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TFAP2C regulates transcription in human naive pluripotency by opening enhancers

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Abstract

Naive and primed pluripotent human embryonic stem cells bear transcriptional similarity to pre- and post-implantation epiblast and thus constitute a developmental model for understanding the pluripotent stages in human embryo development. To identify new transcription factors that differentially regulate the unique pluripotent stages, we mapped open chromatin using ATAC-seq and found enrichment of the activator protein-2 (AP2) transcription factor binding motif at naive-specific open chromatin. We determined that the AP2 family member TFAP2C is upregulated during primed to naive reversion and becomes widespread at naive-specific enhancers. TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. Additionally, we identify a previously undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for TFAP2C binding. Taken together, TFAP2C establishes and maintains naive human pluripotency and regulates OCT4 expression by mechanisms that are distinct from mouse.

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Fig. 1: Determination of regulatory elements specific to the naive and primed states in humans.
Fig. 2: Most naive-specific ATAC peaks are present in other naive human cells and the human embryo.
Fig. 3: TFAP2C is highly enriched over naive-specific open chromatin in humans.
Fig. 4: TFAP2C−/− cells differentiate in naive media.
Fig. 5: Ectopic expression of TFAP2C partially rescues the TFAP2C−/− phenotype.
Fig. 6: TFAP2C−/− cells survive in 5iLAF in 5% O2 conditions but do not transition to naive state.
Fig. 7: Identifying direct regulatory targets of TFAP2C.
Fig. 8: A TFAP2C+ intronic enhancer of OCT4.

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Change history

  • 07 September 2018

    In the version of this Article originally published, Supplementary Table 8 was duplicated as Supplementary Table 6. Supplementary Table 6 has now been amended with the correct file.

References

  1. Niakan, K. K., Han, J., Pedersen, R. A., Simon, C. & Pera, R. A. Human pre-implantation embryo development. Development 139, 829–841 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Okamoto, I. et al. Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature 472, 370–374 (2011).

    Article  PubMed  CAS  Google Scholar 

  3. Petropoulos, S. et al. Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos. Cell 165, 1012–1026 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Sahakyan, A. et al. Human naive pluripotent stem cells model X chromosome dampening and X inactivation. Cell Stem Cell 20, 87–101 (2017).

    Article  PubMed  CAS  Google Scholar 

  5. Kunath, T. et al. Developmental differences in the expression of FGF receptors between human and mouse embryos. Placenta 35, 1079–1088 (2014).

    Article  PubMed  CAS  Google Scholar 

  6. Nakamura, T. et al. A developmental coordinate of pluripotency among mice, monkeys and humans. Nature 537, 57–62 (2016).

    Article  PubMed  CAS  Google Scholar 

  7. Deglincerti, A. et al. Self-organization of the in vitro attached human embryo. Nature 533, 251–254 (2016).

    Article  PubMed  CAS  Google Scholar 

  8. Blakeley, P. et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human.Cell 158, 1254–1269 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Huang, K., Maruyama, T. & Fan, G. The naive state of human pluripotent stem cells: a synthesis of stem cell and preimplantation embryo transcriptome analyses. Cell Stem Cell 15, 410–415 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Pastor, W. A. et al. Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory. Cell Stem Cell 18, 323–329 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Song, L. et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res 21, 1757–1767 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Ji, X. et al. 3D chromosome regulatory landscape of human pluripotent cells. Cell Stem Cell 18, 262–275 (2016).

    Article  PubMed  CAS  Google Scholar 

  17. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  PubMed  CAS  Google Scholar 

  18. Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Buecker, C. et al. Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 14, 838–853 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Auman, H. J. et al. Transcription factor AP-2gamma is essential in the extra-embryonic lineages for early postimplantation development. Development 129, 2733–2747 (2002).

    PubMed  CAS  Google Scholar 

  23. Winger, Q., Huang, J., Auman, H. J., Lewandoski, M. & Williams, T. Analysis of transcription factor AP-2 expression and function during mouse preimplantation development. Biol. Reprod. 75, 324–333 (2006).

    Article  PubMed  CAS  Google Scholar 

  24. Werling, U. & Schorle, H. Transcription factor gene AP-2 gamma essential for early murine development. Mol. Cell. Biol. 22, 3149–3156 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Hoffman, T. L., Javier, A. L., Campeau, S. A., Knight, R. D. & Schilling, T. F. Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution. J. Exp. Zool. B Mol. Dev. Evol. 308, 679–691 (2007).

