Supporting Information

Li et al. 10.1073/pnas.1617802113 SI Materials and Methods transfection. Cells were transfected with reporter constructs TOP- Plasmids. Mutant Tet3 (H950D and Y952A) was generated by or FOP-Flash using Lipofectamine 2000 (Invitrogen). extracts PCR using Pfu polymerase, Dpn I treatment, and transformation were prepared 48 h after transfection. The luciferase activity was (Stratagene). The ORF of mouse Tet3, mutant Tet3 (H950D and evaluated by using the Dual-Luciferase Reporter Assay System ’ Y952A), Sfpr4, Pcdha4, and Pcdha7 were cloned into pPyCAGIP (Promega) according to the manufacturer s recommendations. vector (60). qRT-PCR. qRT-PCR was performed by using Universal SYBR Green Generation of Tet3 KO and Tet1/2/3 TKO mESCs. Tet3 KO and Tet1/2/3 Master Mix (Roche) and analyzed by a StepOne Plus real-time PCR ’ TKO mouse ESCs were generated from mice (C57BL/6 back- system (Applied Biosystems), according to the manufacturer sin- ground) bearing the individual floxed alleles (Fig. S1C) (14, 18), structions, and the data were normalized for Gapdh expression. followed by excision of the floxed by transient expression of The primers used for qRT-PCR are listed in Dataset S4. Cre recombinase. Immunohistochemistry. Immunohistochemistry was performed as mESC Culture and Differentiation. mESCs were maintained on mi- described (61) with the primary antibodies described below. For ∼ tomycin C-treated mouse embryonic fibroblasts (MEFs; feeders) in statistical analysis, 300 cells were examined for each experiment, standard medium (61). Neural differentiation in SFEB culture was which was repeated four times. Mouse embryos were fixed over- night in 4% (wt/vol) paraformaldehyde, saturated with 20% (wt/vol) performed as described (31) with a minor modification. Briefly, μ mESCs were dissociated to single cells in 0.25% trypsin–EDTA sucrose, and frozen in O.C.T. embedding medium, and 8- msec- (Invitrogen). Dissociated ESCs (1 × 105 cells per mL) were seeded tions were prepared on a cryotome. The following antibodies were onto a nonadhesive bacterial-grade dish and cultured in Knockout used: anti-Sox1 (no. 4194; Cell Signaling), anti-Sox2 (NL20181V; DMEM supplemented with 10% (vol/vol) KSR, 2 mM glutamine, R&D), anti-cTnT (CT3; Hybridoma Bank), anti-T (Brachyury) (AF2085; R&D), anti-Gata4 (sc-1237; Santa Cruz), anti-Tbx6 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol (AF4744; R&D), anti-Isl1 (NL1837R; R&D), anti-Foxa2 (2-ME). On day 2, the medium was changed to N2B27 medium (IC2400G; R&D), and anti–Active-β-Catenin (05-665; Millipore) (62). The medium was changed every other day. The day on which generated against the peptide 36-44, HSGATTTAP, dephosphory- ESCs were seeded to differentiate is defined as differentiation day lated at Ser-37 and Thr-41. 0. Because Tet1/2/3 TKO mESCs did not differentiate well under SFEB culture in N2B27 medium, they were differentiated by using Whole-Transcriptome RNA-Seq. Total RNA was extracted by using an the SFEB method in 10% (vol/vol) KSR medium continuously. RNeasy Plus Mini Kit (Qiagen). Multiplexed libraries were con- For nonpermissive neural differentiation, dissociated ESCs (1 × 5 structed by using a SOLiD Total RNA-seq kit. Libraries were 10 cells per mL) were seeded onto an ultralow attachment culture sequenced on the SOLiD 5500 platforms according to the man- dish (Corning) and cultured in Knockout DMEM supplemented ufacturer’s instructions. Approximately 10–15 million uniquely with 15% (vol/vol) FBS, 2 mM glutamine, 0.1 mM nonessential mapped reads per sample were generated. Reads were aligned amino acids, and 0.1 mM 2-ME for 4 or 7 d. to the mouse genome (mm9; NCBI37) by using Tophat. DE-Seq was performed to identify differentially expressed . GO Tet1 Generation of Tet1/2/3-Deficient Mice. The was inactivated biological process analysis was performed by using DAVID Bio- – by targeting its 8 10 encoding part of the catalytic domain informatics Resources 6.7 (https://david.ncifcrf.gov/). Pathway anal- Tet2 The Tet3 (18). targeting was described (14). gene was in- yses were performed by using Ingenuity Pathway Analysis software. activated by targeting exon 2 (18). The deleted allele bears a frame-shift that results in a truncated (Fig. S1B). ChIP-Seq. ChIP using anti-Tet3 antibody (gift of Guoliang Xu, The strains of Zp3-Cre and Stra8-Cre mice used in this study are Institute of Biochemistry and Cell Biology, Shanghai, China) FVB/N-TgN(Zp3-Cre)3Mrt and Tg(Stra8-Cre)1Reb/J (Jackson (16) was performed in NPCs. To generate NPCs, mESCs were Laboratories) respectively. Zp3-Cre and Stra8-Cre are exclusively differentiated in SFEB culture conditions [10% (vol/vol) KSR] expressed in growing oocytes and spermatogonia, respectively (63, for 5 d, followed by adherent culture for 6 d in N2B27 with 10 ng/mL 64). We generated Tet1/2/3 triple-deficient mice by crossing Zp3- EGF and basic FGF. ChIP assays were carried out as described fl/fl Cre and Stra8-Cre mice with Tet1/2/3 mice to generate mice in (5). Briefly, chromatin was sheared by using truChIP High which expression of all three Tet was abrogated in oocytes Cell Chromatin Shearing Kit with Nonionic Shearing Buffer fl/fl and sperm, respectively. The progeny of Tet1/2/3 Zp3-Cre female (Covaris). Chromatin fragments from two biologically independent fl/fl and Tet1/2/3 Stra8-Cre male lack all three Tet proteins beginning at NPCs were immunoprecipitated by using anti-Tet3 antibody. the zygotic stage. Tet1/2/3 triple-deficient mice were on C57BL/6 ChIP-seq libraries were constructed by using a 5500 SOLiD background. Fragment 48 Library Core Kit. Libraries were sequenced on the SOLiD 5500 platforms, according to the manufacturer’s in- Whole-Mount in Situ Hybridization. Embryos from E9.25 were dis- structions. Sequencing reads were mapped against mm9 by using sected, fixed at 4 °C overnight in 4% (wt/vol) paraformaldehyde/ Bowtie. Tet3-enriched regions were identified by MACS peak- PBS, dehydrated through a series of increasing methanol con- calling software (Version 1.4.2) (66). We had performed pulldowns centrations, and stored at −20 °C. Whole-mount in situ hybrid- by using control IgG antibody, but no chromatin was recovered. ization was performed as described (65). Tet3 probe was amplified Therefore, sequencing reads from input were used as negative from mouse cDNA (GenBank accession no. NM_183138.2) and controls in MACS. The statistical cutoff used for identifying corresponds to the nucleotide 569–1,443 region. Tet3-binding sites was P < 0.0005. Genomic distribution of Tet3- bound sites was performed by using the cis-regulatory element Luciferase Reporter Assay. β-catenin/TCF reporter assay was per- annotation system (67). The distribution of Tet3-binding sites to formed as described (61). Briefly, cells were seeded at a density of TSS was undertaken by using ChIPseek (68). De novo motif 1 × 105 per well into a 24-well tissue-culture plate 24 h before discovery was performed by using the findMotifsGenome.pl

