Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases

Nature Cell Biology volume 22, pages 1223–1238 (2020)Cite this article

Subjects

Abstract

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF–MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF–MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: An inhibitor screen for factors that promote the PSC naive state identifies a distinct role for Mediator kinase activity.
Fig. 2: Positive effect of long-term treatment of CDK8/19i on PSC self-renewal and pluripotency.
Fig. 3: Positive effect of long-term treatment of CDK8/19i on the self-renewal and pluripotency of human PSCs.
Fig. 4: Gene expression in mouse and human PSCs adapted to 2i or CDK8/19i.
Fig. 5: CDK8/19i regulates the phosphoproteome and RNA Pol II similar to 2i-naive pluripotency, but not DNA methylation.
Fig. 6: 2i and CDK8/19i exert similar effects on Mediator, RNA Pol II loading and enhancer activity.
Fig. 7: 2i and CDK8/19i hyperactivate naive-state enhancer activity, conferring resistance to enhancer destabilization.
Fig. 8: CDK8 expression in vivo and the role of Mediator during mouse preimplantation development.

Similar content being viewed by others

Data availability

RNA-seq and ChIP–seq data are available from the GEO database under accession numbers GSE112208 and GSE127186. The MS proteomics data are available from the ProteomeXchange Consortium/PRIDE repository under the dataset identifier PXD009200. Details on the published datasets used in Fig. 4e,k are provided in Supplementary Table 3. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. Heinz, S., Romanoski, C. E., Benner, C. & Glass, C. K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Allen, B. L. & Taatjes, D. J. The Mediator complex: a central integrator of transcription. Nat. Rev. Mol. Cell Biol. 16, 155–166 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jeronimo, C. & Robert, F. The Mediator complex: at the nexus of RNA Polymerase II transcription. Trends Cell Biol. 27, 765–783 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Soutourina, J. Transcription regulation by the Mediator complex. Nat. Rev. Mol. Cell Biol. 4, 262–274 (2017).

    Google Scholar 

  7. Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Fant, C. B. & Taatjes, D. J. Regulatory functions of the Mediator kinases CDK8 and CDK19. Transcription 2, 76–90 (2019).

    Article  Google Scholar 

  9. Chen, H. et al. A pan-cancer analysis of enhancer expression in nearly 9000 patient samples. Cell 173, 386–399 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Clark, A. D., Oldenbroek, M. & Boyer, T. G. Mediator kinase module and human tumorigenesis. Crit. Rev. Biochem. Mol. Biol. 50, 393–426 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Poss, Z. C. et al. Identification of mediator kinase substrates in human cells using cortistatin a and quantitative phosphoproteomics. Cell Rep. 15, 436–450 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Knuesel, M. T., Meyer, K. D., Bernecky, C. & Taatjes, D. J. The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function. Genes Dev. 23, 439–451 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. van de Peppel, J. et al. Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. Mol. Cell 19, 511–522 (2005).

    Article  PubMed  Google Scholar 

  14. Pelish, H. E. et al. Mediator kinase inhibition further activates super-enhancer-associated genes in AML. Nature 526, 273–276 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gonzalez, D. et al. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator. Proc. Natl Acad. Sci. USA 111, 2500–2505 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bancerek, J. et al. CDK8 kinase phosphorylates transcription factor STAT1 to selectively regulate the interferon response. Immunity 38, 250–262 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Galbraith, M. D., Donner, A. J. & Espinosa, J. M. CDK8: a positive regulator of transcription. Transcription 1, 4–12 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hackett, J. A. & Surani, M. A. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 15, 416–430 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Galonska, C., Ziller, M. J., Karnik, R. & Meissner, A. Ground state conditions induce rapid reorganization of core pluripotency factor binding before global epigenetic reprogramming. Cell Stem Cell 17, 462–470 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Weinberger, L., Ayyash, M., Novershtern, N. & Hanna, J. H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155–169 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Wu, J. & Izpisua Belmonte, J. C. Dynamic pluripotent stem cell states and their applications. Cell Stem Cell 17, 509–525 (2015).

    Article  CAS  PubMed  Google Scholar 

  23. Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu, X. et al. Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming. Nat. Methods 11, 1055–1062 (2017).

    Article  Google Scholar 

  25. Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bhagwat, A. S. et al. BET bromodomain inhibition releases the Mediator complex from select cis-regulatory elements. Cell Rep. 15, 519–530 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Di Micco, R. et al. Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes. Cell Rep. 9, 234–247 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Finley, L. W. S. et al. Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity. Nat. Cell Biol. 5, 565–574 (2018).

    Article  Google Scholar 

  29. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Ficz, G. et al. FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13, 351–359 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dale, T. et al. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat. Chem. Biol. 11, 973–980 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. 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  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shahbazi, M. N. et al. Pluripotent state transitions coordinate morphogenesis in mouse and human embryos. Nature 552, 239–243 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chen, H. et al. Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency. Nat. Commun. 6, 7095 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, J. et al. Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. Nature 516, 405–409 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Stirparo, G. G. et al. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast. Development 145, dev158501 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. 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  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wu, J. et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell 168, 473–486 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wu, J. et al. Stem cells and interspecies chimaeras. Nature 540, 51–59 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. Gifford, W. D., Pfaff, S. L. & MacFarlan, T. S. Transposable elements as genetic regulatory substrates in early development. Trends Cell Biol. 23, 218–226 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Göke, J. et al. Dynamic transcription of distinct classes of endogenous retroviral elements marks specific populations of early human embryonic cells. Cell Stem Cell 16, 135–141 (2015).

