High-resolution genome-wide analyses have revealed that culture environments impose distinctive transcriptional and epigenomic properties on mouse ES cells. In total some 13,000 genes are transcribed at above background levels (>0.2 RPKM). The corollary of this is that around half of genes are effectively inactive. Therefore, undifferentiated ES cells do not show promiscuous gene expression or global transcriptional hyperactivity (Efroni et al., 2008
). Nonetheless, the pluripotent transcriptome displays a broad bandwidth; more than 25% of active genes show 2-fold or greater differences between 2i and serum. Around 1,400 genes, predominantly associated with metabolic processes, are upregulated in 2i. In contrast, KEGG analysis points to decreased expression in 2i of components that might drive differentiation, such as cell communication, mitogen-activated protein kinase (MAPK), and transforming growth factor β (TGFβ and Wnt) pathways. Most strikingly, many ectodermal and mesodermal specification genes that exhibit significant expression in serum are repressed in 2i. Low to absent lineage-affiliated gene expression indicates that multilineage priming is not an intrinsic feature of self-renewing ES cells. Upregulation of such genes in serum suggests that metastability may be an induced condition rather than an inherent property of pluripotent cells.
Some endodermal genes such as Hex
retain low-level expression in 2i. This may reflect the potential to generate extraembryonic endoderm (Canham et al., 2010
). High levels of Prdm14, which has been reported to repress extraembyronic endoderm transcription factors (Ma et al., 2011
), may prevent full activation of this program.
Importantly, ES cells transferred between 2i and serum switch their transcriptional profile. Thus a significant component of previously described ES cell signatures reflects an induced serum response. However, critical pluripotency factors are transcribed at similar or only slightly higher levels in 2i. The pluripotency repressors Tcf3 (Wray et al., 2011
) and components of the NuRD complex (Kaji et al., 2006
) are also expressed at comparable levels. A subset of SCM factors are specifically upregulated in serum, including the Id
genes that are induced by BMPs or fibronectin and are thought to directly counter the effects of Erk activation (Ying et al., 2003a
). Increased Eras, shown to be important for ES cell propagation (Takahashi et al., 2003
), and factors such as Sall4, Lin28, and Utf1, may also contribute to reinforcing self-renewal in the face of differentiation stimuli.
The conflict between pluripotency factors and lineage specifiers results in metastability and incipient differentiation in serum. It is suggested that this “precarious balance” (Loh and Lim, 2011
) may reflect the circumstance in egg cylinder epiblast cells. However, serum stimulation is an artifactual scenario that may be far from representative of the spatiotemporal precision of inductive stimuli in the embryo. To access postimplantation definitive lineages, ES cells should pass through a phase equivalent to egg cylinder epiblast (Rossant, 2008
). Consistent with this, Fgf5
is upregulated in EBs prior to definitive germ layer markers. From 2i ES cells and the Rex1GFP-positive fraction of serum ES cells, this process follows similar kinetics. Therefore although serum induces transcriptional and epigenetic changes and associated metastability, developmental potential within the Rex1-positive compartment is not fundamentally altered. This is substantiated by the capacity of ES cells from either condition to contribute extensively to chimaeras. However, a significant proportion of cells in serum lose expression of Rex1
and of core pluripotency factors such as Nanog
. They are developmentally more advanced and should be considered distinct from ES cells even though they retain Oct4 (Smith, 2010
Expression of many genes associated with metabolic and biosynthetic processes is enriched in 2i. This is likely in large part a response to absence of serum constituents, loss of MAPK signaling, and inhibition of GSK3 and indicates that ES cells have adaptable metabolomic capacity. Probably as a consequence of low c-Myc, the cell-cycle inhibitors p16, p19, and p21 are upregulated in 2i, even at the protein level (D). Nonetheless, ES cells continue to proliferate rapidly, reflecting their freedom from cyclin checkpoint control (Burdon et al., 2002; Stead et al., 2002
). These features can explain the robust expansion of ES cells independent of serum factors and likely underlie their latent tumorigenicity (Chambers and Smith, 2004
In 2i and serum H3K4me3 peaks are globally similar in number and intensity. In contrast, there is a striking difference in the pattern of H3K27me3 deposition. This mark is present as a lawn across intergenic regions and inactive genes (C). However, elevated deposits at promoters of repressed genes are greatly diminished in 2i. The majority of these genes show reduced rather than increased transcription in 2i. This promoter-specific diminution in H3K27me3 is common to multiple ES cell lines. The majority of these genes show reduced rather than increased transcription in 2i. Ezh2 is localized less intensely at promoters in 2i, which may underlie the selective reduction in H3K27me3. Global levels of H3K27me3 are similar in 2i and serum. Indeed, H3K27me3 is increased at satellites in 2i, indicating that these may serve as a sink. Notably, there is no change in H3K9me3 over satellites (C).