Li et al. www.pnas.org/cgi/content/short/1617802113 1of9 function of the Homer software package (69) using default pa- using BS-Seeker2 (71). The primer sequences are shown in rameter settings. Tet3-specific peaks within 100 kb flanking the Dataset S4. TSS were used for motif analysis. Single-Embryo RNA Sequencing. CTL and Tet1/2/3 TKO embryos BS-Seq. BS-seq was performed as described (70). Briefly, genomic were collected at E6.5. Single embryos were lysed and converted DNA was extracted by using PureLink Genomic DNA mini Kit into double-stranded cDNA by using the SMARTer ultralow (Invitrogen) and treated with sodium bisulfite (MethylCode Bi- RNA kit for Illumina sequencing (Clontech). A total of 1 ng of sulfite Conversion Kit; Invitrogen). The PCR amplicons were cDNA for each sample was used for preparing libraries by using generated by using a PyroMark PCR kit (Qiagen) and quantified by the Nextera XT DNA sample preparation kit (IIlumina). The using a Quant-iT PicoGreen dsDNA reagent (Invitrogen). The good quality of the prepared libraries was validated by using the PCR amplicon was generated by using a PyroMark PCR kit Bioanalyzer high-sensitivity DNA kit (Agilent). Libraries were μ sequenced on Illumina HiSeq 2500 platforms according to the (Qiagen). PCR amplicons were then mixed together for 1 g final ’ – quantity and used for the library preparation using NEBNext manufacturer s instructions. Approximately 10 15 million DNA Library Modules for illumine platform (NEB). The final uniquely mapped reads per sample were generated. Reads were aligned to the mouse genome (mm9; NCBI37) by using Tophat. libraries were then combined together and quantified by using a DE-Seq was performed to identify differentially expressed genes. KAPA library quantification kit for Illumina (KAPA Biosystems), GO biological process analysis was performed by using DAVID then sequenced on Miseq (300 bp, paired end; Illumina). The data Bioinformatics Resources 6.7 (https://david.ncifcrf.gov/). are based on thousands of sequence reads per amplicon. To monitor bisulfite conversion efficiency, a 210-bp spike-in control Statistical Analysis. All values are shown as means ± SD. To de- was generated by using unmethylated lambda DNA (Promega) as termine the significance between groups, comparison was made by a template and added to the genomic DNA to a final ratio of using Student’s t test. For all statistical tests, the 0.05 confidence 0.5%. Based on C-to-T conversion efficiencies of spike-in controls, level was considered statistically significant. In all figures, * de- the average bisulfite conversion efficiencies were >99.5%. Reads notes P < 0.05 and ** denotes P < 0.01 in a two-tailed Student’s were aligned to the genome and used to measure methylation t test.

