Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.
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Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981).
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Zwaka, T. P. & Thomson, J. A. A germ cell origin of embryonic stem cells? Development 132, 227–233 (2005).
Niwa, H. How is pluripotency determined and maintained? Development 134, 635–646 (2007).
Tang, F. et al. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 6, 468–478 (2010).
Hackett, J. A. & Surani, M. A. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 15, 416–430 (2014).
Smith, Z. D. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012).
Habibi, E. et al. Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13, 360–369 (2013).
Boroviak, T., Loos, R., Bertone, P., Smith, A. & Nichols, J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat. Cell Biol. 16, 516–528 (2014).
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).
Leitch, H. G. et al. Naive pluripotency is associated with global DNA hypomethylation. Nat. Struct. Mol. Biol. 20, 311–316 (2013).
Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012).
Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).
Bortvin, A. et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673–1680 (2003).
Casanova, E. et al. Pramel7 mediates LIF/STAT3-dependent self-renewal in embryonic stem cells. Stem Cells 29, 474–485 (2011).
Cinelli, P. et al. Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3. BMC Dev. Biol. 8, 57 (2008).
Burdon, T., Stracey, C., Chambers, I., Nichols, J. & Smith, A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev. Biol. 210, 30–43 (1999).
Kunath, T. et al. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134, 2895–2902 (2007).
Stavridis, M. P., Lunn, J. S., Collins, B. J. & Storey, K. G. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development 134, 2889–2894 (2007).
Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007).
Bostick, M. et al. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 317, 1760–1764 (2007).
Liu, X. et al. UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9. Nat. Commun. 4, 1563 (2013).
Hotton, S. K. & Callis, J. Regulation of cullin RING ligases. Annu. Rev. Plant Biol. 59, 467–489 (2008).
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).
Grice, G. L. & Nathan, J. A. The recognition of ubiquitinated proteins by the proteasome. Cell. Mol. Life Sci. 73, 3497–3506 (2016).
Kobe, B. & Kajava, A. V. The leucine-rich repeat as a protein recognition motif. Curr. Opin. Struct. Biol. 11, 725–732 (2001).
Nishiyama, A. et al. Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 502, 249–253 (2013).
Arita, K., Ariyoshi, M., Tochio, H., Nakamura, Y. & Shirakawa, M. Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism. Nature 455, 818–821 (2008).
Achour, M. et al. UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity. Biochem. Biophys. Res. Commun. 390, 523–528 (2009).
Gelato, K. A. et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol. Cell 54, 905–919 (2014).
Chu, V. T. et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16, 4 (2016).
Schmidt, C. S. et al. Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state. PLoS ONE 7, e52629 (2012).
Sakaue, M. et al. DNA methylation is dispensable for the growth and survival of the extraembryonic lineages. Curr. Biol. 20, 1452–1457 (2010).
Savic, N. et al. lncRNA maturation to initiate heterochromatin formation in the nucleolus is required for exit from pluripotency in ESCs. Cell Stem Cell 15, 720–734 (2014).
Bonapace, I. M. et al. Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry. J. Cell Biol. 157, 909–914 (2002).
Kalkan, T. & Smith, A. Mapping the route from naive pluripotency to lineage specification. Phil. Trans. R Soc. B 369, 20130540 (2014).
Tai, C. I. & Ying, Q. L. Gbx2, a LIF/Stat3 target, promotes reprogramming to and retention of the pluripotent ground state. J. Cell Sci. 126, 1093–1098 (2013).
Huang, C. & Qin, D. Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells. J. Genet. Genomics 37, 441–449 (2010).
Weidgang, C. E. et al. TBX3 directs cell-fate decision toward mesendoderm. Stem Cell Rep. 1, 248–265 (2013).
Fouse, S. D. et al. Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation. Cell Stem Cell 2, 160–169 (2008).
Sharif, J. et al. Activation of endogenous retroviruses in Dnmt1−/− ESCs involves disruption of SETDB1-mediated repression by NP95 binding to hemimethylated DNA. Cell Stem Cell 19, 81–94 (2016).
van Baren, N. et al. PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukaemia cells. Br. J. Haematol. 102, 1376–1379 (1998).
Ikeda, H. et al. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 6, 199–208 (1997).
van ’t Veer, L. J. et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530–536 (2002).
