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NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers

Nature volume 529, pages 403–407 (2016)Cite this article

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Abstract

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice1, where its precise role is yet unclear2,3,4. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate5. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells6,7,8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

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Figure 1: Nanog induces PGCLCs in EpiLCs.
Figure 2: Loss of Prdm1 and Nanog affects PGCLC specification.
Figure 3: Competence for PGCLCs versus reversion to ES cells.
Figure 4: Context-dependent NANOG binding in ES cells/EpiLCs.

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Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus under accession number GSE71933.

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Acknowledgements

We thank H. Leitch for ES cell lines, C. Lee for help with animal husbandry, H. Niwa for vectors and conditional Sox2-knockout ES cells, N. Miller, R. Walker and A. Riddell for FACS sorting and J. Bauer for analysis of microarray data. K.M. was supported by the Japan Society for the Promotion of Science (JSPS) Institutional Program for Young Researchers Overseas Visits. U.G. was supported by a Marie Skłodowska-Curie and a Newton Trust/Leverhulme Trust Early Career fellowship. J.J.Z. was a recipient of a Wellcome Trust PhD Studentship (RG44593). T.K. was supported by a JSPS Fellowship for research abroad. This research was supported by Gurdon Institute core grants from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492), and a grant from the Wellcome Trust to M.A.S. (WT096738).

Author information

Author notes

  1. Kazuhiro Murakami and Ufuk Günesdogan: These authors contributed equally to this work.

Authors and Affiliations

  1. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK

    Kazuhiro Murakami, Ufuk Günesdogan, Jan J. Zylicz, Walfred W. C. Tang, Roopsha Sengupta, Toshihiro Kobayashi, Shinseog Kim, Richard Butler & M. Azim Surani

  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK

    Kazuhiro Murakami, Ufuk Günesdogan, Jan J. Zylicz, Walfred W. C. Tang, Roopsha Sengupta, Toshihiro Kobayashi, Shinseog Kim & M. Azim Surani

  3. Wellcome Trust Medical Research Council Stem Cell Institute, University of Cambridge,, Tennis Court Road, Cambridge, CB2 1QR, UK

    Kazuhiro Murakami, Ufuk Günesdogan, Jan J. Zylicz, Walfred W. C. Tang, Roopsha Sengupta, Toshihiro Kobayashi, Shinseog Kim, Sabine Dietmann & M. Azim Surani

  4. Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Hyogo, Japan

    Kazuhiro Murakami

  5. Laboratory for Molecular and Cellular Biology, Faculty of Advanced Life Science, Hokkaido University, Kita21 Nishi11, Kita-ku, Sapporo, 001-0021, Hokkaido, Japan

    Kazuhiro Murakami

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  1. Kazuhiro Murakami
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Contributions

K.M. and U.G. designed and performed experiments, and wrote the paper; W.W.C.T. designed and carried out the luciferase assays; NANOG ChIP experiments were carried out by J.J.Z., while R.S. performed immunofluorescence analysis; T.K. and S.K. designed and carried out the chimaera experiments; S.D. performed bioinformatic analysis; R.B. developed the ‘Object Scan’ plugin; M.A.S. supervised the project, designed experiments and wrote the paper. All authors discussed the results and contributed to the manuscript.

Corresponding author

Correspondence to M. Azim Surani.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 GOF-GFP as a reporter for PGCLCs; Dox-inducible transgene system.

a, Experimental design for the induction of PGCLCs in vitro5. b, Representative brightfield/GFP images of male/female GOF-GFP ES cells, day-2 EpiLCs, day-2 and day-4 cytokine-induced PGCLCs. Scale bar, 200 μm. D, day. c, FACS analysis for GFP with samples shown in b. SSC, side scatter. d, Simplified scheme of PiggyBac- (PB 5′TR and PB 3′TR) based plasmids for transgene overexpression using the Tet-On system. The rtTA protein activates the minimal promoter (hCMV*-1) driving the expression of the cDNA of interest only in the presence of doxycycline (Dox). e, Proof-of-principle experiment to test the Dox-inducible expression of a transgene during the sequential differentiation of PGCLCs from ES cells. GOF-GFP ES cells carrying PiggyBac based Dox-inducible mCherry expression plasmids were differentiated into day-2 EpiLCs and then induced into PGCLCs with cytokines in plus or minus Dox conditions. Representative brightfield, GFP and mCherry images are shown 12 h after aggregation. Scale bar, 200 μm. f, Representative FACS analysis for GFP and mCherry of day-2 cytokine-induced PGCLCs from male/female GOF-GFP ES cells carrying a Dox-inducible mCherry transgene. Most cells express mCherry after Dox addition.