    Article  PubMed  Google Scholar 

  44. Grow, E. J. et al. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature 522, 221–225 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Theunissen, T. W. et al. Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 19, 502–515 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Eckersley-Maslin, M. A. et al. MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs. Cell Rep. 17, 179–192 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kolodziejczyk, A. A. et al. Single cell RNA-sequencing of pluripotent states unlocks modular transcriptional variation. Cell Stem Cell 17, 471–485 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fidalgo, M. et al. Zfp281 coordinates opposing functions of Tet1 and Tet2 in pluripotent states. Cell Stem Cell 19, 355–369 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bulut-Karslioglu, A. et al. Inhibition of mTOR induces a paused pluripotent state. Nature 540, 119–123 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Boroviak, T. et al. Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev. Cell 35, 366–382 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Blakeley, P. et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3613–3613 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  55. 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  CAS  PubMed  Google Scholar 

  56. Xue, Z. et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 500, 593–597 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Guo, G. et al. Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Rep. 6, 437–446 (2016).

    Article  CAS  Google Scholar 

  59. Chan, Y. S. et al. Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 13, 663–675 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  61. von Meyenn, F. et al. Impairment of DNA Methylation maintenance is the main cause of global demethylation in naive embryonic stem cells. Mol. Cell 62, 848–861 (2016).

    Article  Google Scholar 

  62. Yagi, M. et al. Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 548, 224–227 (2017).

    Article  CAS  PubMed  Google Scholar 

  63. Choi, J. et al. Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells. Nature 548, 219–223 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Di Stefano, B. et al. Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells. Nat. Methods 15, 732–740 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Leitch, H. G. et al. Naive pluripotency is associated with global DNA hypomethylation. Nat. Struct. Mol. Biol. 20, 311–316 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

  67. Khan, S. A., Audergon, P. N. C. B. & Payer, B. X-chromosome activity in naive human pluripotent stem cells—are we there yet? Stem Cell Investig. 4, 54–54 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Williams, L. H. et al. Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks. Mol. Cell 58, 311–322 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Respuela, P. et al. Foxd3 Promotes exit from naive pluripotency through enhancer decommissioning and inhibits germline specification. Cell Stem Cell 18, 118–133 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Westerling, T., Kuuluvainen, E. & Makela, T. P. Cdk8 is essential for preimplantation mouse development. Mol. Cell. Biol. 27, 6177–6182 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Yamanaka, Y., Lanner, F. & Rossant, J. FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development 137, 715–724 (2010).

    Article  CAS  PubMed  Google Scholar 

  74. Nichols, J., Silva, J., Roode, M. & Smith, A. Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development 136, 3215–3222 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Lopes Novo, C. et al. Long-range enhancer interactions are prevalent in mouse embryonic stem cells and are reorganized upon pluripotent state transition. Cell Rep. 22, 2615–2627 (2018).

    Article  Google Scholar 

  76. Di Stefano, B. et al. The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis. Cell Stem Cell 25, 622–638 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Factor, D. C. et al. Epigenomic comparison reveals activation of ‘seed’ enhancers during transition from naive to primed pluripotency. Cell Stem Cell 14, 854–863 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Postlmayr, A., Dumeau, C. E. & Wutz, A. Cdk8 is required for establishment of H3K27me3 and gene repression by Xist and mouse development. Development 147, dev175141 (2020).

  79. Feldmann, A., Dimitrova, E., Kenney, A., Lastuvkova, A. & Klose, R. J. CDK-Mediator and FBXL19 prime developmental genes for activation by promoting atypical regulatory interactions. Nucleic Acids Res. 48, 2942–2955 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Bradner, J. E., Hnisz, D. & Young, R. A. Transcriptional addiction in cancer. Cell 168, 629–643 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Diéguez-Hurtado, R. et al. A Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka. Genesis 49, 36–45 (2011).

    Article  PubMed  Google Scholar 

  82. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Porter, D. C. et al. Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities. Proc. Natl Acad. Sci. USA 109, 13799–13804 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Lynch, C. J. et al. The RNA polymerase II factor RPAP1 is critical for mediator-driven transcription and cell identity. Cell Rep. 22, 396–410 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Plaisier, S. B., Taschereau, R., Wong, J. A. & Graeber, T. G. Rank–rank hypergeometric overlap: identification of statistically significant overlap between gene-expression signatures. Nucleic Acids Res. 38, e169 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Castro-Diaz, N. et al. Evolutionally dynamic L1 regulation in embryonic stem cells. Genes Dev. 28, 1397–1409 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Papadopoulou, T., Kaymak, A., Sayols, S. & Richly, H. Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation. Cell Cycle 15, 1479–1493 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Xiang, L. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537–542 (2020).

    Article  CAS  PubMed  Google Scholar 

  89. Broude, E. V. et al. Expression of CDK8 and CDK8-interacting genes as potential biomarkers in breast cancer. Curr. Cancer Drug Targets 15, 739–749 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Smith, T. MacFarlan, Z. Izsvák, M. Ko, J. Hanna, D. Grégoire and U. Hibner for gifts of reagents; N. Prats and L. César Fernández for assistance, as well as staff at the CNIO and IRB core facilities. M.N.S. was funded by a Leverhulme Trust early career fellowship. Work in the laboratory of M.Z.-G. was funded by the Wellcome Trust (098287/Z/12/Z) and the European Research Council (ERC) (669198). I.C. was funded by the Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and European Social Fund. I.A. and P.S. were supported by the Fondation pour la Recherche Medicale (DEQ20170336757), Infrastructure Nationale en Biologie et Santé INGESTEM (ANR-11-INBS-0009), IHU-B CESAME (ANR-10-IBHU-003), LabEx REVIVE (ANR-10-LABX-73), LabEx DEVweCAN (ANR-10-LABX-0061) and LabEx CORTEX (ANR-11-LABX-0042) of University of Lyon within the programme ‘Investissements d’Avenir’ (ANR-11-IDEX-0007). Research by J.P., S.M. and C.B.-A. was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2013-44267-R) and by the CNIO. Work in the laboratory of D.F. was funded by the Institut National du Cancer (PLBIO10-068 and PLBIO15-005) and the Ligue National Contre le Cancer (EL2018.LNCC/DF). Work in the laboratory of N.M. was funded by the ERC, under the European Union Horizon 2020 research and innovation programme (StG-2014–640525_REGMAMKID), the Spanish Association Against Cancer (AECC/LABAE16006), Carlos III Health Institute (Red TerCel, CardioCel, RD16/0011/0027), Ministry of Economy and Competitiveness (MINECO) projects SAF2017–89782-R, SAF2015–72617-EXP and RYC-2014–16242, and the CERCA/Government of Catalonia (2017 SGR 1306). Work in the laboratory of S.O. was funded by SAF2013–44866-R from MINECO Spain. Work in the laboratory of M.F.F. was funded by Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892, to M.F.F. and A.F.F.); the ISCIII-Subdireccion General de Evaluación y Fomento de la Investigación and Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131, to A.F.F.); IUOPA (to M.I.S.); and the Asturias Regional Government (GRUPIN14–052, to M.F.F.). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Work in the laboratory of M.S. was funded by the CNIO, the IRB and by grants from Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), Botin Foundation, Banco Santander (Santander Universities Global Division), laCaixa Foundation and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Tumour Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Cian J. Lynch, Raquel Bernad, Isabel Calvo, Noelia Alcazar, Maribel Muñoz-Martín & Manuel Serrano