Tsumura, A. et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006).
Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).
Johnson, W. E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).
Le, S., Josse, J. & Husson, F. FactoMineR: an R package for multivariate analysis. J. Stat. Softw. 25, 1–18 (2008).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Hermann, M., Cermak, T., Voytas, D. F. & Pelczar, P. Mouse genome engineering using designer nucleases. J. Vis. Exp. 86, 50930 (2014).
The authors thank D. Bär for technical assistance, W. Piwko and O. Shakhova for discussions and reagents and J. vom Berg, D. Korkmaz and M. Tarnowska for embryo isolation. We acknowledge assistance provided by the Functional Genomic Center Zurich, especially C. Aquino and L. Opitz. This work was supported by the Olga Mayenfisch Foundation (to P.C. and R.S.), Julius Müller Stiftung (to R.S.), the Novartis Foundation for Medical-Biological Research (to P.C.), the Theodor und lda Herzog-Egli Foundation (to P.C. and R.S.), the Sassella Stiftung (P.C.), the Stiftung für Wissenschaftliche Forschung an der Universität Zürich (to P.C. and R.S.), the Helmut Horten Stiftung (to L.P.), Krebsliga Schweiz (KFS-3497-08-2014 to R.S.), the Swiss National Science Foundation (31003A_173056 and 31003A-152854 to R.S., 323530-133905 to F.A.W., 31003A-166370 to L.P.), the UBS-Promedica Stiftung (to R.S.) and the Forschungskredit of the University of Zurich (to U.G., E.V. and D.D.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Expression of Pramel7 in E14, Uhrf1−/− and DNMT-TKO ESCs grown in serum/LIF or 2i conditions. Pramel7 mRNA levels were measured by qRT-PCR and normalized to Actin mRNA. Average values of two independent experiments, lines represent means. (b) Western blot showing expression levels of Pramel7 and UHRF1 in E14, DNMT-TKO, Uhrf1−/− ESCs and P7 ESCs cultured in serum/LIF and 2i conditions detected with Pramel7 and UHRF1 antibodies. Tubulin is shown as loading control. Representative of 3 independent experiments. Unprocessed original scans of immunoblots are shown in Supplementary Figure 6.
Correlation of P7-ESC gene expression to the early embryo stages. PCA analysis (dimensions 2 and 3) of P7-ESCs, embryonic stages E3.5 and E4.5, ESCs cultured in serum/LIF or 2i from data set published in9.
(a,b) COBRA analysis showing extensive demethylation of H19/Igf2 ICR and KvDMR1 (an intronic CpG island within the KCNQ1 gene) in P7-ESCs. Bisulfite converted DNA was amplified with bisulfite specific primers and methylation content was assessed by digestion with BstUI that recognizes CGCG sequences. Digestion of BstUI serves as indicator of the presence of methylated sequences which were resistant to C to U conversion. Bisulfite sequencing of KvDMR1 (b) supports the lack of DNA methylation measured by COBRA assay. (c) Example of changes in transcription of two imprinting regulated genes in P7-ESCs. Experiments in a and b were repeated independently twice.
(a) Western blot showing expression levels of DNMT1, 3a and 3b in E14 and P7-ESCs cultured in serum/LIF. Tubulin is shown as loading control. (b) Tet1, Tet2 and Tet3 expression levels measured by RT-qPCR. Values were normalized to Actin mRNA and represent the average of two independent experiments. Average values of two independent experiments. Unprocessed original scans of immunoblots are shown in Supplementary Figure 6.
(a) Representative images of ESCs grown in 2i condition and upon 5 days of differentiation. Upon differentiation methylation defective Uhrf1−/− and DNMT-TKO ESCs formed less differentiated and more compact colonies as in the case of P7-ESCs. Scale bar: 200 μm. Representative of 2 independent experiments. (b) Expression of pluripotency-associated genes Nanog, Oct4 and Rex1 in three P7 reconverted clones. Values were measured by qRT-PCR, normalized to actin mRNA and represented as fold change relative to the expression in E14-ESCs. Average values of two independent experiments.
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Graf, U., Casanova, E., Wyck, S. et al. Pramel7 mediates ground-state pluripotency through proteasomal–epigenetic combined pathways. Nat Cell Biol 19, 763–773 (2017). https://doi.org/10.1038/ncb3554
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