Extended Data Figure 2 Nanog but not Oct3/4 induces GFP+ cells from competent EpiLCs.

a, qPCR analysis of transgenic Nanog expression 24 h after Dox addition in male/female GOF-GFP ES cells. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01. Related to Fig. 1a. b, Representative brightfield/GFP images of male day-2 PGCLCs induced from GOF-GFP day-2 EpiLCs; plus Dox for Nanog expression. Scale bar, 200 μm. Related to Fig. 1a. D, day. c, Representative FACS analysis of male day-4 PGCLCs (shown in Fig. 1a) induced from GOF-GFP day-2 EpiLCs; plus Dox for Nanog expression. SSC, side scatter. d, Representative brightfield/GFP images of female day-2 and day-4 PGCLCs induced from GOF-GFP day-2 EpiLCs; plus Dox for Nanog expression. Scale bar, 200 μm. Related to Fig. 1a. e, FACS analysis for GFP with samples shown in d. Related to Fig. 1a. f, qPCR analysis of transgenic Oct3/4 expression 24 h after Dox addition in male ES cells. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01. g, Expression of Oct3/4 (unlike Nanog) does not result in the induction of GFP+ cells. PGCLC induction from female GOF-GFP EpiLCs; plus Dox for Oct3/4 or Nanog expression. Representative brightfield/GFP images at day 4. Scale bar, 200 μm. h, FACS analysis for GFP with samples shown in g.

Extended Data Figure 3 The transcriptomes of day-4 Nanog- and cytokine-induced PGCLCs are highly similar.

a, 100–200 ng ml−1 of Dox in EpiLCs results in NANOG expression levels similar to ES cells as shown by western blot analysis for NANOG and α-tubulin (α-TUB) with GOF-GFP ES cells and day-2 EpiLCs 24 h after PGCLC induction (EpiLC aggregations) with Nanog (+Dox). For gel source data, see Supplementary Fig. 1. b, PGCLC induction with 100 or 700 ng ml−1 Dox (for Nanog expression) with noggin from GOF-GFP EpiLCs. Representative brightfield/GFP images at day 4. GFP+ cells are induced in both conditions. Scale bar, 200 μm. c, Physiological (equivalent to ES cells) or higher levels of Nanog induce PGCLCs with comparable efficiency. FACS for GFP at day 4 of PGCLC induction with 100 or 700 ng ml−1 Dox (for Nanog expression) with noggin from GOF-GFP or Prdm1-GFP EpiLCs. D, day; SSC, side scatter. d, Alternative representation of qPCR data for Nanog, Prdm1 and Tfap2c shown in Fig. 1c. The induction of these genes in plus cytokine conditions appears less evident, when compared with plus Dox conditions. The data were log2-scaled, which allows a better comparison. e, qPCR analysis of female GOF-GFP cells. GFP+ cells were FACS-sorted. Note the upregulation of PGC markers but not of the ES-cell marker Klf4. ΔΔCt mean values ± s.d.; n = 3 biological replicates. Colour code is shown in d. Related to Fig. 1c. f, qPCR analysis of male Prdm1-GFP cells. GFP+ cells were FACS-sorted. Note the upregulation of PGC markers but not of the ES-cell marker Klf4. ΔΔCt mean values ± s.d.; n = 3 biological replicates. Colour code is shown in d. Related to Fig. 1c. g, The transcriptomes of Nanog- and cytokine-induced PGCLCs are highly similar. Scatter plot showing the correlation of microarray data of ES cells, FACS-sorted day-4 PGCLCs induced by cytokines or Nanog with noggin. R indicates the Pearson correlation coefficient. n = 2 biological replicates; related to Fig. 1d. h, Nanog- and cytokine-induced PGCLCs cluster together as shown in unsupervised hierarchical clustering of microarray data described in g. Related to Fig. 1d. i, Heat map showing the expression levels of selected genes from microarray data described in g. Related to Fig. 1d. j, Nanog-induced day-4 PGCLCs are closely related to cytokine-induced day-6 PGCLCs. PCA analysis with published microarray data sets5 (cross-platform comparison; see Methods for details). Note that the separation of ES cell samples is probably due to differences in genomic background and culture conditions.