  2. Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    Cian J. Lynch, Raquel Bernad, Isabel Calvo, Noelia Alcazar, Maribel Muñoz-Martín & Manuel Serrano

  3. ProteoRed-ISCIII Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Ana Martínez-Val & Javier Muñoz

  4. Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Marta N. Shahbazi & Magdalena Zernicka-Goetz

  5. MRC Laboratory of Molecular Biology, Biomedical Campus, Cambridge, UK

    Marta N. Shahbazi

  6. Department of Medical Sciences and Institute of Biomedicine (iBiMED), University of Aveiro, Aveiro, Portugal

    Sandrina Nóbrega-Pereira

  7. Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Carmen Blanco-Aparicio, Sonia Martinez & Joaquin Pastor

  8. Pluripotency for Organ Regeneration, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute for Science and Technology (BIST), Barcelona, Spain

    Carolina Tarantino, Elena Garreta & Nuria Montserrat

  9. Maintenance of Transcriptional Repression by Polycomb Proteins, Institut Curie, Paris, France

    Laia Richart-Ginés

  10. Bioinformatics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Osvaldo Graña-Castro & Gonzalo Gómez-Lopez

  11. Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, INSERM U1208, Bron, France

    Irene Aksoy & Pierre Savatier

  12. Transgenic Mice Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Sagrario Ortega

  13. IGMM, University of Montpellier, CNRS, Inserm, Montpellier, France

    Susana Prieto, Elisabeth Simboeck, Alain Camasses & Daniel Fisher

  14. Bioinformatics-Biostatistics Unit, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    Camille Stephan-Otto Attolini

  15. Cancer Epigenetics and Nanomedicine Laboratory, Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Institute of Oncology of Asturias (IUOPA), ISPA-Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Oviedo, Spain

    Agustin F. Fernandez, Marta I. Sierra & Mario F. Fraga

  16. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Barcelona, Spain

    Nuria Montserrat

  17. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain

    Nuria Montserrat & Manuel Serrano

  18. Division of Biology and Biological Engineering, Caltech, Pasadena, CA, USA

    Magdalena Zernicka-Goetz

Authors
  1. Cian J. Lynch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Raquel Bernad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Martínez-Val
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marta N. Shahbazi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sandrina Nóbrega-Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Isabel Calvo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Carmen Blanco-Aparicio
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carolina Tarantino
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Elena Garreta
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Laia Richart-Ginés
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Noelia Alcazar
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Osvaldo Graña-Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Gonzalo Gómez-Lopez
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Irene Aksoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Maribel Muñoz-Martín
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Sonia Martinez
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Sagrario Ortega
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Susana Prieto
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Elisabeth Simboeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Alain Camasses
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Camille Stephan-Otto Attolini
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Agustin F. Fernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Marta I. Sierra
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Mario F. Fraga
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Joaquin Pastor
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Daniel Fisher
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Nuria Montserrat
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Pierre Savatier
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Javier Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Magdalena Zernicka-Goetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Manuel Serrano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.J.L. designed and performed most of the experiments with mouse cells and embryos, contributed to bioinformatics data analysis and cowrote the manuscript. R.B. designed and performed most of the experiments with human cells, and provided general experimental support. A.M.-V. performed proteomic and bioinformatics analysis. M.N.S. performed embryo experiments, immunofluorescence and data analysis. S.N.-P., I.C., L.R.-G., N.A. and M.M.-M. contributed to experimental work and data analysis. C.T. and E.G. contributed to research with human PSCs and performed differentiation, immunofluorescence and confocal analysis of these experiments, supervised by N.M.; O.G.-C., G.G.-L. and C.S.-O.A., contributed to bioinformatics analyses. C.B.-A., S.M. and J.P. selected, synthesized and characterized small-molecule inhibitors. S.O. provided reagents, contributed to experimental design and supervised mouse embryo research. I.A. and P.S. performed human–rabbit interspecies chimaera and STAT3 assays. S.P., E.S., A.C. and D.F. generated the CDK8-KO mouse, provided reagents and performed additional inhibitor analyses. A.F.F., M.I.S. and M.F.F. performed DNA methylation analysis. P.S., D.F., J.M. and M.Z.-G. provided reagents, discussion and revisions. M.S. designed and supervised the study, secured funding, analysed the data and cowrote the manuscript. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Manuel Serrano.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 An Inhibitor Screen for Promotion of ES Naïve State identifies a distinct role for Mediator kinase activity.

