The mammalian TET enzymes catalyze DNA demethylation. While they have been intensely studied as major epigenetic regulators, little is known about their physiological roles and the extent of functional redundancy following embryo implantation. Here we define non-redundant roles for TET1 at an early postimplantation stage of the mouse embryo, when its paralogs Tet2 and Tet3 are not detectably expressed. TET1 regulates numerous genes defining differentiation programs in the epiblast and extraembryonic ectoderm. In epiblast cells, TET1 demethylates gene promoters via hydroxymethylation and maintains telomere stability. Surprisingly, TET1 represses a majority of epiblast target genes independently of methylation changes, in part through regulation of the gene encoding the transcriptional repressor JMJD8. Dysregulated gene expression in the absence of TET1 causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice. Collectively, our study highlights an interplay between the catalytic and non-catalytic activities of TET1 that is essential for normal development.
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We thank G. Daley and A. Yabuuchi (Harvard Medical School) for generating the Tet1 GT mouse. SMARTer RNA–seq and amplicon sequencing were performed by K. Coeck at the VIB Nucleomics Core (KU Leuven) under the expert guidance of P. Verhasselt, R. Janky, W. Van Delm and S. Derveaux. WGBS and oxWGBS were performed at Novogene, and data were analyzed by Y.S. Li. We also thank L. Vermeire and L. Umans for technical guidance in mouse embryo manipulation and protocols for WISH, and H. Zhao for help with bioinformatic analyses. We are grateful for suggestions from M. Wilkinson and C. Verfaillie in the critique of this manuscript. This work was supported by Fonds voor Wetenschappelijk Onderzoek (FWO) Research Foundation–Flanders grants G.0C56.13N and G.0632.13N, Ministerie van de Vlaamse Gemeenschap and Marie Curie Career Integration grant PCIG-GA-2012-321658 (K.P.K.), US NIH grant R35 CA210043 (A.R.) and ERC Consolidator Grant award CHAMELEON 617595 (D.L.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Temporal patterns of 5hmC and mapping of the insertion cassette in the Tet1 gene-trap allele.
(a) 5hmC immunohistochemistry on longitudinal sections of wild-type embryos at E7.5–8.5. Scale bars, 100 μm. Epi, epiblast or embryonic ectoderm; ExE, extraembryonic tissues; VE, visceral endoderm; M, mesoderm; HF, head fold; Ch, chorion; Am, amnion. (b) Schematic representation of the Tet1 gene from exons 2 to 13 (E2 to E13; white boxes) and the position of the gene-trap cassette insertion. En2 intr1, engrailed-2 intron 1; SA, splice acceptor; β-geo, beta-galactosidase–neomycin resistance fusion gene; pA, poly(A). (c) Genome-wide coverage plot of targeted locus amplification (TLA) sequencing results confirms a single gene-trap event at the Tet1 locus on chromosome 10. (d) UCSC Genome Browser view of TLA read coverage over the Tet1 intron, between exon 2 and exon 3, at Chr10:62330560–62338166 (NCBI37/mm9). Note that the reads are mapped to the minus strand. Informative reads (black boxes) flanking regions with no coverage (deletions) or indicating fusion events with the gene-trap cassette are shown below, aligned to the genomic location. A read sequence fragmented into two parts of divergent sequence directionality (red arrows) is shown joined by a curve to indicate an inversion event. Genomic sections corresponding to all read fragments are labeled by lettered green bars shown above the track. (e) Southern blot analysis of genomic DNA harvested from wild-type (no bands detected) and RRG140 ESCs, using a probe for the β-geo sequence (position marked by the red line in h), suggesting integration of three copies of the gene-trap cassette. (f) Quantitative gene copy dosage measurement of the lacZ gene in genomic DNA harvested from the RRG140 cell line and from Tet1GT/wt mice (generation N2–3), indicating three copies of the gene-trap cassette per haploid genome. Similar results were obtained with Tet1GT/wt mice of further backcross generations N6–8 (data not shown). Error bars, s.e.m. of technical replicates of the cell line and biological replicates of mice (n = 8). (g) Sequences of fusion regions determined by Sanger sequencing of a long-range PCR product amplified using a forward primer in region A and a reverse primer in the gene-trap En2 region, confirming the fusion event at the site of insertion and a deletion between regions A and D. (h) Schematic reconstruction of the genome rearrangements at the Tet1 gene-trap insertion site inferred from the results of c–g. Arrows indicate the direction of transcription. RV, EcoRV restriction site. An asterisk refers to a possibly truncated third tandem copy of the integrated cassette of <4 kb.
Supplementary Figure 2 Expression of Tet1 in Tet1GT/wt embryos and phenotypic analyses of Tet1-deficient mouse strains.