Extended Data Figure 4 Nanog-induced PGCLCs show hallmarks of PGC development.

a–c, Immunofluorescence analysis of PGC markers in GFP+ cells induced by Nanog from male (a) and female (b) GOF-GFP and male Prdm1-GFP (c) EpiLCs shows expression of BLIMP1, PRDM14, AP-2γ and TET1, enrichment of H3K27me3 and 5hmC and a decrease of H3K9me2 intensity; DAZL is detected in some cells on day 6. Arrowheads and dashed lines highlight single or cluster of GFP+ cells. n = 2 biological replicates. Scale bar, 10 μm. Quantification in c was scale normalized. Two-sided/unpaired t-test: S, significant (P ≤ 0.01); n = number of cells analysed. Related to Fig. 1e.

Extended Data Figure 5 Functional analysis of Nanog-induced PGCLCs.

a, Experimental design (for b, c) for the derivation of EGCLCs. PGCLCs were induced with cytokines or by Nanog (+Dox) from male or female GOF-GFP EpiLCs carrying a constitutively active Kusabira-Orange reporter. On day 4, aggregations were dissociated and cultured on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells in PGC selection medium (LIF, SCF, bFGF, retinoic acid) for 5 days. After the selection, selected colonies were dissociated and transferred into ES-cell medium (2i/LIF). b, Experiment was performed as shown in a. Left panel shows representative images of proliferating GFP+ cells after 3 days of PGC selection. Right panel shows established EGCLCs after three passages in 2i/LIF. c, EGCLCs derived from day-4 PGCLCs by Nanog expression were injected into blastocysts, resulting in high contribution to chimaeras at E9.5 as shown by Kusabira-Orange expression. d, Experimental design (for e–g) for generating chimaeras. PGCLCs were induced from a GOF-GFP ES cell line expressing a fluorescent VENUS reporter constitutively and Nanog upon Dox addition (TVN2 cell line). On day 4, aggregations were dissociated and SSEA1+ and CD61+ cells were sorted by FACS, injected into morulae and analysed on E9.5. e, Representative brightfield, GFP/VENUS images of GOF-GFP or TVN2 cells during PGCLC induction by cytokines or Nanog (+Dox). Scale bars, 100 μm. D, day. f, FACS profile for SSEA1+ and CD61+ PGCLCs on day 4 induced as described and shown in d, e. g, ES cells but not PGCLCs contribute efficiently to chimaeras. ES cells or FACS-sorted Nanog-induced SSEA1+/CD61+ PGCLCs at day 4were injected into morulae and representative brightfield/VENUS images from chimaeras at E9.5 are shown.

Extended Data Figure 6 Prdm1−/− abrogates PGCLC induction by Nanog; Nanog and the WNT pathway act independently.

