Both Erk and Smad2 phosphorylation was induced in PiCs upon addition of either bFGF or activin (Figure3F), and mCherry+/eGFP+(yellow) and mCherry/eGFP+(green) cells increased at the expense of mCherry+/eGFP(red) cells (FigureS3E)

Both Erk and Smad2 phosphorylation was induced in PiCs upon addition of either bFGF or activin (Figure3F), and mCherry+/eGFP+(yellow) and mCherry/eGFP+(green) cells increased at the expense of mCherry+/eGFP(red) cells (FigureS3E). in ESCs In this article, Minchiotti, De Cesare, and colleagues show that the relative levels of two physiological metabolites, vitamin C andl-proline, direct ESCs toward naive or reversible primed says of pluripotency, placed between the naive/2i and FGF/activin says, and highlight a pivotal role of metabolic-epigenetic crosstalk in the developmental continuum. == Introduction == Pluripotency is transiently induced during the early stages of mammalian embryo development. Blastocyst stem cells progress from a naive/ground state to a primed state of pluripotency before lineage commitment (Martinez Arias et al., 2013). Two distinct pluripotent stem cells, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), are considered as their in vitro counterparts. ESCs and EpiSCs differ with respect to their morphology, metabolism, DNA methylation levels, transcription profiles, and growth factors requirement (Weinberger et al., 2016). Pluripotency states are unstable and thus difficult to stabilize in vitro. Indeed, ESC cultures consist of heterogeneous cells dynamically fluctuating between different pluripotent says (Hayashi et al., 2008, Toyooka et al., 2008). EpiSCs frequently lose the (Z)-SMI-4a primed state, acquiring features of late pre-gastrula embryos (Wu and Izpisua Belmonte, 2015). Known molecular determinants of such plasticity are mainly transcription factors, while the role of metabolism has been largely unexplored until recently. (Z)-SMI-4a Indeed, it has now become evident that metabolites, including amino acids, work as key regulators of pluripotent stem cell plasticity and behavior. For instance, it has been shown that ESC self-renewal depends onl-threonine (Wang et al., 2009), while ESC identity is regulated byl-proline (l-Pro) availability (Casalino et al., 2011, Comes et al., 2013, D’Aniello et al., 2015, Washington et al., 2010). Moreover, several metabolites act as epigenetic signals (Blaschke et al., 2013, Comes et al., 2013, Shyh-Chang et al., 2012), thus defining a regulatory network among metabolism, epigenetic modification, and pluripotency, knowledge of which is still limited (Harvey et al., 2016). Here we provide evidence that pluripotency is finely controlled by the mutual availability of two physiological metabolites, vitamin C (VitC) andl-Pro, and propose that naive and early primed pluripotency says can be captured in vitro by exploiting the epigenetic activity of these metabolites. == Results == == Vitamin C andl-Proline Induce Opposite Effects on DNA Methylation in ESCs == Recent evidence from our laboratory demonstrates that ESCs suffer a highly specific intrinsic shortage of the nonessential amino acidl-Pro, which induces the amino acid stress response pathway (D’Aniello et al., 2015). Exogenously providedl-Pro alleviates this stress condition and converts round-shaped ESCs into flat-shaped pluripotent stem cells. This phenotypic transition is fully reversible, either afterl-Pro withdrawal or by addition of VitC Nr4a1 (Casalino et al., 2011, Comes et al., 2013), raising the hypothesis that these two metabolites play antagonistic roles in controlling ESC identity. VitC is the most relevant naturally occurring reducing agent and enhances the catalytic activity of 2-oxoglutarate/Fe(II)-dependent dioxygenases, including TET DNA demethylases, and thereby contributes to epigenetic regulation, cell differentiation, and reprogramming (Hore et al., 2016, Krishnakumar and Blelloch, 2013, Monfort and Wutz, 2013). We thus reasoned that VitC andl-Pro might regulate pluripotency by exerting opposite effects on the dynamics of DNA methylation. We first quantified DNA methylation levels in ESCs treated withl-Pro VitC at different time points by liquid chromatography followed by mass spectrometry (LC-MS) (Figures 1A and 1B). VitC supplementation led to a rapid and sustained increase of 5-hydroxymethylcytosine (5hmC) and reduced 5-methylcytosine (5mC) levels. Conversely, supplementall-Pro increased 5mC and reduced 5hmC levels, and its effect was fully counteracted by VitC (Figure 1B). We then compared genome-wide methylation profiling of ESCs l-Pro and ESCs, VitC, at days 2 and 5 of treatment. Reduced representation bisulfite sequencing (RRBS) analysis identified 1, 000 differentially methylated regions (DMRs) distributed throughout all chromosomes (Figure 1C andTable S1). The methylation levels of individual CpGs were highly correlated (r > 0. 9) (Z)-SMI-4a between the different groups (Figures S1A and S1B). Interestingly, the (Z)-SMI-4a majority of DMRs (77% at day 2 and 96% at day 5) that were hypomethylated in VitC-treated ESCs were conversely hypermethylated inl-Pro-treated ESCs (Figures 1D andS1C), thus indicating that VitC andl-Pro modulate methylation at the same DNA regions in an opposite manner. Of note, a high fraction of DMRs (50%) lay in promoters (mostly HCPs) and 20% in enhancers (Figures.