(a) X-gal staining (blue) of preimplantation Tet1GT/wt embryos. (b) Whole-mount X-gal staining of TetGT/wt postimplantation E5.5–10.5 embryos. (c) Whole-mount X-gal staining of E5.5–10.5 Tet1GT/GT embryos from early (N ≤ 3) backcross generations. (d) Sagittal sections of stained Tet1GT/GT embryos from early (N ≤ 3) backcross generations. (e) Whole-mount X-gal staining of E8.0–9.5 Tet1GT/GT embryos from late (N ≥ 6) backcross generations. (f) Sagittal sections of stained Tet1GT/GT embryos from late (N ≥ 6) backcross generations. (g) Schematic representation of the Tet1 targeted mutation in the Tet1tm1koh line, showing the insertion of a floxed reporter cassette at the ATG start codon of exon 2 (E2). NLS, nuclear localization signal; BGHpA, bovine growth hormone polyadenylation; pA, polyadenylation. (h) Whole-mount X-gal staining of an E6.5 Tet1lacZ/wt embryo obtained from crossing wt with Tet1lacZ/wt mice. Epi, epiblast or embryonic ectoderm; ExE, extraembryonic tissues; EPC, ectoplancental cone; M, mesoderm; HF, head fold; FB, future brain; NT, neural tube; Ch, chorion; Am, amnion; Al, allantois; NE, neuroepithelium.
Supplementary Figure 3 Quality control of low-input RNA–seq and analysis of differentially expressed genes in the epiblast.
(a) Unsupervised clustering on a Spearman correlation matrix using normalized counts of all sample libraries shows segregation of epiblast (Epi) and extraembryonic ectoderm (ExE) samples. (b) RPKM of markers for epiblast (Oct4, Nanog, Otx2 and Fgf5), ExE (Tead4 and Elf5) and visceral endoderm (VE) (Afp). In Epi samples, epiblast markers (Oct4, Nanog, Otx2 and Fgf5) were detected with high read counts and trophoblast markers (Elf5 and Tead4) were absent. In ExE samples, epiblast markers were absent and trophoblast markers were highly expressed. Reads for the VE marker AFP were only detected at low levels in 4 of the 23 samples, indicating that the dissection had effectively eliminated VE cells within experimental limits. (c) RPKM of Tet1 and RPM of the bacterial β-galactosidase gene (lacZ). The dotted line represents the hypothetical threshold level for Tet1 WISH detection and for lacZ X-gal staining detection. (d) Venn diagram of differentially expressed (DE) genes in the Epi using P < 0.001. Gene names highlighted in red are those in DE1 or DE2 at FDR < 0.05 (Fig. 2b). (e,f) Methylation analysis by clonal Sanger sequencing of bisulfite conversions at Lefty2 (e) and Tet1 (f) in E6.25 wt and Tet1GT/GT Epi. The boxed regions indicate the subset further analyzed by high-throughput amplicon sequencing in Figure 2h,i. Independent biological replicate pools were used to generate Sanger (shown here) and amplicon (Fig. 2i) sequencing data at Tet1 loci. Open and filled circles represent unmethylated and methylated (5mC + 5hmC) CpGs, respectively. The percentages below indicate average methylation levels of all CpGs over the sequences analyzed. TET1 ChIP–seq tracks are binding peaks profiled in mouse ESCs18; the y-axis denotes Tet1 ChIP-seq peaks detected using an antibody generated against a C-terminal epitope normalized to IgG ChIP-seq control. Paired Student’s t test, *P < 0.05.
(a) Venn diagram of DE genes in the ExE using P < 0.001. Gene names highlighted in red are those in DE1 or DE2 defined using FDR < 0.05 (Fig. 3c). (b) RPKM values of selected ExE DE genes. ANOVA, *P <0.05, **P < 0.01. (c) Results of IPA analysis of DE genes between Tet1GT/GT and wt (DE1, P < 0.05), showing enrichment in the oxidative phosphorylation pathway. Genes upregulated significantly in Tet1GT/GT relative to wt are shown in shades of pink through red (higher color intensity with increasing fold change). A double border in red indicates a group or complex that shows significant collective dysregulation of constituent genes. Genes in gray show fold changes with P > 0.05. Genes in white are part of the pathway but had no fold change (P =1) in DESeq2 analysis. (d) qRT–PCR of Tet transcript copies normalized to Gapdh copies. Error bars, s.e.m. of biological replicates of wt ExE (n = 4; from Fig. 3b) and of independent culture passages of TSCs (n = 3). The dotted line indicates background levels. (e) Methylation analysis by clonal Sanger sequencing of bisulfite conversions at the Xaf1 locus in E6.25 wt and Tet1GT/GT ExE. The same bisulfite-treated DNA pool was used for high-throughput amplicon sequencing analysis of the boxed region to generate the data shown in Figure 3i. Results are presented as described in Supplementary Figure 3e,f. (f–h) Methylation analysis by amplicon sequencing of bisulfite conversions at ExE DE gene loci, indicated by bold horizontal bars below gene structure schematic. Percentages for average methylation levels of all CpGs over each sequence analyzed are shown for wt (in blue) and Tet1GT/GT (in black). Paired Student’s t test, *P < 0.05.