a, PGCLC induction with cytokines or Nanog (+Dox) from Prdm1−/− ES cells (Prdm1−/−; Nanog). Representative brightfield images of day-4 and day-6 aggregations. Scale bar, 200 μm. Related to Fig. 2a. D, day. b, Loss of Prdm1 abrogates PGCLCs induced by NANOG as shown by qPCR analysis of mutant (Prdm1−/−; Nanog) compared with control (Prdm1+/+; Nanog) cells with Nanog (+Dox) or cytokines (+cyto). Unsorted samples were used for analysis. Note that the data shown in Fig. 2a was combined with additional qPCR data on cells at day 6 of PGCLC induction. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; *P < 0.05. Related to Fig. 2a. c, Nanog does not affect cell proliferation rate of Prdm1−/−; Nanog cells. Immunofluorescence staining for the mitotic marker H3S10ph in Prdm1−/−; Nanog cells at day 6 of PGCLC induction; plus Dox for Nanog expression. Scale bar, 10 μm. Two-sided/unpaired t-test; NS, not significant (P > 0.01); n = estimated number of cells (see Methods for details). Related to Fig. 2a. d, Induced expression of Nanog results in an increased number of cell death of Prdm1−/−; Nanog cells. Immunofluorescence stainings of the DNA double-strand-break marker γH2AX in Prdm1−/−; Nanog cells at day 6 of PGCLC induction; plus Dox for Nanog expression. Scale bar, 10 μm. Two-sided/unpaired t-test; s, significant (P ≤ 0.01); n = estimated number of cells (see Methods for details). Related to Fig. 2a. e, Experimental design (for f–i) to test the interdependence of Nanog and the WNT pathway for PGCLC induction. Prdm1-GFP ES cells were sequentially differentiated into PGCLCs plus or minus tankyrase inhibitor XAV939, which causes the degradation of β-catenin25; plus Dox for Nanog expression. f, XAV939 does not affect the morphology and proliferation of day-2 EpiLCs. Representative brightfield/GFP images of day-2 EpiLCs induced from GOF-GFP ES cells with 1 μM XAV939. Scale bar, 200 μm. g, qPCR of day-2 EpiLCs treated with XAV939 as shown in e, f. The expression of Nanog and of the EpiLC markers Dnmt3a and Dnmt3b are not affected by XAV939. Brachyury, the downstream target of WNT, is most efficiently repressed with 1 μm XAV939. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; *P < 0.05. h, The efficiency of PGCLC induction by cytokines but not by Nanog (+Dox) is markedly reduced upon XAV939 addition. PGCLCs were induced from 1 μM XAV939-treated day-2 EpiLCs. Representative FACS analysis for GFP with cells at day 4 of PGCLC induction. SSC, side scatter. i, XAV939 does not affect the induction of PGC marker expression in Nanog-induced PGCLCs. Gene expression analysis by qPCR with FACS-sorted Nanog-induced day-4 PGCLCs plus or minus 1 μM XAV939. Mean ΔΔCt values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; *P < 0.05; NS, not significant.

Extended Data Figure 7 Sox2 inhibits PGCLC induction by Nanog.

a, Prdm1 and Tfap2c are upregulated and Prdm14 is downregulated in Sox2-knockout (KO) ES cells from published microarray data26. **P < 0.01; *P < 0.05. b, Experimental design for the western blot shown in Fig. 3d. Conditional Sox2-knockout ES cells carrying transgenes for Dox-inducible Nanog expression were treated with Dex to induce a Sox2-knockout and/or Dox for Nanog expression for 2 days. c, Experimental design for the qPCR analysis shown in d. Sox2-knockout ES cells: conditional Sox2-knockout ES cells carrying transgenes for Dox-inducible Nanog expression were treated plus or minus Dex for 2 days; Sox2-knockout day-1 EpiLCs: ES cells were cultured in 2i/LIF medium with Dex for 1 day and in bFGF/activin A (ActA) medium with Dex for one more day; Sox2-knockout day-2 EpiLCs: ES cells were transferred into bFGF/activin A medium containing Dex for 2 days. D, day. d, Loss of Sox2 results in upregulation of Prdm1 and Tfap2c and downregulation of Prdm14 in ES cells, day-1 and day-2 EpiLCs; qPCR analysis following Sox2-knockout (+Dex). ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; *P < 0.05. Experimental design is shown in c. p., parental. Related to Fig. 3e. e, Experimental design for the qPCR analysis shown in Fig. 3e. Sox2-knockout ES cells, day-1 or day-2 EpiLCs were generated as described in c, and subsequently induced into PGCLCs plus or minus Nanog (+/−Dox). f, Western blot for NANOG, SOX2 and α-tubulin (α-TUB) in GOF-GFP ES cells carrying Dox-inducible transgenes for Nanog, Sox2 or Nanog/Sox2 (+Dox for 24 h). Related to Fig. 3f. For gel source data, see Supplementary Fig. 1. g, Time-course qPCR analysis showing Nanog and Sox2 expression kinetics during PGCLC induction. PGCLCs were induced from GOF-GFP EpiLCs; +100 or 700 ng ml−1 Dox for Nanog/Sox2 expression. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Related to Fig. 3f. h, Time-course western blot for NANOG, SOX2 and α-tubulin (α-TUB) showing NANOG and SOX2 protein kinetics during PGCLC induction. PGCLCs were induced from GOF-GFP EpiLCs; +100 or 700 ng ml−1 Dox for Nanog/Sox2 expression. For gel source data, see Supplementary Fig. 1. Related to Fig. 3f. i, FACS analysis for GFP at day 4 of PGCLC induction from GOF-GFP or Prdm1-GFP EpiLCs; plus Dox for Nanog, Sox2 or Nanog/Sox2 expression. Related to Fig. 3f.