a, FACS: Percent cells Nanog-GFPhigh in indicated treatments with serum-free KSR/LIF culture. Mean±SD, n=4 independent experiments. b, Western blot: CDK8-target STAT1-Ser727-Phosphorylation; n=3 experiments with indicated cell lines. STAT1-Ser727P induction by 3h Interferon-γ, ± simultaneous 1 µM CDK8/19i. c–e, FACS histograms: mouse Nanog-GFP knockin reporter PSC previously adapted to 2i or CDK8/19i, tested at intervals. Decreased proportion of Nanog-GFPHigh cells indicates loss of naïve state. c, Changes similar by 2i-removal or CDK8/19i-removal. d, 2i protects Nanog-GFPHigh cells longer than CDK8/19i following LIF removal. e, Only [CDK8/19i+2i] protects Nanog-GFPHigh naïve-cells completely following JAK-STAT inhibition. Representative of n=2 experiments. f, Western blots: lentiviral shRNA-mediated knockdown of CDK8, CDK19 or Cyclin C (CCNC) in mouse PSC. Efficient shRNAs (red). Representative of n=2 experiments. g, Pluripotency marker mRNA expression (qRT-PCR) in mouse PSCs following 7d lentiviral shRNA-mediated knockdown of CDK8, CDK19, or CCNC. Data are the mean of two experiments. h, Upper schematic: inducible-CDK8-knockout. 4-hydroxy-tamoxifen (4OHT)- inducible Cre drives excision of Exon2. Lower: PCR confirmation of CDK8 Exon2 deletion using indicated primers. Mouse Cdk8(fl/fl)-RERT-Cre iPS (n=3–4 independent clones) treated 6d with 0.5 µM 4OHT. i, Indel mutation in one mouse CDK19-KO iPS clone, induced by indicated CRISPR guide-RNA against CDK19 Exon1, using lentiviral CRISPR-Cas9. Indel is 10 bp deletion at predicted CRIPSR target site, generating a frameshift immediately downstream of ATG start codon. j, Western blots: 4-OHT-inducible 8Cdk8-knockout mouse iPS clones as in (h). Schematic summarizes generation of these cells. Data representative of 4 experiments with n=3 iPS clones. k, Western blots: 4-OHT-inducible CDK8/19-double-knockout mouse iPS clones, generated as in (J). CDK8-knockout confirmed at protein level after 4-OHT-inducible Cre treatment. CDK19 protein was undetectable in PSCs, but readily detectable in intestinal organoid controls. Arrow indicates CDK19, confirmed by CRISPR-knockout as shown; *non-specific band. Data: CDK8/19-knockout with n=10 independent iPS clones. l, Left, Western blot: CDK8/19-double knockout (dKO) iPSCs ± empty-vector, catalytically-active CDK8 wild-type (WT), or CDK8 Kinase-Dead (KD). Right, bright field images: mouse iPS lines as indicated. Arrows: naïve-like colony morphology in cells expressing CDK8-WT plus treatment with CDK8/19i. Importantly, CDK8/19-dKO iPS with empty vector do not respond to CDK8/19-inhibitor. Images representative n=3 iPS clones. Scale bars 100 µm. m, Re-expression of CDK8-WT in null background rescues response to CDK8/19i. Left, overview. Right, mRNA expression of naïve pluripotency markers (qRT-PCR; Mean, 2 experiments) in CDK8/19-dKO mouse iPS with empty-vector, or catalytically-active CDK8-WT, in serum/LIF or CDK8/19i.

Source data

Extended Data Fig. 2 Positive effect of long- term CDK8/19i on mammalian ES self- renewal and pluripotency.

a, Cell morphology and alkaline phosphatase staining of WT mouse EpiSC infected with pMSCV-Empty or pMSCV-CDK8-KD, plus 7d/1 passage in either EpiSC media (Fgf2/ActivinA/fibronectin; Methods) or serum/LIF ES media (see schematic). Images representative of day 7 after pMSCV infection and selection, n=2 cell experiments. Scale bars 100 µm. b, FACS: maintenance of pluripotency markers in mouse PSCs. Percentage of double- positive Nanog-GFP+/ICAM1+ PSCs following 3 weeks culture in control serum/LIF, 2i- naïve, or CDK8/19i. Data: Mean, n=2 cell experiments. c, Immunofluorescence: TFE3 expression and localization in mouse PSCs adapted to control serum/LIF, 2i-naïve, or CDK8/19i, as in (b). Scale bar 10 µm. d, Embryoid body (EB) differentiation with beating cardiac centre demonstrates developmental capacity of mouse PSCs previously adapted to indicated conditions. Data: Mean±SD, n=3 cell experiments, two technical replicates each. e, Differentiation in vitro demonstrates developmental capacity of mouse PSCs previously adapted to control, 2i or CDK8/19i conditions. PSC differentiation was by LIF-removal or LIF-removal plus retinoic acid, and assessed by qRT-PCR to show loss of pluripotency (Oct4) and induction of differentiation (Brachyury, T). Data: Mean, 2 experiments. f, Teratoma assay in vivo demonstrates developmental capacity of mouse PSCs previously adapted to CDK8/19i conditions. Three embryonic germ layers confirmed in teratomas using histology (H+E stain; upper panels), and staining for germ layer markers: NESTIN (ectoderm), VIMENTIN (mesoderm), and Alpha-feto-protein (AFP, endoderm). Data representative of n=6 teratomas. Scale bar 200 µm. g, Brightfield images showing colony morphology in 3 human PSC lines in primed state (upper panels), or 14d treatment with CDK8/19i. Images representative n=5 independent experiments. Scale bar 100µm. h, Brightfield and live-cell GFP-fluorescence images of human iPS cells (HERVH-GFP reporter) in primed conditions, or following 14d treatment with indicated media cocktails. To derive and maintain the 2i p38iJNKi condition. Images representative of n=5 independent experiments. Scale bar 100 µm. i, FACS analysis of pluripotency markers in human PSCs (HERVH iPS or WIBR3 ES), following 3 weeks adaption to indicated culture conditions, as in (g, h). Data represent one experiment with n=2 independent PSC lines. Primed or CDK8/19i PSCs were routinely passaged in bulk using collagenase. In contrast, for the 2i p38iJNKi condition, cytometric sorting was required to select the top 10% HERVH-GFP cells at each passage, for 3 passages, before fixing the cells 4d after third passage/selection- round.

