Lynch, Cian J. and Bernad, Raquel and Martínez-Val, Ana and Shahbazi, Marta N. and Nóbrega-Pereira, Sandrina and Calvo, Isabel and Blanco-Aparicio, Carmen and Tarantino, Carolina and Garreta, Elena and Richart-Ginés, Laia and Alcazar, Noelia and Graña-Castro, Osvaldo and Gómez-Lopez, Gonzalo and Aksoy, Irene and Muñoz-Martín, Maribel and Martinez, Sonia and Ortega, Sagrario and Prieto, Susana and Simboeck, Elisabeth and Camasses, Alain and Stephan-Otto Attolini, Camille and Fernandez, Agustin F. and Sierra, Marta I. and Fraga, Mario F. and Pastor, Joaquin and Fisher, Daniel and Montserrat, Nuria and Savatier, Pierre and Muñoz, Javier and Zernicka-Goetz, Magdalena and Serrano, Manuel (2020) Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases. Nature Cell Biology, 22 (10). pp. 1223-1238. ISSN 1465-7392. https://resolver.caltech.edu/CaltechAUTHORS:20201012-163632801
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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.
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| Additional Information: | © 2020 Nature Publishing Group. Received 02 May 2019. Accepted 07 August 2020. Published 28 September 2020. 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. 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. Author 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. The authors declare no competing interests. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Record Number: | CaltechAUTHORS:20201012-163632801 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Official Citation: | 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 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Deposited By: | George Porter | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Deposited On: | 13 Oct 2020 16:16 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Last Modified: | 13 Oct 2020 16:16 |
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