Extended Data Figure 8 Sox2 positively affects cell proliferation rate of cytokine-induced PGCLCs.

a, Sox2 increases the number of GFP+ cells induced by BMP4 alone. Representative FACS analysis for GFP at day 4 of PGCLC induction from Prdm1-GFP EpiLCs; plus Dox for Sox2 expression. D, day; SSC, side scatter. b, Sox2 does not affect the upregulation of PGC markers in cytokine-induced PGCLCs. qPCR analysis of FACS-sorted GFP+ cells induced by BMP4 and/or Sox2 (+Dox). ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. ES cells were used as a reference for P values (two-sided/unpaired t-test): **P < 0.01; *P < 0.05. c, Time-course FACS analysis of GFP+ cells after PGCLC induction with BMP4 and plus or minus Nanog or Sox2 (+/−Dox). The number of GFP+ cells at day 2 of PGCLC induction with or without Sox2 expression is comparable, but increased with Nanog. After day 2, PGCLCs induced by BMP4 with Sox2 or Nanog increase their proliferation rate.

Extended Data Figure 9 Nanog shows a cell-type-specific binding pattern and induces Prdm1, Prdm14 and Tfap2c.

a, Time-course qPCR for Prdm1, Prdm14 and Tfap2c between 1–48 h after PGCLC induction with cytokines from GOF-GFP EpiLCs. ΔΔCt mean values ± s.d.; n = 3 technical replicates. Related to Fig. 4a. b, Prdm1 alone can induce the expression of Tfap2c. GOF-GFP EpiLCs with combinations of Dox-inducible transgenes encoding Prdm1, Prdm14 and/or Nanog plus or minus Dox for 6 h were analysed by qPCR. The expression of Prdm1, Prdm14 and/or Nanog is upregulated in the corresponding EpiLCs upon Dox addition. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; *P < 0.05. D, day. c, To acquire sufficient numbers of cells for ChIP-seq studies, GOF-GFP day-1 or day-2 EpiLCs (~1 × 106 cells per 6-cm plate) with Dox-inducible Nanog transgenes were aggregated in low-binding plates plus Dox to induce PGCLCs. qPCR analysis of day-1 and day-2 EpiLCs after 3 h with 100 or 200 ng ml−1 Dox is shown. The addition of 200 ng ml−1 of Dox results in Nanog expression levels comparable to ES cells after 3 h. ΔΔCt mean values ± s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. Two-sided/unpaired t-test: **P < 0.01; NS, not significant. D, day. d, NANOG ChIP-seq analysis shows genomic distribution of NANOG in GOF-GFP ES cells and day-1 EpiLCs plus Nanog (+Dox) for 3 h. ‘Distal’ refers to intergenic peaks, which are within ±50 kb of an annotated coding gene, while those further away are categorized as ‘intergenic’. Related to Fig. 4b. e, De novo motif analysis with NANOG ChIP-seq data. Shown are the top five matches of the de novo motifs to known motifs. The analysed cell types show enrichment for the NANOG and SOX motifs. ES cells show additional enrichment for pluripotency motifs, while EpiLCs show a different set of motif enrichment. f, Day-2 EpiLC-specific (D2 EpiLCs +Dox 3 h) NANOG-bound enhancers become more enriched for H3K27ac than ES-cell-specific NANOG-bound enhancers in cytokine-induced day-2 PGCLCs as compared to ES cells. Contour plots showing differential binding of NANOG in day-2 EpiLCs versus ES cells (x-axis) compared to the differential enrichment of H3K27ac in day-2 PGCLCs30 versus ES cells9 (y-axis). g, NANOG binds enhancers that are enriched for H3K27ac in day-1/2 EpiLCs (D1/2 EpiLCs +Dox 3 h). A subset of enhancers, however, becomes more enriched for H3K27ac in cytokine-induced day-2 PGCLCs30 as compared to day-2 EpiLCs30. h, NANOG might contribute to the activation of enhancers associated with germline genes. Scatter plots show differential gene expression analysis between day-6 PGCLCs5 and day-2 EpiLCs5 (y-axis), and differential H3K27ac enrichment between day-2 PGCLCs30 and day-2 EpiLCs30 (x-axis) on NANOG-binding sites. The top 40% of NANOG peaks were associated with the nearest gene in a 200-kb window. Highlighted are candidate enhancers, which are associated with germline genes and become activated (H3K27ac-enriched) in PGCLCs.

