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Pramel7 mediates ground-state pluripotency through proteasomal–epigenetic combined pathways

A Corrigendum to this article was published on 01 August 2017

This article has been updated

Abstract

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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Figure 1: Expression of Pramel7 in ESCs induces a gene signature similar to ICM.
Figure 2: Expression of Pramel7 induces hypomethylation of embryonic stem cells.
Figure 3: Pramel7 associates with UHRF1 and components of the Cullin 2 RING E3 ubiquitin ligase (CRL) complex.
Figure 4: Pramel7 expression affects UHRF1 stability and leads to UHRF1 degradation via the 26S-proteasome pathway.
Figure 5: Pramel7 LRRs and UHRF1-SRA and RING domains are implicated in Pramel7–UHRF1 interaction and UHRF1 stability.
Figure 6: Pramel7 is required for ICM.
Figure 7: Pramel7 maintains the pluripotent state by repressing DNA methylation through regulation of UHRF1 stability.
Figure 8: Forced expression of Pramel7 affects the stable terminally differentiated state.

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  • 21 June 2017

    In the version of this Article originally published, the following affiliation was omitted for Sarah Wyck: Clinic of Reproductive Medicine, University of Zurich, Winterthurerstrasse 260, CH–8057 Zurich, Switzerland. This has been corrected in the online version of the Article.

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Acknowledgements

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.).

Author information

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Authors

Contributions

U.G. performed most of the experiments, contributed to experimental design and data analysis and wrote the manuscript. E.A.C. performed yeast two-hybrid screening. S.W., D.D. and E.V. performed methylation analysis. R.S., D.D. and M.W.S. analysed transcriptome and methylation data. M.J.O. performed RNA-seq and PCA analysis. E.A.C., F.A.W. and S.S.P. generated and analysed Pramel7 ESCs. J.L., J.S., H.K. and J.W. contributed to UHRF1 plasmids and UHRF1-KO ESCs. G.A.W. contributed to data analysis. M.G. and L.P. performed the ubiquitylation analysis. P.C. and P.P. generated and analysed Pramel7 knockout embryos; P.C. designed most of the experiments; R.S. and P.C. jointly directed the study and wrote the manuscript.

Corresponding authors

Correspondence to Raffaella Santoro or Paolo Cinelli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 (Related to Fig. 1).

(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.

Supplementary Figure 2 (Related to Fig. 1).

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.

Supplementary Figure 3 (Related to Fig. 2).

(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.

Supplementary Figure 4 (Related to Fig. 2).

(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.

Supplementary Figure 5 (Related to Fig. 7 and 8).

(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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