    Article  PubMed  CAS  Google Scholar 

  26. Guttormsen, J. et al. Disruption of epidermal specific gene expression and delayed skin development in AP-2 gamma mutant mice. Dev. Biol. 317, 187–195 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Qiao, Y. et al. AP2gamma regulates neural and epidermal development downstream of the BMP pathway at early stages of ectodermal patterning. Cell Res. 22, 1546–1561 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Schemmer, J. et al. Transcription factor TFAP2C regulates major programs required for murine fetal germ cell maintenance and haploinsufficiency predisposes to teratomas in male mice. PLoS ONE 8, e71113 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Niakan, K. K. & Eggan, K. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Dev. Biol. 375, 54–64 (2013).

    Article  PubMed  CAS  Google Scholar 

  30. Xie, W. et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134–1148 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Tsankov, A. M. et al. Transcription factor binding dynamics during human ES cell differentiation. Nature 518, 344–349 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Yan, L. et al. Single-cell RNA-seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131–1139 (2013).

    Article  PubMed  CAS  Google Scholar 

  33. Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Tesar, P. J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).

    Article  PubMed  CAS  Google Scholar 

  35. Xie, H. & Simon, M. C. Oxygen availability and metabolic reprogramming in cancer. J. Biol. Chem. 292, 16825–16832 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. Guo, G. et al. Epigenetic resetting of human pluripotency. Development 144, 2748–2763 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. McLean, C. Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

    PubMed  CAS  Google Scholar 

  39. Eloranta, J. J. & Hurst, H. C. Transcription factor AP-2 interacts with the SUMO-conjugating enzyme UBC9 and is sumolated in vivo. J. Biol. Chem. 277, 30798–30804 (2002).

    Article  PubMed  CAS  Google Scholar 

  40. Wong, P. P. et al. Histone demethylase KDM5B collaborates with TFAP2C and Myc to repress the cell cycle inhibitorp21(cip) (CDKN1A). Mol. Cell. Biol. 32, 1633–1644 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Magnusdottir, E. et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat. Cell Biol. 15, 905–915 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Braganca, J. et al. Human CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) 4, a new member of the CITED family, functions as a co-activator for transcription factor AP-2. J. Biol. Chem. 277, 8559–8565 (2002).

    Article  PubMed  CAS  Google Scholar 

  43. Braganca, J. et al. Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED2. J. Biol. Chem. 278, 16021–16029 (2003).

    Article  PubMed  CAS  Google Scholar 

  44. Bamforth, S. D. et al. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat. Genet. 29, 469–474 (2001).

    Article  PubMed  CAS  Google Scholar 

  45. Kuckenberg, P. et al. The transcription factor TCFAP2C/AP-2gamma cooperates with CDX2 to maintain trophectoderm formation. Mol. Cell. Biol. 30, 3310–3320 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Lee, S. H., Kwon, J. W., Choi, I. & Kim, N. H. Expression and function of transcriptional factor AP-2ɣ in early embryonic development of porcine parthenotes. Reprod. Fertil. Dev. 28, 1197–1205 (2015).

    Article  CAS  Google Scholar 

  47. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  PubMed  CAS  Google Scholar 

  48. Fogarty, N. M. E. et al. Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Kalinka, A. T. & Tomancak, P. The evolution of early animal embryos: conservation or divergence?. Trends Ecol. Evol. 27, 385–393 (2012).

    Article  PubMed  Google Scholar 

  50. Irie, N. & Kuratani, S. Comparative transcriptome analysis reveals vertebrate phylotypic period during organogenesis. Nat. Commun. 2, 248 (2011).

    Article  PubMed  CAS  Google Scholar 

  51. Liu, X. et al. Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming. Nat. Methods 14, 1055–1062 (2017).

    Article  PubMed  CAS  Google Scholar 

  52. Diaz Perez, S. V. et al. Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin. Hum. Mol. Genet. 21, 751–764 (2012).