Extended Data Figure 10 Nanog induces PGC-like fate: a model.

a, ChIP-seq data tracks9,30,39 at the Prdm1 and Prdm14 loci for NANOG in ES cells, day-1 and day-2 EpiLCs (EpiLCs were collected after 3 h with 200 ng ml−1 Dox for Nanog expression). Boxed are putative enhancer elements. The Prdm1 enhancer is enriched for H3K4me1 in day-2 EpiLCs and gains H3K27ac in PGCLCs. The Prdm14 enhancer shows enrichment for H3K4me1 in ES cells and EpiLCs and becomes enriched for H3K27ac in ES cells and PGCLCs but not in EpiLCs. Note that these enhancer marks follow the expression pattern of Prdm1 or Prdm14, respectively. D, day; RPM, reads per million. Related to Fig. 4d. b, ChIP-qPCR validation of NANOG ChIP-seq data with GOF-GFP day-2 EpiLCs before and 3 h after PGCLC induction by Nanog expression (+Dox). NANOG is enriched at putative enhancer regions, which are close to Prdm1 and Prdm14. Error bars indicate s.d.; n = 4 values obtained from two technical replicates from each of two biological replicates. c, ES cell lines with luciferase reporter plasmids with a genomic region, which does not show any enhancer marks and NANOG binding, and indicated Dox-inducible transgenes served as a negative control. Luciferase activity, measured in ES cells, day-2 EpiLCs and 24 h after PGCLC induction (EpiLC aggregations), was normalized to protein quantity (luc/pro). Mean values ± s.d.; n = 6 values obtained from two technical replicates from each of two biological replicates. d, Biological replicate experiment for the luciferase assay with the Prdm1 enhancer as shown and described in Fig. 4e. Luciferase activity, measured in ES cells, day-2 EpiLCs and 24 h after PGCLC induction, was normalized to protein quantity (luc/pro). Mean values ± s.d.; n = 3 technical replicates. Colour code is shown in c. Reference for P values (two-sided/unpaired t-test): EpiLC aggregations minus Dox; **P < 0.01; *P < 0.05. e, Biological replicate experiment for the luciferase assay with the Prdm14 enhancer as shown and described in Fig. 4g. Luciferase activity, measured in ES cells, day-2 EpiLCs and 12/24 h after PGCLC induction, was normalized to protein quantity (luc/pro). Mean values ± s.d.; n = 3 technical replicates. Colour code is shown in c. Reference for P values (two-sided/unpaired t-test): EpiLC aggregations minus Dox 24 h; **P < 0.01; *P < 0.05. f, Model showing the role of NANOG during PGCLC induction in vitro. Day-1 EpiLCs are not competent to become PGCLCs, but retain the capability to revert to an ES-like state via 2i/LIF and/or Nanog overexpression. Day-2 EpiLCs differentiate into PGCLCs upon Nanog expression. NANOG binds to putative enhancer elements of Prdm1 and Prdm14 to activate their transcription, which is sufficient to induce the PGCLC fate. This effect can be antagonized by SOX2, which co-binds the Prdm1 enhancer.

Supplementary information

Supplementary Figure 1

This file contains uncropped scans of Western blot gels. (PDF 321 kb)

Supplementary Table 1

This table contains oligonucleotide sequences. (XLSX 11 kb)

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Murakami, K., Günesdogan, U., Zylicz, J. et al. NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers. Nature 529, 403–407 (2016). https://doi.org/10.1038/nature16480

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