In 2i only around 1,000 genes have both H3K4me3 and H3K27me3 marks, which argues against bivalency as a master epigenetic blueprint. Nonetheless, most of the remaining bivalent genes can be classified as developmental. In serum, more genes are bivalent due to acquisition of H3K27me3. Surprisingly, this is accompanied by a slight overall increase in expression, although the majority remain silent or transcribed at very low levels (A). It is conceivable that although the local levels of PRC2 and H3K27me3 are reduced in 2i, they remain sufficient to repress transcription. It should be noted, however, that ES cells lacking PRC2, PRC1, or both are viable and show derepression of lineage-specific markers to only a low level (Leeb et al., 2010
). Our findings are thus in line with genetic evidence that polycomb is not a central mechanism for silencing gene expression in the naive state and only becomes critical during differentiation.
RNA polymerase pausing has been identified by GRO-seq analysis (Min et al., 2011
) at variable extents at many genes in ES cells cultured in serum. Our findings indicate that pausing is more prevalent in 2i than serum. Induction of c-Myc in serum may facilitate pause release at some loci. This is consistent with recent evidence that Myc function is unnecessary in naive ES cells but required in serum (Hishida et al., 2011
). However, many of the genes whose expression is most markedly upregulated in serum, including germ layer specification factors, are not reported Myc targets. Therefore additional mechanisms are likely to control pause release in ES cells.
In mammals pluripotent cells harbor the germline and most pluripotency factors are also key players in germ cell specification and differentiation. It is interesting therefore that in Caenorhabditis elegans
, germline development is dependent on transcriptional pausing mediated at the level of pTEFb antagonism by Pie-1 and Pgc, respectively (Nakamura and Seydoux, 2008
). This raises the question of whether naive ES cells might contain an analogous factor that interferes with pTEFb to suppress transcriptional elongation. It will also be revealing to determine whether Erk signaling may cause activation of pTEFb (Fujita et al., 2008; Lee et al., 2010
Recruitment and pausing of RNA polymerase II with lack of consolidated H3K27me3 silencing may constitute a potentiated template for induction of lineage-specific transcription programs. Pausing may serve to minimize the effects of noise and ensure rapid, coordinated, and synchronous gene induction in response to developmental cues or extrinsic stimuli (Boettiger and Levine, 2009; Nechaev and Adelman, 2011
). Recent studies also indicate that Pol II pausing inhibits nucleosome assembly (Gilchrist et al., 2010
) and could thereby influence histone modification profile. Interestingly, in Xenopus
embryos H3K27me3 is not deposited during zygotic gene activation but is acquired later and associated with spatial restriction of gene expression (Akkers et al., 2009
). In the mouse ICM, H3K27me localization has not been determined, but various epigenetic silencing components appear to be expressed at low levels (Tang et al., 2010
Collectively the observations reported here yield insights into the molecular underpinning of naive pluripotency and revise previous assumptions derived from analysis of heterogeneous and metastable serum-treated cultures. The findings provoke questions about the regulation of gene expression in pluripotent cells and the process of lineage specification. Transcriptional potentiation through promoter proximal pausing may play a major role in the establishment and stable maintenance of naive pluripotency. Currently there is great interest in isolating human pluripotent stem cells in a naive state (Hanna et al., 2010; Wang et al., 2011
). The distinctive transcriptome and epigenome characteristics of ground state mouse ES cells may provide a valuable criterion against which to measure such claims. In addition, these data sets provide a benchmark resource for analysis and modeling of gene expression control during self-renewal and in the transition from naive pluripotency to lineage commitment.