Supplementary Figure 5 Methylation changes in Tet1-deficient primed epiblast-like cells examined by dot blot and DNA immunoprecipitation.
(a) qRT–PCR for Tet1 in wt, het and KO ESCs detected using primers spanning exons 12–13 (left) or exons 3–4 (right). Error bars, s.e.m. of biological replicates (n = 3 cell lines). (b) Western blot analysis using a TET1 antibody. (c,d) 5mC dot blot quantification (c) and mass spectrometry measurements (d) of genomic DNA from wt, het and KO ESCs cultured in serum + LIF (SL), in ground state (2iL) and converted to EpiLCs. The red circles in c denote KO male line 12 showing higher levels of 5mC as compared to the other two KO female lines analyzed in parallel. Error bars, s.e.m. (n = 3) of biological replicate lines. (e) Unsupervised hierarchical heat map clustering of the 1,000 most variant peaks from the hMeDIP (left) and MeDIP (right) data sets of EpiLC lines. Red boxes denote KO samples. The sex of each line is indicated below the sample name. (f,g) MA plots of differentially hydroxymethylated regions (DHMRs) (f) and differentially methylated regions (DMRs) (g) in Tet1 KO EpiLCs as compared to wt or het. FDR < 0.05. (h) MA plot of DMRs at CpG islands. FDR < 0.1. (i) Normalized MeDIP–seq read counts in wt, het and KO EpiLCs (left) and distribution of MeDIP peaks gained in KO relative to all peaks (right) among high- (HCP), intermediate- (ICP) and low- (LCP) density CpG promoters. (j) MA plots of 18,762 5hmC peaks on collated hMeDIP data (left) and 1,355 5mC peaks on collated MeDIP data (right) that were significantly gained (red) and reduced (green) in KO 2iL cells compared to wt or het using FDR < 0.05. (k) MA plot of DHMRs with loss of 5hmC in KO EpiLCs superimposed over DHMRs in 2iL ESCs. (l) MA plot of DMRs with gain of 5mC in KO EpiLCs superimposed over DMRs in 2iL ESCs. het, Tet1GT/wt; KO, Tet1GT/GT.
Supplementary Figure 6 Methylation and hydroxymethylation changes in Tet1-deficient primed epiblast-like cells examined by whole-genome bisulfite and oxidative bisulfite sequencing.
(a) Percentage of methylated cytosines (mCs) among all cytosines covered by ≥5 reads in WGBS and oxWGBS. (b) Distribution of mCs in CG, CHH and CHG contexts. (c) Distribution of methylated CpG (mCpG) frequencies by 10% 5mC level bins at all mCpGs with coverage ≥10. HMR, highly methylated region; LMR, lowly methylated region; UMR, unmethylated region. (d) Distribution of methylated CHH or CHG by 5mC level bins at all mCs with coverage ≥10. (e) 5mC levels of CpGs distributed within annotated genomic features in wt (line 19) and KO (line 2) EpiLC samples. (f) Distribution of statistically validated hmCs identified in CG, CHH and CHG contexts. (g) Distribution of hmCpG frequencies by hmC level bins at mCpG sites with coverage >10. The total 5hmCpG number of each sample is shown in brackets. (h) Distribution of hmCpG frequencies by 5mC level bins at mCpG sites with coverage ≥5. The total 5hmCpG number of each sample is shown in brackets. (i) hmC levels at mCpGs distributed within annotated genomic features in wt and KO EpiLC samples. Below, a whisker plot representing 5hmC levels at CGIs for all samples. (j) Heat map showing 5mC levels within 100-bp bins spanning ±5-kb regions centred at TET1 ChIP–seq binding peaks.
(a) Significantly enriched GO terms associated with up- or downregulated genes identified in RNA–seq of KO relative to wt EpiLCs. (b) List of the most significantly enriched GO terms associated with DMR-related genes from both WGBS (top) and oxWGBS (bottom) analyses. (c) Top 20 KEGG pathways associated with DMR-related genes from both WGBS (top) and oxWGBS (bottom) analyses. Dot size is proportional to the number of DMR-related genes in each pathway; colors reflect the range of q values (corrected P values) and the rich factor represents the degree of enrichment.