Source data

Extended Data Fig. 3 Self-renewal, genomic stability, and gene expression analysis in mouse and human PSCs in CDK8/19i.

a, Single cell clonogenicity: human PSCs in primed or CDK8/19i conditions. Cells were FACS-selected for top or bottom 5% HERVH-GFP intensity, seeded at clonal density in primed or CDK8/19i culture (±p38iJNKi) for 7d (individual colonies arise separately), then Alkaline phosphatase stained (inset, example colonies) to visually score maintenance of pluripotent status. Data: n=10 fields of view, multiple colonies per view, one experiment. b, Western blots: naïve pluripotency marker KLF17 in primed or CDK8/19i conditions. SMC1: nuclear internal loading control; n=4 human PSC lines. c, Mean pluripotency marker mRNA expression levels (qRT-PCR) in each of n=5 human PSC lines, in primed or CDK8/19i conditions >14d. d, Karyotyping indicates genomic stability: Human PSC lines (n=5), 16–19 passages in primed or CDK8/19i conditions. Inset, representative example: karyotype maintenance. e, Rank-Rank Hypergeometric Overlap (RRHO):85 RNA-seq differential expression in mouse PSCs adapted to 2i-naïve or CDK8/19i conditions, versus serum/LIF (n=3 biological replicates; n=12,629 genes). Genes arranged by magnitude-change, then assessed for overlap by RRHO sliding window of 100 genes. Colour intensity: -log10 p- value after Benjamini-Yekutieli correction of hypergeometric overlap. f, Overlap and hypergeometric significance of differentially-expressed mRNAs in mouse PSCs in 2i or CDK8/19i, versus control KSR/LIF (serum-free conditions) RNA- seq; n=3 biological replicates; FDR<0.05. g, Pluripotency marker and LINE L1 repeat RNA expression (qRT-PCR) in mouse PSCs cultured as in (e). Data: n=3 experiments, Mean±SD, t-test, unpaired, two-tailed, *P<0.05. h, Effect of 2i or CDK8/19i on LINE L1 super-family expression (RNA-seq) in mouse PSCs cultured as in (e). Data: Mean, n=3 biological replicates, for each LINE L1 family, arranged by evolutionary age, which also reflects transcriptional activity and regulatory mechanisms86. 2i and CDK8/19i similarly regulate the youngest and most transcriptionally-active families (calculations, notes: Source Data). i, Dot plot: RNA-seq expression, in mouse PSCs, cultured as in (e). Pluripotency markers (red, n=18); 2C-fluctuation markers (green, n=112);46,47,82 lists: Source Data). Below: effect of 2i or CDK8/19i (current study), or CDK8-knockdown87, on these genesets. Significance: GSEA FDR q-values<0.25, indicated. j, Western blots, mouse PSCs. Markers of pluripotency, or 2C-fluctuation (ZSCAN4). Representative: n=2 experiments. k, l, RNA expression: 2C-fluctuation markers (qRT-PCR), mouse PSCs cultured as in (e), or after 10d CDK8/19i-withdrawal (l). Data: Mean, n=2 experiments. m, FACS quantification, percent fluorescencehigh cells in 2C-fluctuation in two independent mouse PSC 2C-reporter lines, cultured as in (e). Induction of 2C- fluctuation: inducible-Kdm1a-knockout46, or 48h TSA46. Data: Mean±SD, n=3 experiments.

Source data

Extended Data Fig. 4 Comparison of CDK8/19i and 2i in the current work versus published studies.

a, b, FACS quantification, percent fluorescencehigh cells in 2C-fluctuation: n=2 independent mouse PSC 2C-reporter lines46,82, cultured as indicated. 2i and CDK8/19i repress the 2C-fluctuation, reversible by 2–10d inhibitor-removal. Data: Mean, n=3 experiments. c, Bright field or fluorescencehigh cells in 2C-fluctuation: n=2 independent mouse PSC 2C-reporter lines46,82, cultured as indicated. Induction of 2C-fluctuation: 7d inducible- Kdm1a-knockout46, or 48h TSA46. d, e, Overlaps between RNAseq published datasets versus current study in mouse PSCs adapted to 2i or CDK8/19i versus control serum/LIF. Differentially expressed mRNAs in current study (FDR<0.01 and 2-fold change), versus three published studies23,48,49 (d), or, versus developmental stage-specific marker genesets from preimplantation mouse embryos52 (e). (D,E) Overlap hypergeometric significance, and number of genes changing in same direction, are reported below each 2-way comparison. f, Pluripotency (NANOG, POU5F1/OCT4, KLF4, CDH1/E-cadherin) or differentiation (CDH2, NESTIN) marker expression (qRT-PCR) in human PSCs adapted to indicated conditions. Mean±SD, n=1–3 biological experiments. g, Human embryo stage-specific developmental genesets (scRNA-seq), defining pre- implantation naïve epiblast (n=242 genes) and post-implantation primed epiblast (n=620 genes), can distinguish human PSCs between naïve and primed pluripotent states in vitro by up/down-regulation39,53,55 (listed in: Source Data). RNA-seq expression of these genesets is shown in the current study in human PSCs adapted to indicated conditions. Tukey box plots; box reflects 25th -75th percentile; horizontal line is median; white-cross indicates mean. Data: n=3 biological replicates, Mean± SD, t-test, unpaired, two-tailed, ****P < 0.0001. h, i, Human embryo lineage-specific genesets38 defining early/late pre-implantation naïve epiblast (n=22/24 genes), and late primitive endoderm (n=50 genes), were assessed by GSEA in our human PSC cultured in 2i-naïve or CDK8/19i. h, 1.1 μM CDK8/19i, one PSC line. i, 0.4 μM CDK8/19i, 4 PSC lines. Significance indicated by GSEA FDR q-values<0.25, and up/down-regulation (red arrows), in each panel. j, Human embryo lineage-specific scRNA-seq genesets88, defining pre-implantation naïve epiblast (n=417 genes), primitive endoderm (n=83 genes), or trophectoderm (n=111 genes), were assessed by GSEA in our human PSC cultured in 0.4 μM CDK8/19i versus primed culture (n=4 PSC lines). Significance indicated by GSEA FDR q- values<0.25, and up/down-regulation (red arrows), in each panel.

