Knockout of Dnmta and Dnmt

Knockout of Dnmta3 and Dnmt3b in ESCs leads to progressive hypomethylation, supporting the notion that de novo activity is required to maintain 5mC in ESCs, possibly to offset persistent 5hmC conversion (Chen et al., 2003). However, importantly, Dnmt3a/b null ESCs only exhibited an ∼10%–20% reduction in global 5mC after five passages compared with >2-fold in 2i (Jackson et al., 2004; Chen et al., 2003; Leitch et al., 2013), suggesting that ground-state conditions must activate additional DNA demethylation processes independent of Dnmt3a/b repression. Indeed, Tet1/Tet2 null ESCs used here exhibit a partial block to DNA demethylation in 2i conditions, supporting an important role for 5hmC conversion. Nonetheless, a significant degree of hypomethylation was still observed in DKO ESCs, implying that DNA demethylation is not reliant on 5hmC per se. Thus, a dual model may operate whereby repression of de novo methylation activity is sufficient to drive hypomethylation at slow kinetics, whereas the presence of abundant 5hmC activity may enhance both the rate and the extent of demethylation, particularly at CpG-dense regions that are preferential TET-binding sites, such as the Dazl promoter (Williams et al., 2012). Notably, though, base-resolution data reveal that 5hmC is detectable to some extent throughout the ESC genome (Yu et al., 2012), and thus, 5hmC may play at least a subordinate role in promoting hypomethylation genome wide. Indeed, we observed a partial suppression of demethylation at introns (Cul1) and CpG-poor promoters (Elf5, Essrb, and Capn9) in DKO ESCs in addition to CpG-dense regions (Dazl and Rhox9). This model of synergistic demethylation mechanisms is reminiscent of reprogramming phases in PGCs and the preimplantation embryo, where significant global 5hmC conversion has been reported, and that coincides with periods of suppressed maintenance and/or de novo methylation activity (Hackett et al., 2013; Kagiwada et al., 2013; Iqbal et al., 2011; Pastor et al., 2013). The functional consequences of global hypomethylation in 2i conditions are difficult to disentangle from the direct effects of ground-state pluripotency. However, one possible outcome is a relaxation on epigenetic barriers imposed by DNA methylation. For example, hypermethylation of the Elf5 promoter has been reported to act as an epigenetic barrier to prevent ESCs in serum from entering into extraembryonic lineages (Ng et al., 2008). Because we observed rapid and dramatic DNA demethylation and activation of Elf5 Q-VD(OMe)-OPh after switching ESCs to 2i (Figure S4), this should enable these ESCs to contribute to extraembryonic tissues and thus be functionally “totipotent.” Indeed, we found that ESCs carrying a reporter for constitutive-Venus expression and cultured in 2i robustly contributed to extraembryonic tissues and that this property occurred soon after the switch in culture conditions (unpublished data), a finding supported by a recent study (Morgani et al., 2013). Thus, global hypomethylation in 2i may contribute to generating a less-restricted in vitro state, which is closer to functional totipotency, possibly through demethylation of Elf5. Additionally, DNA hypomethylation per se appears to promote homogeneity among sister cells after ESC division and may therefore directly promote self-renewal (Jasnos et al., 2013). In summary, we have established the 5mC and 5hmC profiles of distinct PSCs and revealed that DNA demethylation during transition to ground-state pluripotency is directed by synergistic TET-mediated 5hmC and PRDM14-directed repression of Dnmt3a/Dnmt3b/Dnmt3L. The globally hypomethylated state of cells in 2i may be an in vitro correlate to the preimplantation epiblast or migrating PGCs, and thus, transfer to 2i conditions may provide a tractable system for mechanistic studies into developmental epigenetic transitions.
Experimental Procedures
Acknowledgments
Introduction Pluripotent stem cells (PSCs) are heterogeneous under self-renewing conditions in culture (Enver et al., 2009; Graf and Stadtfeld, 2008; Martinez Arias and Brickman, 2011) and during embryonic development (Chazaud et al., 2006). This heterogeneity extends not only to the expression of pluripotency factors such as NANOG, REX1, and STELLA (Chambers et al., 2007; Hayashi et al., 2008; Singh et al., 2007; Toyooka et al., 2008), but also to lineage-specific factors such as HEX, HES1, and GATA6 (Canham et al., 2010; Kobayashi et al., 2009; Singh et al., 2007). Variations in gene expression are transient and reversible, indicating that PSCs alternate between different cell states. Although the function and molecular mechanisms underpinning this heterogeneity are unclear, it appears to be influenced by variations in the activity of signaling pathways at the single-cell level. WNT, BMP, NODAL, and FGF signaling through their downstream effectors has been implicated in contributing to PSC heterogeneity and serves to prime cells for differentiation when transiently activated (Galvin-Burgess et al., 2013; Price et al., 2013). As an example, heterogeneity can be significantly reduced when murine PSCs are cultured in the presence of small-molecule compounds that block ERK and GSK3 signaling (2i media) (Marks et al., 2012; Wray et al., 2011; Ying et al., 2008). In human embryonic stem cells (hESCs), suppression of WNT activity reduces signaling heterogeneities and the sporadic expression of developmental regulators such as BRACHYURY (Blauwkamp et al., 2012; Singh et al., 2012). Together, these observations indicate that signaling heterogeneities reflect alternate cell states that represent different differentiation potentialities.