Li et al. www.pnas.org/cgi/content/short/1617802113 2of9 Fig. S1. Tet3 mediates neuroectoderm and cardiac mesoderm cell fate determination in mESCs. (A) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels on E6.5, E7.5, E8.5, and E9.5. For E6.5 and E7.5, three embryos are pooled. Data are shown as mean ± SD (n = 3). (B) RNA-seq data for Tet1, Tet2, and Tet3 mRNA expression in E6.75 and E7.5 embryos. RPKM, reads per kilobase of transcript per million mapped reads. (C, Left) Schematic view of targeting scheme for deletion of exon 2 in the endogenous Tet3 locus. (C, Right) Genotyping of Tet3 fl/fl and Tet3 KO mESCs. Bands for floxed (C, Upper) and Tet3 KO (C, Lower) alleles are shown. Genotyping primer sequences are provided in Dataset S4. (D) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels in WT and Tet3 KO mESCs. Data are shown as mean ± SD (n = 3). (E) Schematic representation of Tet3 and catalytically inactive TET3 HxD mutant. Cys-rich, cysteine-rich; DSBH, double-stranded β-helix. *P < 0.05; **P < 0.01.

Li et al. www.pnas.org/cgi/content/short/1617802113 3of9 Fig. S2. Tet3-dependent transcriptional programs during mESC differentiation. (A) Heat map of genes related to neuroectoderm differentiation, which were significantly activated by Tet3, and genes related to mesoderm differentiation, which were significantly repressed by Tet3. Red, high expression; blue, low expression using Z-score values normalized. (B and C) Signaling pathway analysis (using Ingenuity Pathway Analysis software) of genes differentially expressed (at least twofold change) in Tet3 KO relative to WT mESCs on days 6 (B) and 10 (C) of SFEB culture. The statistically significant canonical pathways are listed according to their P value (−Log10) (blue bars), and the ratio of differentially expressed genes found in each pathway over the total number of genes in that pathway (ratio) is shown by orange squares. The threshold line corresponds to a P value of 0.01. Note the predominance of the Wnt/β-catenin signaling pathway (red asterisks) at both 6 and 10 d. (D and E) Tet3 ChIP-seq peaks in NPCs were enriched in gene promoters (D) and gene bodies (E) above the genomic background by using randomly generated peaks.

Li et al. www.pnas.org/cgi/content/short/1617802113 4of9 Fig. S3. Tet3 regulates mESC differentiation by modulating Wnt/β-catenin signaling. (A) Heat map of genes related to neuroectoderm differentiation, which were repressed in the absence of Tet3 and whose expression was restored by the addition of Dkk1 to the culture. Red, high expression; blue, low expression using Z-score values normalized. (B) Heat map of genes involved in mesoderm differentiation, which were activated in the absence of Tet3 and whose ex- pression was inhibited by addition of Dkk1 to the culture. Red, high expression; blue, low expression using Z-score values normalized.

Li et al. www.pnas.org/cgi/content/short/1617802113 5of9 ie al. et Li www.pnas.org/cgi/content/short/1617802113

Fig. S4. Tet3 regulates mESC differentiation through Pcdha cluster. (A) University of California Santa Cruz Genome Browser snapshots showing Tet3 binding sites in Pcdha. Gene tracks are shown at the bottom. The y axis of binding profiles denotes the numbers of sequence tag reads. (B) qRT-PCR analysis of transcripts of Pcdha in Tet3 KO mESCs on day 6 of SFEB culture. The transcript level in WT cells was set as 1. Data are shown as mean ± SD (n = 3). All bars are <1 with P < 0.05. (C–H) BS-seq showing the percentage of 5mC+5hmC at each CpG site in the promoter regions of Pcdha4 (C), Pcdha9 (D), Pcdha12 (E), Pcdhac1 (F), Pcdha8 (G), and Pcdhac2 (H) in WT and Tet3 KO mESCs on day 6 of SFEB culture. The positions of CpG sites are indicated relative to the TSS. Data are shown as mean ± SD (n = 2). *P < 0.05. (I) TOP/FOP-Flash luciferase reporter assay after transient expression of vector, Pcdha4 or Pcdha7 in mESCs induced to differentiate by withdrawal of LIF plus addition of 6of9 0.1 μM all-trans retinoic acid (RA) for 4 d. Data are shown as mean ± SD (n = 3). **P < 0.01. Fig. S5. Cardiac progenitor marker Isl-1 expression is increased in the Tet3 KO heart. (A) qRT-PCR analysis of the transcript level of Isl1 of whole hearts from E18.5 WT and Tet3 KO embryos. Data are shown as mean ± SD (n = 3). (B and C) Immunostaining for Isl1 in transverse (B) and sagittal section (C) of heart from E18.5 WT and Tet3 KO embryos. Nucleus staining: DAPI (blue). **P < 0.01. (Scale bars: B, 100 μM; C, 62.5 μM.)