(a) Expression of Zscan4 in different cell states. Error bars, s.e.m. (n = 3 independent cultures) of each biological replicate. (b) Telomere length measurement by flow-FISH using Cy3-conjugated telomere probe in three wt and three KO ESC lines, cultured in 2iL medium for >5 passages. Percentages of cells with shorter, normal or longer telomeres are indicated with s.d. Two technical replicates are superimposed (light and dark blue lines). The values of subtelomere length fractions are shown below as the mean ± s.e.m. of biological triplicates (n = 3 cell lines). For telomere length data on EpiLCs, refer to Figure 5e,f. KO, Tet1GT/GT.
Supplementary Figure 9 Base-resolution methylation changes at TET1-bound sites and rescue of TET1 expression.
(a) IGV tracks at specific target genes, representing ChIP–seq peaks for TET1 binding and WGBS and oxWGBS methylation levels in EpiLC lines. Green bars indicate the location of CpG islands. Boxes demarcate TET1-bound regions. Regions showing the presence of 5hmC in wt EpiLCs are highlighted in blue. Regions that are lowly methylated in wt but gain methylation in KO are highlighted in red. (b) Western blot for TET1 in HEK293T cells expressing full-length TET1 from the pEF1 or PiggyBac (PB) vector. (c) 5hmC dot blot of genomic DNA extracted from HEK293T cells transfected with a PB vector expressing full-length TET1 or a mutant with base substitutions at the catalytic residues (mHxD). (d) Western blot for TET1 in ESC clones stably transfected with a PB vector expressing full-length TET1 or catalytic mutant TET1 (mHxD). The western blot shows TET1 expression with and without doxycycline induction, with leakiness of the tetO-CMV minimal promoter activity in the absence of doxycycline. HEK293T cells transfected with a PB vector containing full-length TET1, and also wt and KO ESCs, were used as controls. KO, Tet1GT/GT.
Supplementary Figure 10 Analysis of differentially expressed genes in epiblast-like cells on the CD1 background.
Heat map clustering of genes differentially expressed in three wt and three KO EpiLCs of CD1 background. The sex of each line is indicated below the sample name. (b) Overlap of DE genes from RNA–seq of EpiLCs derived on the CD1 background in comparison to data obtained from B6 lines. Gene names highlighted in red are DE genes common in B6 Epi and EpiLCs. (c) GO terms associated with DE genes in KO versus wt EpiLCs on the B6 and CD1 backgrounds. KO, Tet1GT/GT.
(a) Western blot on subcellular fractions of HEK293T cells overexpressing C-terminal V5/His-tagged JMJD8(1–271), JMJD8(27–271) (ΔN-JMJD8) or empty pEF1-V5/His vector (mock). The purity of nuclear (N) and cytosol (Cy) fractions is indicated by lamin A and tubulin, respectively. WCL, whole-cell lysate. (b) Western blot detection of 6×His and V5 tags in ESC clones overexpressing C-terminally V5/His-tagged JMJD8(27–271) (ΔN-JMJD8; two clones) or empty vector (mock) after conversion to EpiLCs. (c) IGV tracks representing ChIP–seq peaks for JMJD8 binding at target genes. ChIP was performed using V5 or His antibodies in clone 2 expressing JMJD8(27–271) in wt EpiLC, using a mock clone as a negative control. (d) Western blot detection of epitope-tagged ΔN-JMJD8 in two independent doxycycline (DOX)-inducible clones.
Supplementary Figures 1–11 and Supplementary Note. (PDF 3490 kb)
Differential expression analysis of epiblast (Epi) samples by DESeq2. (XLSX 4433 kb)
RPKM values of gene expression in all Epi and ExE samples. (XLSX 7201 kb)
Differential expression analysis of ExE samples by DESeq2. (XLSX 2772 kb)
List of differentially expressed genes in Tet1 KO ExE involved in mitochondrial oxidative phosphorylation. (XLSX 12 kb)
Differentially methylated regions in Tet1 KO EpiLC12 versus wt EpiLC15. (XLSX 2446 kb)
Differential expression analysis of EpiLC samples by DESeq2. (XLSX 3790 kb)
RPKM values of gene expression in all EpiLC samples. (XLSX 5006 kb)
List of primers. (XLSX 15 kb)
BED file of WGBS DMRs with gain of methylation in EpiLC KO12 compared to EpiLC wt15. (TXT 9 kb)
BED file of WGBS DMRs with loss of methylation in EpiLC KO12 compared to EpiLC wt15. (TXT 1 kb)
BED file of oxWGBS DMRs with gain of methylation in EpiLC KO12 compared to EpiLC wt15. (TXT 628 kb)
BED file of oxWGBS DMRs with loss of methylation in EpiLC KO12 compared to EpiLC wt15. (TXT 0 kb)
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Khoueiry, R., Sohni, A., Thienpont, B. et al. Lineage-specific functions of TET1 in the postimplantation mouse embryo. Nat Genet 49, 1061–1072 (2017). https://doi.org/10.1038/ng.3868
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