    Article  PubMed  CAS  Google Scholar 

  53. Pastor, W. A. et al. MORC1 represses transposable elements in the mouse male germline. Nat. Commun. 5, 5795 (2014).

    Article  PubMed  CAS  Google Scholar 

  54. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    Article  PubMed  CAS  Google Scholar 

  56. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS).Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Gkountela, S. et al. The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat. Cell Biol. 15, 113–122 (2013).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the UCLA Broad Stem Cell Research Center (BSCRC) Flow Cytometry core and the UCLA BSCRC High Throughput Sequencing Core for technical assistance. W.A.P. was supported by the Jane Coffin Childs Memorial Fund for Medical Research and a UCLA BSCRC Postdoctoral Training Fellowship. D.C. is supported by a UCLA BSCRC Postdoctoral Training Fellowship. W.L. is supported by the Philip J. Whitcome Fellowship from the UCLA Molecular Biology Institute and a scholarship from the Chinese Scholarship Council. Work was funded by R01 HD079546 (ATC) and a NHMRC project grant APP1104560 (to J.M.P.) and a Sylvia and Charles Viertel Senior Medical Research Fellowships (to J.M.P.). All work with human pre-implantation embryos was funded by UCLA BSCRC and not the National Institute of Health. S.E.J. is a fellow of the Howard Hughes Medical Institute.

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Authors

Contributions

W.A.P., D.C., J.H. R.K., T.J.H., A.L. and X.L. conducted experiments. W.A.P. and W.L. conducted bioinformatics analysis. W.A.P. and A.T.C. wrote the manuscript. J.M.P., S.E.J. and A.T.C. supervised the research.

Corresponding authors

Correspondence to Steven E. Jacobsen or Amander T. Clark.

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Integrated supplementary information

Supplementary Fig. 1 Properties of ATAC-seq libraries and ATAC-seq peaks.

a, Size distribution of distance between paired end reads in ATAC-seq libraries used in this paper. Note the spikes corresponding to helix pitch and the peaks at nucleosome distance, consistent with a successful ATAC-seq library. b, Metaplot of ATAC-seq read density over genes categorized by RPKM. The positive correlation between gene RPKM and ATAC-seq read density near the TSS further validates the ATAC-seq libraries. c, Location of ATAC-seq peak summits in primed and naive cells, and for primed or naive-specific summits, defined by eightfold enrichment in one state relative to the other. Fraction in each category is shown in absolute terms and relative to the genome as a whole. Note that naive and primed-specific peaks are less promoter-enriched than the general sets of primed and naive peaks, reflecting the fact that most promoters are open in both conditions, but enhancer utilization changes more strongly. d, Schematic for assessing the role of a given peak in regulating adjacent genes. All interactions between the peak and the adjacent genes are assessed and binned. Genes with multiple annotated TSS (e.g. Gene C) and TSS with multiple genes (e.g. Gene E and Gene F) are excluded from the analysis. e,f Percentage the time a gene whose transcriptional start site is a given distance from a naive-specific ATAC peak (e) or primed-specific ATAC peak (f) is upregulated or downregulated in the naive state. The left panel shows frequencies for ATAC peaks upstream of the gene TSS and the right panel shows frequencies for ATAC peaks downstream of gene TSS. ATAC peaks are strongly predictive of gene upregulation in in both cases.

Supplementary Fig. 2 ATAC-seq density over naive specific ATAC peaks in blastocysts.

All ATAC-seq or 5mC-seq data are plotted over the naive-specific set defined in 5iLAF (5032 peaks), a subset that contained an AP2 motif but no KLF motif (1054 peaks), a subset that contained a KLF motif but no AP2 motif (1551 peaks) and primed-specific peaks (2562 peaks). a, Blastocyst ATAC-seq reads are strongly enriched over naive-specific ATAC-seq peaks, including the AP2+KLF- and KLF+AP2- motif subsets, but are less enriched over primed-specific peaks. b, DNA methylation from primed and naive cells plotted over different ATAC-seq peak sets. Note that cells cultured in t2iLGö show drops in methylation over all naive-specific peak sets, including the AP2+ peak set, indicating that this same regions are likely to be open chromatin in the t2iLGö conditions. c, DNA methylation from oocyte and blastocyst are plotted relative to ATAC-seq peak sets. Note sharp loss of DNA methylation in blastocyst over the naive-specific sets. d, After fertilization, the paternal genome is demethylated while the maternal genome remains methylated, so thoughout the blastocyst genome the DNA methylation level is very close to half the Oocyte methylation level12. Thus, we plotted (methylation in blastocyst – 50% methylation level in oocyte) to determine regions that have undergone localized demethylation during embryogenesis. Sharp demethylation is observed over naive-specific peak sets, validating their identity as enhancers.

