PRDM14 maintains pluripotency of embryonic stem cells through TET-mediated active DNA demethylation

https://doi.org/10.1016/j.bbrc.2015.08.122Get rights and content

Highlights

  • PRDM14 overexpression promotes ESC self-renewal in a LIF-independent manner.

  • Both TET1 and TET2 are required for the repression of ESC differentiation by PRDM14.

  • Base excision repair pathway is required for the repression of ESC differentiation by PRDM14.

Abstract

Pluripotency and self-renewal of mouse embryonic stem cells (ESCs) depend on a network of transcription factors maintained by exogenous leukaemia inhibitory factor (LIF). PR-domain containing transcriptional regulator 14 (PRDM14), is essential for maintenance of ESC self-renewal when the cells are cultured in serum plus LIF, but not in 2i medium plus LIF. Here, we show that pluripotency of ESCs is maintained by enforced expression of PRDM14 at a high level, as observed in ESCs in 2i plus LIF and developing primordial germ cells in the absence of LIF. Constitutive expression of PRDM14 represses de novo DNA methylation in pluripotency-associated genes, resulting in the maintenance of gene expression after withdrawal of LIF, while also repressing the upregulation of differentiation markers. Further, knockdown of Tet1/Tet2 and administration of base excision repair (BER) pathway inhibitors impairs the PRDM14-induced resistance of ESCs to differentiation. We conclude that, in the absence of LIF, PRDM14 governs the retention of pluripotency-associated genes through the regulation of TET functions in the BER-mediated active demethylation pathway, while acting to exert TET-independent transcriptional repressive activity of several differentiation markers.

Introduction

DNA methylation is formed by the de novo DNA methyltransferases DNMT3A/DNMT3B, and is maintained by the UHRF1/DNMT1 complex during DNA replication [1], [2], [3]. Inhibition of the UHRF1/DNMT1 complex triggers passive dilution of DNA methylation, whereby less 5mC is incorporated into the genome after each subsequent cell division. Recently, it has been shown that oxidation of 5mC by ten-eleven translocation (TET) proteins produces 5-hydroxymethylcytosine (5hmC) [4]. 5hmC is further oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), both of which can be repaired by the base excision repair (BER) pathway to produce unmodified cytosine [5], [6]. Importantly, the level of DNA methylation in the regulatory elements of the pluripotency-associated genes is closely linked to their role in ESC self-renewal and differentiation [7], [8]. In undifferentiated ESCs, the balance between 5mC to 5hmC/5fC/5caC in these genes is shifted toward 5hmC/5fC/5caC, promoting both passive demethylation and BER-dependent active demethylation and also sustained hypomethylation in the cells [9], [10], [11], [12].

Recently, we observed high levels of PR-domain containing transcriptional regulator 14 (PRDM14) in developing primordial germ cells (PGCs) [13] and provided evidence indicating that PRDM14 promotes TET-BER-dependent active DNA demethylation in pluripotency-associated genes, germ cell-specific genes, and imprinted loci in ESCs [14]. Furthermore, PRDM14 represses the expression of de novo DNA methyltransferase Dnmt3a/b/l and induces rapid proliferation, which propagates passive DNA demethylation in ESCs cultured with 2i medium plus LIF [11], [12], [13]. However, the complete biological function of PRDM14-induced TET-BER-dependent active demethylation is largely unknown.

In this study, we sought to elucidate the biological role of PRDM14 during TET-BER-dependent active demethylation. Moreover, we investigated the effects of PRDM14 overexpression on ESC fate in the absence of LIF. In doing so, we revealed that enforced expression of PRDM14 could maintain ESC pluripotency, even in the absence of LIF, through TET-BER-mediated active demethylation.

Section snippets

Cell culture

E14tg2a ESCs were cultured in feeder-free conditions in GMEM (Wako Pure Chemical Industries Ltd.) supplemented with 10% FCS (Invitrogen, Life Technologies), 1 mM glutamine (Wako Pure Chemical Industries Ltd.), nonessential amino acids (Wako Pure Chemical Industries Ltd.), and 0.1 mM 2-mercaptoethanol (Wako Pure Chemical Industries Ltd.). The ESCs were also supplemented with LIF (Wako Pure Chemical Industries Ltd.).

ESCs stably expressing PRDM14 were established from single E14tg2a mouse ESC

High expression of PRDM14 promotes maintenance of ESC capacity in a LIF-independent manner

We previously showed that overexpression of Prdm14, as observed in developing PGCs, promotes TET-BER-dependent active demethylation in ESCs [14]. To investigate the function of TET-BER-dependent demethylation by PRDM14 in the spontaneous differentiation of ESCs induced by LIF-withdrawal, we established PRDM14-overexpressing ESCs (PRDM14-OE ESCs), which mirror the high expression levels observed in ESCs with 2i medium plus LIF and developing PGCs (Fig. 1A). We firstly detected AP activity,

Discussion

Our data shows that PRDM14 promotes TET-BER-mediated active demethylation to sustain hypomethylation of pluripotency-associated genes in ESCs, which is required in order to resist differentiation after LIF withdrawal. Knockdown of Tet1 and Tet2 in serum ESCs also causes downregulation of Tcl1, accompanied with an increase of methylation in the promoter regions of pluripotency-associated genes [7]. Furthermore, the 5fC/5caC intermediate forms of active demethylation are significantly elevated in

Author contributions

N.O. and Y. Seki. designed the study; N.O., N.S., K.I., A.M., A.S. and Y. Seki. performed experiments; N.O. and Y. Seki. collected and analysed data; N.O. and Y. Seki. wrote the manuscript. Y. Suwa edited the manuscript.

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgements

We thank G. Nagamatsu for technical advice. This study was supported by JSPS KAKENHI Grant Number 26112514, 24681040, the Takeda Science foundation, the Sumitomo foundation, the Chemicals Evaluation and Research Institute, and by an Individual Special Research A grant from Kwansei Gakuin University.

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    DNA demethylation can occur through passive or active mechanisms. PRDM14, a PR-domain-containing transcriptional regulator, represses expression of de novo DNA methyltransferases by recruiting Polycomb repressive complexes (PRC2) (Fig. 1) [3,4]. PRDM14 promotes DNA demethylation through an active mechanism by means of Ten-eleven-translocation (TET)-mediated base excision repair (BER) in embryonic stem cells [3], maintaining pluripotency of embryonic stem cells [5] and human primordial germ cells [6], supporting the role of DNA methylation states in Polycomb-dependent 3D genome reorganization [7].

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    5hmC cannot serve as a template for DNA methylation maintenance and can be removed by the base excision repair (BER) pathway (Hackett et al., 2013; Hajkova et al., 2010; Hill, Amouroux, & Hajkova, 2014; Tang et al., 2016). PRDM14-deficient cells display defects in both types of DNA demethylation, since they fail to repress Dnmt3b and Uhrf1 in mPGCs or mPGCLC and do not recruit TET enzymes to target loci in mESCs and mPGCs (Okashita et al., 2014, 2015; Shirane et al., 2016). Together this results in considerable genome hypermethylation compared to wildtype (WT) controls.

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