Stable Methylation at Promoters Distinguishes Epiblast Stem Cells from Embryonic Stem Cells and the In Vivo Epiblasts

Abstract

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.

Comparative FAIRE-seq Analysis Reveals Distinguishing Features of the Chromatin Structure of Ground State- and Primed-Pluripotent Cells

“…S7 and data not shown). Accordingly, the “extinguishing” of many of these accessible regions correlates with increased levels of DNA methylation at these loci in EpiSCs as reported in a recent MethylCap‐Seq analysis . Thus, these events would be predicted to create an epigenetic barrier that prevents reversion to the ground state or selection toward a XEN fate, and to reinforce lineage commitment to the primed epiblast fate.…”

Multi-Omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency

“…The formative state characterized by EpiLCs is transient and cannot be captured using current culture conditions (Hayashi et al, 2011). Our observation that Dnmt3l is transitorily expressed during ESC to EpiLC transition (Figure 7D) and in the epiblast (E4.5-6.5) (Smith et al, 2012) but not in EpiSCs (Veillard et al, 2014) suggests that it could be an excellent marker, besides EpiLCspecific cell surface proteins, to isolate formative pluripotent stem cells from a heterogenous population of pluripotent cells. It will be of future interest to determine whether the formative phase can be captured as a stem cell state in culture, as achieved for naïve ESCs and EpiSCs.…”

A non-catalytic role of TET3 promotes open chromatin and enhances global transcription

“…This is in line with the possibility of TET3 playing a role in zygotic genome activation. Other examples of TET3 upregulation when cells undergo identity changes include exit from pluripotency (Veillard et al 2014), iPS reprogramming (Milagre et al 2017), SCNT reprogramming (Hosseini et al 2016) and PGC specification (Seisenberger et al 2012). In fact, PGCs have recently been shown to exhibit increased RNA levels, elevated transcription, increased cell size and upregulation of ribosomal protein transcripts (Percharde et al 2017b), features that are mirrored in Tet3 expressing ES cells.…”

“…Thus, we interpret the finding that TET3 enhances transdifferentiation from ES to TS-like cells as an ability to expand cellular potency and/or facilitate changes of cell identity. Intriguingly, apart from its roles in early embryonic development TET3 expression changes have also been reported in a number of seemingly unrelated studies: For example, while Tet3 levels are low in both ES and epiblast stem (EpiES) cells, its transcript is temporarily upregulated during differentiation from ES to EpiES cells (Veillard et al 2014). Moreover, Tet3 is higher in embryos produced by somatic cell nuclear transfer compared to in vitro fertilisation in bovine (Hosseini et al 2016), relating its upregulation to somatic reprogramming.…”