Source data

Extended Data Fig. 5 CDK8/19i regulates the phospho-proteome and proteome similar to 2i- naïve pluripotency, but not DNA methylation.

a, Left: Overlap and hypergeometric significance (P-value) of differentially expressed proteins (FDR<0.05), in mouse PSC lines adapted to 2i-naïve or CDK8/19i, versus standard serum/LIF (n=5 lines, displayed individually). Right: table compares overlap in proteins up/down-regulated, per cell line and per condition, to highlight that the positive correlation (proteins changing in same direction) is greater than the negative correlation in all PSC lines. Supplementary Table 4: list of differentially expressed proteins. b, Summary, all proteomic changes, shown per mouse PSC line, adapted to 2i-naïve or CDK8/19i conditions, versus control serum/LIF, as in (a). c, Heatmap: normalized enrichment of biological pathways identified as significantly up/down-regulated (blue/yellow), by GSEA of proteomic changes, shown per cell line, in mouse PSCs cultured as in (a). Significance was confirmed in all pathways shown (GSEA FDR q-values<0.25). d, e, Rank-Rank Hypergeometric Overlap (RRHO):85 differential mRNA expression (X- axis) in mouse PSCs adapted to 2i-naïve conditions (d) or CDK8/19i (e), versus, differential protein expression (Y-axis), for the same genes (n = 5289). Genes arranged by magnitude-change, then assessed for overlap by RRHO sliding window of 100 genes. Colour intensity: -log10 p-value after Benjamini-Yekutieli correction of hypergeometric overlap. f, g, DNA methylation changes (5-methyl-cytosine; pyrosequencing) in n=4 mouse PSC lines adapted to 2i or CDK8/19i. f, Methylation levels at two CpG sites in Major Satellite repeats, shown independently (right), or Mean±SD across the CpG loci (left). g, Methylation levels at four CpG sites in IAP repeats, shown independently (right), or Mean±SD across the CpG loci (left). h, XIST RNA levels in human PSC lines in this study (qRT-PCR). Female (n=3: WIBR3, CB5 and H9) and Male (n=1: D2#2) PSCs display low/undetectable XIST expression compared to control adult female human somatic cells (lung fibroblasts), suggesting X-silencing erosion may have already occurred in parental cells, as previously observed67. Data: n=3 technical replicates. i, Functional analysis of proteins containing a CDK phospho-target motif that displays phosphorylation decrease (FDR<0.05) within 15 min treatment of mouse PSCs with 2i or CDK8/19i. Data: n=2 PSC lines. j, Western blots: ERK1/2 phosphorylation after long-term adaption (3 weeks) of mouse PSCs to serum/LIF, 2i, or CDK8/19i. Above: relative ERK1/2 phospho-levels, normalized by total ERK1/2 levels. k, Western blots: CDK8 kinase-target STAT1-phospho-serine727, in human PSCs, with indicated culture media. l, CDK8 protein levels per cell measured by cytometry in mouse PSCs treated with indicated inhibitors. j–l Representative, n=2 independent experiments.

Source data

Extended Data Fig. 6 Analysis of RNA Pol II genomic distribution and corellation with RNA gene expression.

a, ChIP-seq: RNA Pol II Serine 5 phosphorylation (Ser5P) abundance at all Refseq Transcription Start Sites (TSS; n=28,441), in mouse PSCs, treated as indicated. Left: Heatmap, TSS +/−5 Kb. Right: Metagene average +/−2 Kb. ChIP-seq n=3 pooled replicates. b, ChIP-qPCR: RNA Pol II and histone marks at the Nanog TSS. RNA Pol II and H3K4me3 (active euchromatin) are increased. Data: Mean±SD, n=4 ChIP replicates. c, Re-analysis of published23,69 ChIP-seq: RNA Pol II abundance at all Refseq Transcription Start Sites (TSS; n=28,441), in mouse PSCs, treated as indicated, (similar conditions to current study, compare with Fig. 5h, i). Left: Heatmap, TSS +/−5Kb. Right: Metagene average +/−2Kb. ChIP-seq n=3 pooled replicates. d, Schematic: defining gene regions and Pol II loading ratios used in this study, similar to previous reports68. Lower panel: schematic summarizing results in Fig. 5h, i, where Promoter Loading Index is increased (Promoter/Body). e, f, Rank-Rank Hypergeometric Overlap (RRHO):85 differential mRNA expression (RNA-seq data; Y-axis) in mouse PSCs adapted to 2i-naïve conditions (e; n=10,117) or CDK8/19i (f; n=10,136), versus, differential RNA Pol II abundance at promoter-TSS (ChIP-seq data; X-axis), for the same genes. Genes arranged by magnitude-change, then assessed for overlap by RRHO sliding window of 100 genes. Colour intensity: - log10 p-value after Benjamini-Yekutieli correction of hypergeometric overlap. g, h, Venn diagrams: genes with differential mRNA expression up/down-regulated (green circles; FDR<0.01) in 2i (g) or CDK8/19i (h) overlap significantly with genes where the promoter has the greatest/least change in RNA Pol II abundance (red circles; promoters with fold change > one standard deviation from mean). Overlap significance: hypergeometric test; P-values, and number of genes “n”, indicated in each panel. Genes up (top Venn diagrams), and genes down (lower Venn diagrams), refer to inhibitor-treated cells versus control serum/LIF conditions. i, Genes with the greatest change in RNA Pol II abundance (lower panel; ranked list of promoters by magnitude of RNA Pol II abundance fold-change; n=12,693; ChIP-seq) correlate with the top 100 most differentially expressed mRNAs up/down-regulated in 2i-naïve conditions (upper two panels; RNA-seq). All changes refer to 2i-treated cells versus control serum/LIF conditions.

