison of the expression of Eset bound genes versus non-eset bound genes in ES cells. Two different sets of ES cell expression microarray data (GEO ID: GSE2972 and GSE3766) were used for the analysis. Eset-bound genes showed lower average expression level in independent expression datasets. Supplemental Figure S8. Expression of imprinted genes upon Eset knockdown. Supplemental Figure S9. Kinetics of induction of Tcfap2a and Cdx2 at different timepoints after Eset depletion. ES cells were transfected with control shrna or Eset shrna constructs. The mrna was harvested from day 2 to day 5 after puromycin selection and assessed against control shrna knockdown sample by real-time PCR analysis. Data are represented as mean ± s.d., n = 3. Supplemental Figure S1. Eset and H3K9me3 ChIP-seq profiles at the Tcfap2c gene. (A) Real-time PCR analysis of Tcfap2c expression after knockdown using Eset shrna construct. Control shrna targets luciferase sequence. The levels of the transcripts were normalized against control Luc shrna. 3

24 (B) Scheme of the amplicons (black bars labeled 1 4) used to analyze ChIP-enriched fragments over the Tcfap2c. (C) Eset regulates H3K9me3 and H3K9me2 levels of Tcfap2c. ChIP assays were performed using anti-h3k9me3 (top panel) or anti-h3k9me2 (bottom panel) antibodies with extracts prepared from Eset knockdown (black bars) and control knockdown (white bars) cells. (D) Eset binds to Tcfcp2c. ChIP assays were performed with Eset or control GFP antibodies and DNA samples were measured by real-time PCR using primers targeting amplicons shown in (B). Supplemental Figure S11. Eset and H3K9me3 ChIP-seq profiles at the Cdx2 gene. Supplemental Figure S12. Gene expression in the preimplantation embryo (A) Equivalent amounts of total RNA from freshly harvested embryos at the developmental stages indicated were analyzed for Gapdh, Nanog, Pou5fl, Sall4 and Eset, by reverse transcription and Taqman realtime-qpcr. A system background of Ct=28 was used as a reference. Each value is a mean of 2-4 replicates. (B) Gene expression difference between manually dissected TE and ICM cells. For a specific gene, its expression level in TE cells was compared with that in ICM cells after normalizing to Gapdh expression. Supplemental Figure S13. Defective ICM outgrowth of Eset-depleted blastocysts. Eset shrna transduced blastocysts displayed defective ICM outgrowth after culture in ES medium for four days while normal ICM outgrowth was observed in control shrna transduced blastocysts. The cells were stained with Nanog antibody to identify the pluripotent cells. Control knockdown blastocysts displayed normal ICM outgrowth with Nanog positive 4

25 cells on the trophoblast layer. Eset knockdown blastocysts did not give any Nanog positive cells. Supplemental Figure S14. Embryo aggregation assay to determine the fate of Eset depleted cells (A) Diagrammatic sketch of embryo aggregation assay. After removal of zona pellucida, the 2-cell stage embryo is transfected with shrna plasmid harboring GFP reporter gene. The cells were allowed to develop to 4-cell stage. Two 4-cell embryos were aggregated with a 4- cell embryo transfected either with control or Eset shrna plasmid. The resulting aggregrates were cultured in microwells. Giant blastocysts were formed after two days and the localization of transfected GFP positive cells was traced by fluorescence microscopy. If the gene of interest is involved in suppressing ICM formation, depletion of its transcript by RNAi will lead to preferential incorporation of GFP positive cell into ICM. However, if the gene of interest is involved in suppressing TE formation, depletion of its transcript by RNAi will result in preferential incorporation of GFP positive cells into TE. Depletion of transcript that has no effect on ICM or TE formation will result in random incorporation of the GFP positive cells into ICM or TE. (B) Control or Eset shrna constructs were transfected into 2-cell embryos. Embryo aggregration assay was conducted as described in (A). Fluorescence microscopy was used to visualize the GFP positive cells. (C) Quantitation of GFP positive cells in ICM and TE for three independent embryo aggregation experiments. Supplemental Figure S15. Generation of trophoblast giant cells from Eset-depleted ES cells. 5

26 (A) Trophoblast giant cells were formed from Eset shrna knockdown ES cells, but not from control shrna knockdown ES cells. After three days post infection, FACS was used to isolate GFP positive cells. The cells were then cultured in TS cell medium for five days. Scale bar represents 1 μm. (B) Trophoblast giant cell marker genes Pl1, Pl2 and Plf were induced in the cells shown in (A). Data are represented as mean ± s.d., n = 3. Supplemental Figure S16. Comparison of gene expression profiles of TS cells versus Eset knockdown cells. To define the genes preferentially up-regulated in TS cells, we analyzed publicly available expression datasets for ES cells and TS cells (GEO ID: GSE2766). Top 281 Affymetrix probe sets with higher expression values for TS cells as compared to ES cells were selected using SAM software. A criterion of more than 2 fold (TS cell / ES cell) was used. Unique gene names corresponding to the probe sets IDs were identified and mapped to our Illumina microarray data obtained from 5 days Eset shrna and control shrna knockdown ES cell samples. Genes that displayed absent calls or negative values were removed. Finally, the expression values of 159 non-redundant genes were used to generate the heatmap. Among them, 14 genes (65%) are upregulated with higher average expression level in Eset knockdown cells as compared to the control knockdown cells. 55 genes (35%) are downregulated after Eset knockdown. Thus, majority of TS cell up-regulated genes (about 2/3) are induced when Eset is depleted in ES cells. Supplemental Figure S17. Knockdown of Eset in TS cells (A) TS cell markers Eomes and Cdx2 were reduced, while intermediate diploid trophoblast marker Tpbpa, trophoblast giant cell markers Pl1, Pl2 and Plf were induced in Eset shrna 6

27 lentivirus infected TS cells as compared to the control knockdown TS cells. Data are represented as mean ± s.d., n = 3. (B) Immunofluorescence staining of Eset and control knockdown cells with anti-kip2 antibody. Trophoblast giant cell marker Kip2 was expressed in Eset shrna lentivirus infected TS cells but not expressed in the control shrna lentivirus infected TS cells. Scale bar represents 1 μm. Supplemental Figure S18. Detection of GFP positive cells in the chimeric placental tissues. (A) Fluorescence microscopy was used to visualize the GFP fluorescence of placentas. ES cells were infected by control shrna lentivirus or Eset shrna lentivirus which also harbors a GFP reporter. The GFP-positive cells were FACS sorted two days after infection, and followed by culture for one more day in TS cell medium. These cells were then injected into the 4 to 8 cell stage embryos. These embryos were cultured in KSOM medium till blastocyst stage and then transferred to pseudopregnant mouse to allow the blastocysts to develop in vivo until day 9.5. Eset shrna lentivirus infected GFP positive cells can be found in the chimeric placenta while control shrna lentivirus infected ES cells were absent from the placenta. (B) Immunohistochemical analysis of the placenta shown in (A). GFP positive cells were detected by immunohistochemical staining by DAB (brown coloration) in chimera placenta derived from Eset shrna lentivirus infected cells. Supplemental Figure S19. Co-localization analysis with Oct4, Sox2, Nanog and Suz12 ChIP-seq datasets 7

28 Supplemental Figure S2. Eset occupancy, H3K9me3 and H3K9me2 levels at Cdx2 and Tcfap2a in TS cells (A) Level of Eset transcripts in ES cells and TS cells as measured by real-time PCR analysis. Data are represented as mean ± s.d., n = 3. (B) Eset occupancy at Cdx2 and Tcfap2a in ES cells and TS cells was determined by realtime PCR analysis of Eset ChIP-enriched DNA. Eset binding at Nnat (an imprinted gene) served as a positive control. Data are represented as mean ± s.d., n = 3. (C) No enrichment of H3K9me3 and H3K9me2 levels was detected at Cdx2 and Tcfap2a in TS cells. Supplemental Table S1. Genomic loci and genes showing Eset occupancy Supplemental Table S2. Genomic loci and genes showing Eset-dependent trimethylation Supplemental Table S3. Known and putative imprinted genes showing Eset occupancy and Eset-dependent H3K9me3 8

29 Supplemental Materials and Methods TS cell culture and lentivirus infection TS cells were maintained as described previously (Tanaka et al. 1998). Briefly, mitomycintreated MEFs were used as feeders to support the TS cells in TS cell medium (2% fetal bovine serum, 1 mm Sodium-pyruvate, 5 U ml 1 penicillin, 5 µg ml 1 streptomycin, 5 µm β-mercaptoethanol, 25 ng ml 1 bfgf (Sigma) and 1 µg ml 1 heparin in RPMI164 (Invitrogen). For feeder-free TS cell culture medium, 7% TS cells medium is preconditioned on MEF feeder cells. TS cell differentiation medium contains no heparin and bfgf. The TS cell medium is changed every two days and the TS cells are passaged every fourth day at 1:1 ratio after trypsin treatment. To achieve efficient infection, the TS cells were infected at high MOI immediately after passage. The virus-containing medium was removed after 12 hours and the lentivirus-infected TS cells are maintained in TS cell medium. Immunohistochemical analysis of chimeric placenta tissues The 9.5 dpi placentas were harvested. They were then fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned. The sections were incubated with mouse anti-gfp monoclonal antibody (Santa Cruz) and peroxidase-conjugated mouse IgG using Vectastain ABC kit (Vector laboratories, Inc). The signal was detected by DAB staining followed by counterstaining with hematoxylin. Analysis of Eset bound genes versus non-eset bound genes in ES cells To estimate expression level of Eset target vs non-eset target genes, expression data derived from R1 ES cells (GEO access: GSE2972, MOE43A and B Affymetrix array) were used for analysis. For both arrays, the non-redundant gene symbols were mapped corresponding to RefSeq gene names. Eset-bound genes were defined by the criterion of a binding site within 9

30 5Kbp distance from the TSS. A total of 14,485 genes was recovered from the two arrays. Among them, 1835 were Eset-bound genes. Average expression values for the three replicates were calculated and weighted based on common genes across the arrays. We repeated this analysis using an independent microarray data derived from R1 ES cells (GEO access: GSE3766). This array includes 7,854 non redundant gene names, among them 98 genes were bound by Eset. The average of two replicates was used to compare the expression of Eset bound genes versus non-eset bound genes. 1

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