Fig. S6. The phenotype of Tet3-deficient mESCs is exacerbated by concurrent deficiency of Tet1 and Tet2. (A) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels in WT and Tet1/2/3 TKO mESCs. Data are shown as mean ± SD (n = 3). (B) Growth curves of WT and Tet1/2/3 TKO mESCs. Cells were split every 3 d, and cells were counted. (C and D) qRT-PCR analysis of transcripts of neural maker genes Sox1 and Foxg1 (C), mesoderm marker T (Brachyury), and car- diomyocyte marker genes Nkx2-5, Myh7,andTnnt2 (D). WT or Tet1/2/3 TKO mESCs were cultured in differentiation medium containing 15% (vol/vol) FBS for 7 d. Data are shown as the mean ± SD (n = 3). (E and F) qRT-PCR analysis of transcripts of neural marker genes Sox1 and Foxg1 (E) and cardiac mesoderm marker T(Brachyury), Nkx2-5, Myh7,andTnnt2 (F). WT or Tet3 KO mESCs were differentiated under SFEB culture conditions for 7 d in 10% (vol/vol) KSR. Data are shown as mean ± SD (n = 3). (G–I) qRT-PCR analysis of transcripts of Sfrp4 (G), Pcdha1, Pcdha4, Pcdha9 (H), and Pcdh8 (I). WT or Tet1/2/3 TKO mESCs were differentiated under SFEB culture conditions for 7 d in 10% (vol/vol) KSR. Data are shown as mean ± SD (n = 3). **P < 0.01.

Li et al. www.pnas.org/cgi/content/short/1617802113 7of9 Fig. S7. Triple Tet deficiency promotes the expression of mesoderm-related genes during early embryogenesis. (A) Phase contrast images of WT (Tet1/2/3 fl/fl) and Tet1/2/3 TKO embryos at E6.75. (B) Genome browser snapshots showing RNA-seq reads for targeted exons of Tet1, Tet2, and Tet3. Reads corresponding to deleted Tet1 exons 8–10, Tet2 exons 8–10, and Tet3 exon 2 are specifically absent in Tet1/2/3 TKO embryos. (C) Hierarchical clustering of data for the 69 most significant differentially expressed genes between WT and Tet1/2/3 TKO E6.75 embryos. P < 0.0005; adjusted P value < 0.1. Red, high ex- pression; blue, low expression using Z-score values normalized. (D) GO biological process analysis of up-regulated genes in Tet1/2/3 TKO embryos at E6.75. (E) RNA-seq data for seven mesoderm genes (Six1, Six4, Alx1, Alx3, Bin1, Hoxd13, and Crabp2) involved in regulation of embryonic limb and skeletal devel- opment. *P < 0.05; **P < 0.01.

Li et al. www.pnas.org/cgi/content/short/1617802113 8of9 Fig. S8. TET proteins control the balanced differentiation of NMPs during early embryogenesis in vivo. (A) Phase contrast image of WT and Tet1/2/3 TKO embryo at E7.25–E7.5. (Scale bars: 100 μM.) (B–E) Immunocytochemistry of WT (E7.25) or Tet1/2/3 TKO (E7.5) for Sox2 (B), T (C), Sox2 and T (D), active β-catenin (E). ne, neuroectoderm; no, node; NS, nonspecific; ys, yolk sac. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 3). (F) Immunocy- tochemistry of WT or Tet1/2/3 TKO at E8.25-E8.5 for Foxa2. Due to the failure of neural plate closure, both left and right sides of the embryo are shown in Tet1/ 2/3 TKO embryo sections. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 3). (G) RNA-seq data for Wnt3 and Nodal expression in WT and Tet1/2/3 TKO embryos at E7.25–7.5 shown in A.

Other Supporting Information Files

Dataset S1 (XLSX) Dataset S2 (XLSX) Dataset S3 (XLSX) Dataset S4 (XLSX)

Li et al. www.pnas.org/cgi/content/short/1617802113 9of9