Source data

Extended Data Fig. 7 ChIP-seq for CDK8 and analysis of its genomic distribution.

a, CDK8/19 average ChIP-seq enrichment2,3 density in mouse PSCs at Promoter-TSS regions +/− 2 Kb, n=28,441 TSS (Refseq). b, CDK8/19 binding loci defined in mouse PSCs by ChIP-seq2,3, MACS peak calling, and categorized by functional annotation of the region by HOMER (n=21,703, see Supplementary Table 7). Note: ChIP antibody binds both CDK8 and CDK19, see Methods. Promoter-TSS: TSS+/− 1Kb. Gene Body: Exons, Introns, and transcription termination site TTS +/− 1Kb. Enhancer constituent regions, as defined2,3. c, Percentage of SE-constituent regions2,3 enriched for CDK8/19 binding (see also Supplementary Table 7). d, CDK8 is an integral part of Enhancer-Mediator in mouse PSCs. Pearson correlation Matrix summarizes correlation/co-occupancy between 59 factors in 10627 Enhancers in mouse PSCs, based on comparison of ChIP-seq signal intensity in published datasets. Enhancer loci and ChIPseq data extracted from2,3. The 59 factors indicated are a range of chromatin modifiers and transcription factors. Each square of the matrix represents a comparison between the corresponding pair of factors for their similarity in ChIP signal ranking across the 10,627 enhancer regions, to calculate a r2 correlation of their similarity, where 1.0 = exactly similar. An example of a single correlation between two factors is shown for the Mediator subunit Med1 and CDK8/19 abundance within mouse PSC enhancers, in the upper-right of the panel. Hierarchical clustering groups those factors by similarity in ChIP signal pattern across all 10,627 enhancers. Thus, high correlation between two factors (red), indicates co- enrichment to similar levels and at the same set of enhancers, which is suggestive of functional co-operation. Co-enrichment patterns for subunits and co-factors of the Mediator, RNA Pol II and Cohesin complexes can be observed (indicated), consistent with their reported combinatorial roles at enhancers. CDK8/19 clusters most closely with the Mediator complex and other critical regulators of enhancer function. See Methods, Supplementary Table 7, and Source Data for analysis of the published ChIP datasets and enhancer loci defined by2,3.

Source data

Extended Data Fig. 8 RNA Pol II and CDK8 genomic distribution. 2i and CDK8/19i hyper- actívate naïve-state enhancer activity.

a–c, Gene ontology enrichment and functional annotation of CDK8/19-target genes identified by the single-nearest gene to each CDK8/19 binding site in mouse PSCs (ChIP-seq, n=21,703 peaks: Supplementary Table 7) using GREAT analysis90. Data: -log 10 binomial P-value with Bonferroni correction for multiple hypothesis testing indicating the significance of each gene ontology category. d, Metagene enrichment in the indicated genomic regions for CDK8/19 or RNA Pol II abundance, as determined by ChIP-seq in mouse PSCs above. Genomic regions were defined in groups by the CDK8/19 peak intensity as defined in (a). e, Identification of mouse PSC super-enhancer (SE) loci specific to pre-implantation naïve epiblast, or post-implantation primed epiblast. Enhancer loci were extracted from the Prestige Database77. The SEs in naïve or primed epiblast were first identified, and then any SEs common to a panel of 16 somatic tissues (threshold: 1 bp overlap) were subtracted (Methods). Enhancer loci lists: Source Data. f, RNA Pol II abundance in mouse PSC primed-specific super-enhancers (on left, n=1005), or naïve-specific super-enhancers (on right, n=1424), as defined in (e). RNA Pol II levels are significantly higher in 2i or CDK8/19i conditions versus serum/LIF control: t-test, unpaired, two-tailed, Welchs correction, **P<0.01; ****P<0.0001). Tukey box plot centre lines show median values, box limits represent upper and lower quartiles, and whiskers show 1.5× interquartile range. g, qRT-PCR: pluripotency marker genes and naïve-specific eRNA71 abundance in mouse PSC at short time intervals after withdrawal of 2i or CDK8/19i from culture. Data: Mean±SEM, n=3 experiments. h, Rank-Rank Hypergeometric Overlap (RRHO)85: heatmap shows differential mRNA expression (RNA-seq) of the single-nearest target genes (n=3,553; GREAT analysis) identified for all PSC enhancers2,3 (n=10,627) in 2i-naïve conditions (X-axis) or CDK8/19i (Y-axis), compared to control serum/LIF conditions. The enhancer-target mRNA expression changes are arranged by magnitude, then assessed for overlap by RRHO sliding window of 100 genes. Colour intensity: -log10 P value after Benjamini- Yekutieli correction of hypergeometric overlap. Highly significant overlap along the diagonal indicates similar regulation of enhancer-target gene mRNA expression in 2i and CDK8/19i. i, FACS: NANOG and OCT4 protein expression following 7d treatment with 500 nM BRD4i(JQ1) in CDK8/19-dKO iPS clones expressing pMSCV-Empty or pMSCV-CDK8- Kinase-Dead (CDK8-KD). Representative of n=3 cell experiments. j, qRT-PCR: expression of naïve marker genes following 48h treatment with 500 nM BRD4i(JQ1). CDK8/19-dKO iPS ± CDK8-KD were cultured in 2i or standard serum/LIF, as indicated. Mean±SD, n=3 independent clones.