Supplementary Fig. 3 TFAP2C deficient CRISPR lines were generated and are phenotypically normal in the primed state.

a, Diagram indicating mutations observed in the two TFAP2C−/− deficient lines used in this paper. Note that both mutations cause frame shifts. b, Control and TFAP2C−/− lines are karyotypically normal. c,d,c, Expression of OCT4 (c), NANOG (d), SOX2 (e) in control and TFAP2C−/− hESCs cultured in primed conditions. The OCT4 blot is representative of 3 independent experiments. f, TRA-1-85 positive (human) cells were gated and stained for the pluripotency markers TRA-1-61 and TRA-1-80. Data represent 1 out of 3 independent experiments with similar results. g, Expression of lineage markers in primed hESCs and in embryoid bodies formed from control and TFAP2C−/− primed hESCs (n = 4 biological replicates for primed, WT EB and TFAPC2−/− EB) Mean +/− standard error is shown. Note loss of pluripotency markers upon EB differentiation and gain of neural and non-neural ectoderm markers, with a neural bias in TFAP2C−/−. Uncropped Western blot images are available in Supplementary Fig. 9. Source data for g is in Supplementary Table 8.

Supplementary Fig. 4 TFAP2C deficient hESCs undergo rapid neural differentiation in 5iLAF.

a,b Untransfected UCLA1 hESCs, a Control line nucleofected in parallel with the TFAP2C−/− lines, and TFAP2C−/− lines 1 and 2 are treated with 5iLAF media for 3 days (a) and 5 days (b). The TFAP2C−/− colonies show formation of morphologically distinct, flat colonies by 3 days. Scale bar indicates 100 μm. c, Top statistically enriched GO terms of genes upregulated in the TFAP2C−/− cells, calculated using a hypergeometric test with adjustment for multiple hypothesis testing33. All terms are consistent with neural differentiation of the TFAP2C−/− of cells. Terms specific to neural identity are colored blue. d, ATAC-seq peaks specific to WT cells relative to TFAP2C−/−cells after five days in 5iLAF show strong enrichment for AP2 motifs and for factors involved in pluripotency (OCT4, SOX, KLF), consistent with a loss of pluripotency in these cells. e, ATAC-seq peaks specific to TFAP2C−/− relative to WT cells after five days in 5iLAF show enrichment for motifs corresponding to such neural factors as SOX (e.g. SOX1) and ZIC (e.g. ZIC1). Motif enrichment was calculated using a cumulative binomial distribution19. f, Schematic for targeting generation of Tfap2c−/− and Tfap2a−/− Tfap2c−/− mESCs. Note that all deletions either induce frame shifts or delete splice sites. g, Western blot for Tfap2c in control and Tfap2c−/− lines. Representative of 2 independent experiments. Uncropped Western blot images are available in Supplementary Fig. 9.

Supplementary Fig. 5 Ectopic expression and withdrawal of TFAP2C.

a, RPKM for core and naive pluripotency factors upon TFAP2C induction in primed cells. Note that overexpression of TFAP2C does not result in upregulation of naive pluripotency markers. n = 1 replicate for all samples. b, Metaplot of TFAP2C ChIP-seq data upon TFAP2C induction in primed state. When TFAP2C is overexpressed in primed conditions, it primarily hones to regions of conserved openness in primed and naive cells (left pane) rather than naive-specific ATAC-seq peaks (right panel). c, Ectopic expression of TFAP2C rescues morphological abnormality found in TFAP2C−/− upon reversion in 5iLAF. Scale bar indicates 100 μm. d, Thirteen days after withdrawal of TFAP2C, very few colonies are visible in the sample in which doxycycline was withdrawn compared with the sample in which it remained. Scale bar indicates 100 μm. Results are representative of two independent experiments. e. Quantification of reduced cell number upon withdrawal compared with sample in which doxycycline treatment continued. f, Shift toward primed SSEA4+ phenotype in sample in which doxycycline had been withdrawn. Source data for a is in Supplementary Table 8.

Supplementary Fig. 6 TFAP2C deficient hESCs in 5% O2 show a shift toward primed phenotype.

a, Second reversion of control and TFAP2C−/− cells. Bright-field images of cells are shown. Cells were then sorted and TRA-1-85+ (human) cells were gated into SSEA4- negative and positive populations. In this reversion many SSEA4- cells were apparent, but subsequent RNA-seq indicated that they showed low expression of pluripotency genes and elevated neural gene expression, indicating that they were differentiated rather than naive. Scale bar indicates 100 μm. b, Expression (RPKM) of pluripotency markers, naive-specific markers, and primed markers of control and TFAP2C−/− cells relative to primed controls. Ratio is shown for n = 2 biologically independent replicates (Control cells, TFAP2C−/− SSEA4-) or n = 3 replicates TFAP2C−/− SSEA4+ over n = 2 primed control biological independent replicates. c, Principle component analysis of RNA-seq from all pluripotent datasets. Embryoid body differentiation, day 5 5iLAF TFAP2C−/− cells, and 5% O2 reversion 2 TFAP2C−/− SSEA4- cells were excluded because they diverged strongly due to loss of pluripotency. Blue dots: TFAP2C−/− cells in 5% O2 show a shift toward primed-like gene expression relative to control cells. Brown dots: no shift toward naive identity is observed in cells which overexpress TFAP2C in primed media conditions. Green dots, rescue of TFAP2C−/− cells with doxycycline-inducible TFAP2C is indicated by partial shift toward naive identity. d, e, Average RPKM in the indicated cell type is plotted for all genes previously determined6 to be downregulated (d) or upregulated (e) upon the transition from pre-implantation to post-implantation epiblast in primates. The box and whiskers are plotted by the Tukey method. f, Immunofluorescent images of KLF17 and TRA-1-60 staining in control and TFAP2C−/− cells cultured in t2iLGöY. Data represent 1 of 2 independent experiments with similar results. Scale bars, 20μm. g, Ratio of KLF17 expression in t2iLGöY/primed conditions for WT control and TFAPC2−/− cells. Data are for n = 2 control and n = 3 TFAP2C−/− biologically independent replicates in both naive and primed conditions. Source data for b is in Supplementary Table 8.

Supplementary Fig. 7 Identification of TFAP2C direct targets.

a, Frequency with which gene whose transcriptional start site is a given distance from a TFAP2C ChIP-seq peak summit that overlaps with a region of conserved openness in naive and primed cells is positively or negatively regulated by TFAP2C. Note that for this subset of TFAP2C ChIP peaks, there is almost no predictive effect. This indicates that TFAP2C primarily mediates its regulatory role in the context of TFAP2C-dependent open chromatin sites. b, Distance of sites in a, to nearest transcription start site. Note that many are promoter associated. c-e ATAC-seq and ChIP-seq data are shown in the viscinity of c, KLF5 d, NANOG e, FGF4. The control low O2 track is the SSEA4- population, the TFAP2C−/− low O2 is the SSEA4+ population.

Supplementary Fig. 8 OCT4 Intron Elements 1 and 2.

a, Regulatory elements in the proximity of OCT4 locus. The conservation track shows one dot for each base, with the degree of conservation in placental mammals indicated by the value in the Y-axis. Note higher conservation over regulatory elements and coding sequence. b, Sashimi plot showing splice events over POU5F1 locus in naive hESCs. Some RNA-seq reads are observed over the POU5F1 Intron Element 1, possibly enhancer RNA or intronic RNA, but there are no splice events linking these to OCT4 transcripts. This indicates that the Intron Element 1 is not an alternative TSS. c, Normal karyotype in intron enhancer deletant and WT control cells. d, Western blot for OCT4 in Control and ΔIntron enhancer 1 in primed conditions. Uncropped Western blot images are available in Supplementary Fig. 9.

Supplementary Fig. 9 Uncropped Western Blot images.

Uncropped images of all Western blots are shown, as well as the approximate extent of the cropped region. Note that because it was our procedure to stain target and loading control simultaneously, cross-reactive secondary antibody sometimes causes an H3 band to appear in target blot.

Supplementary information

Supplementary Information

Supplementary Figs 1–9 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Description of all cell populations used for next generation sequencing libraries

Supplementary Table 2

Human ATAC and ChIP peak sets used in analysis

Supplementary Table 3

Description of embryos used for ATAC-seq

Supplementary Table 4

Murine ATAC peak sets used in analysis

Supplementary Table 5

RPKM of all RNAseq-samples and differentially regulated gene sets

Supplementary Table 6

Genes within 50 kb of TFAP2C-dependent regulatory element

Supplementary Table 7

GREAT analysis of TFAP2C-dependent regulatory elements

Supplementary Table 8

Statistical source data

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Pastor, W.A., Liu, W., Chen, D. et al. TFAP2C regulates transcription in human naive pluripotency by opening enhancers. Nat Cell Biol 20, 553–564 (2018). https://doi.org/10.1038/s41556-018-0089-0

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