Source data

Extended Data Fig. 9 CDK8 expression in vivo and the role of Mediator during mouse preimplantation development.

a, CDK8 and CDK19 mRNA relative expression levels in PSCs, as detected by RNAseq in 5 mouse datasets23,49,50,51 and 6 human datasets32,34,39,58,66 (including the current study). Mean±SD of independent RNAseq replicates in each published study (see: n replicates indicated in panel; data calculation, see: Source Data). b, Immunofluorescence for CDK8 protein levels during mouse preimplantation development from 1-Cell to early blastocyst stage (E3.5), representing one set of embryos. Scale bars 25μm. c, CDK8/19-inhibition blocks embryo development at 1–2 Cell stage. Day E0.5 zygotes were harvested from females and immediately cultured in vitro in KSOM ± CDK8/19i for 2 days, with assessment of their developmental progression by visual inspection of cell number and morphology at intervals. Data represents n=30 embryos per condition, across two independent experiments. d, e, CDK8 mRNA expression levels in specific embryo stages and lineages during mouse preimplantation development. CDK8 mRNA expression declines until blastocyst stage, both in mouse and human pre-implantation embryos. In (d), mean data values from published microarray studies (Methods). In (e), CDK8 mRNA expression levels detected by RNA-seq in specific embryo stages and lineages during mouse preimplantation development; data from52, Mean±SD, n=2–3 replicates per time point, significance assessed by one-way ANOVA unpaired T-test. *P<0.05; **P<0.01. (see Source Data). f, CDK8 mRNA expression levels during mouse or human embryo pre-implantation development, as detected by microarray in published datasets. Mouse: https://www.ncbi.nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS812:96726_at Human: https://www.ncbi.nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS3959:1553112_s_at (g) Schematic showing example of FACS gating strategy in this study. DAPI was added to live cell suspension 2mins before analysis, as a live/dead discriminator. Gates 1, 2, and 3, sequentially act to exclude cell doublets, and debris, thus selecting live single cells for analysis of Nanog-GFP profile in mouse PSCs.

Source data

Extended Data Fig. 10 Cyclin C expression localization during mouse preimplantation development.

a, Immunofluorescence: CDK8, OCT4, and F-ACTIN in mouse early embryos from E4.5 to E5.5. Scale bars 20 μm. Images representative of n=3 experiments. b, c, Immunofluorescence (b): cyclin C protein levels during mouse development from preimplantation blastocyst stage (E4.5) to post-implantation cylinder stage (E5.5). Co-staining with OCT4 to mark epiblast, and GATA6, to mark primitive endoderm at E4.5 and its maturation into post-implantation visceral endoderm. c,, Cyclin C nuclear-cytoplasmic ratio was quantified and plotted, where each data point represents the mean Nuc-Cyto ratio for epiblast cells of one embryo (Source Data). As internal control, the Nuc-Cyto ratio for OCT4 was also quantified. Nuclear abundance of cyclin C increases in epiblast cells during development from E4.5 to E5.5. In contrast, OCT4 nuclear-cytoplasmic ratio does not follow this pattern. This implies that cyclin C pattern is not related to staining or imaging artefacts. b,, representative examples shown; n=2 experiments. c,, data: Mean±SD, T-test, unpaired, two-tailed, P-values and number of embryos “n” is indicated. d, Western blot: cyclin C localization by sub-cellular localization. Nuclear and cytoplasmic fractions were prepared from mouse cells across developmental spectrum: naïve (adapted to 2i), primed (adapted to serum/LIF), or epiblast-like stem cells (abbreviated EpiLC; derived by treating PSCs for 48 h with EpiSC media50) (Methods). Relative abundance of nuclear cyclin C is greater in primed state EpiLC and in serum/LIF conditions, compared to 2i-naïve. Data represent n=2 experiments. e, Summary: The developmental requirement for CDK8 activity mirrors its embryonic expression pattern. Maxima in CDK8 expression coincide with a requirement for development around the zygote-morula and post-implantation stages. Between these two periods, a transient minima in CDK8 expression occurs during emergence of naïve epiblast, where CDK8 function appears dispensable. We suggest that CDK8/19 chemical inhibition in vitro mimics CDK8 downregulation during pre-implantation development in vivo, providing mechanistic insight on how naïve pluripotency may arise in the embryo: (i) CDK8/19 is required during zygote-to-morula development, where its expression is high. (ii) During morula-to-blastocyst pre-implantation development, CDK8 expression declines, and nuclear cyclin C decreases. This coincides with the emergence of E4.5 pre-implantation naïve epiblast and, accordingly, CDK8/19 inhibition does not interfere with naïve epiblast specification. In contrast to MEK inhibition, CDK8/19 inhibition does not affect the epiblast/PE lineage segregation. (iii) During the subsequent developmental transition of pre-implantation naïve epiblast to post-implantation primed state, CDK8 expression becomes increased and CDK8/19 activity is required for morphogenic events.

Source data

Supplementary information

Supplementary Information

Characterization of novel small molecule inhibitors, related to Fig. 1, and additional qPCR plots and teratoma immunofluorescence images related to Fig. 2.

Reporting Summary

Supplementary Tables

Supplementary Table 1: summary and comparison of inhibitors used in this study. Supplementary Table 2: RNA-seq analysis in mouse PSCs adapted to culture in Serum/LIF, 2i or CDK8/19i conditions. Supplementary Table 3: RNA-seq analysis in human PSCs adapted to culture in Primed, 2i or CDK8/19i conditions. Supplementary Table 4: whole-proteome analysis in five ES lines in control serum/LIF, 2i or CDK8/19i. Supplementary Table 5: immediate phosphorylation site changes in mouse PSCs treated with 2i or CDK8/19i at T = 15 min. Supplementary Table 6: ChIP–seq of RNA Pol II in mouse ES cells in control serum/LIF, 2i or CDK8/19i. Supplementary Table 7: analysis of CDK8/19 ChIP–seq and enhancer regions. Supplementary Table 8: reagents, primers and antibodies used in this study.

Supplementary Dataset

Supporting data for the Supplementary Information.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 1

Unprocessed images.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed images.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed images.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed images.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed images.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed images.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

Source Data Extended Data Fig. 10

Unprocessed images.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lynch, C.J., Bernad, R., Martínez-Val, A. et al. Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases. Nat Cell Biol 22, 1223–1238 (2020). https://doi.org/10.1038/s41556-020-0573-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-020-0573-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing