Transcriptional and epigenetic control in mouse pluripotency: lessons from in vivo and in vitro studies

Available online at www.sciencedirect.com

ScienceDirect Transcriptional and epigenetic control in mouse pluripotency: lessons from in vivo and in vitro studies Ehsan Habibi and Hendrik G Stunnenberg Pluripotent cells were first derived from mouse blastocysts several decades ago. Since then, our knowledge of the molecular events that occur in the pre-implantation embryo has been vastly progressing. The emergence of epigenetics has revolutionized stem cell and developmental biology and further deepened our understanding of the underlying molecular mechanisms controlling the early embryo development. In particular, the emergence of massive parallel sequencing technologies has opened new avenues and became indispensable tools in modern biology. Additionally, development of new and exciting techniques for genome manipulation (TALEN and CRISPR/Cas9) and in vivo imaging provide unique opportunities to perturb and trace biological systems at very high resolution. Finally, recent single-cell — omics combined with sophisticated computational methodologies allow accurate, quantitative measurements for deconvolution of cellular variation in complex cell populations. Collectively, these achievements enabled the detailed characterization and monitoring of various cell states and trajectories during early stages of embryonic development. Here we review recent studies of the transcriptional and epigenetic changes during very early stages of mouse embryo development and compare these with pluripotent cells grown in vitro under different culture conditions. We discuss whether the in vitro cell states have an ‘epiphenocopy’ in the embryo and refine our understanding of the circuitries controlling pluripotency and lineage commitment during early stages of mouse development.

Address Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, 6500HB Nijmegen, The Netherlands Corresponding authors: Habibi, Ehsan ([email protected]), Stunnenberg, Hendrik G ([email protected])

Current Opinion in Genetics & Development 2017, 46:114–122 This review comes from a themed issue on Cell reprogramming Edited by Jianlong Wang and Miguel Esteban http://dx.doi.org/10.1016/j.gde.2017.07.005 0959-437/ã 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Embryonic development is a highly orchestrated series of events that requires a tight spatiotemporal control of gene expression. During a narrow time window in early Current Opinion in Genetics & Development 2017, 46:114–122

development, cells obtain the potential to generate all differentiated cell types. This transient developmental state first appears in the totipotent zygote having the highest differentiation potential that becomes gradually restricted towards the blastocyst. Cell fate specification starts between the 8-cell and 16-cell stages (E2.75) when the first segregation occurs and two populations are established, the ICM (inside cells) and TE (outside cells) [1]. In the early blastocyst stage, the second segregation of ICM into pre-implantation epiblast and extraembryonic hypoblast (primitive endoderm (PrE)) is triggered by the stochastic expression of FGF4 or its receptor FGFR2 [2,3]. Naı¨ve epiblast cells are considered as the pluripotent ground state and are the forerunner of all embryonic lineages [4]. During embryonic development, pluripotent cells differentiate following an autonomous program. Depending on the positional information and juxtacrine/paracrine signals that are received from the environment in a timedependent manner, cells rewire downstream gene regulatory circuits to accordingly remodel their regulatory architectures. Pluripotency in naı¨ve epiblast cells is a very transient feature and is progressively resolved. Therefore, to derive and maintain cells in a particular state such as the naı¨ve pluripotency cells in vitro, this autonomous differentiation program needs to be paused by sustaining an intrinsic transcription factor (TF) circuitry that is achieved by a continuous flow of inputs from extrinsic cues. ICM-derived ESCs are able to propagate indefinitely, a property called ‘self-renewal’ while retaining their pluripotent state. Impressively, after derivation and extensive in vitro expansion, these ESCs are still capable to efficiently contribute to embryonic development upon their implanting into the early embryo [5]. Although ESCs have a limited potential to generate extraembryonic lineages, a recent study showed that derivation of stem cells using a specific chemical cocktail allows to maintain cells in a totipotent-like state [6]. These stem cells, so-called extended pluripotent stem (EPS) cells, are able to contribute to both embryonic and extra-embryonic lineages in both human and mouse chimera’s. Over the past few years, many efforts have been undertaken to establish and optimize in vitro culture conditions [5,7–9,10]. The extent to which these in vitro-grown ESCs preserve the characteristics of their in vivo counterparts strongly depends on the culture medium which either stimulates or blocks different extrinsic signaling www.sciencedirect.com

Transcriptional and epigenetic control in pluripotency Habibi and Stunnenberg 115

pathways. Originally, ESCs were derived and maintained on fibroblast feeder cells in serum-supplemented medium. The main signaling molecules in this condition are LIF, the autocrine factor FGF4, and BMP4 which trigger JAK/STAT3, PI3K, MAPK/ERK and SMAD pathways [11]. Although LIF is generally regarded as a self-renewal regulator, paradoxically when together with FGF4 it can promote differentiation through the activation of MAPK/ERK signaling [12–15]. It has been shown that BMP is able to counteract the stimulatory effect of LIF and FGF signaling via SMAD-dependent upregulation of DUSP9 which in turn dephosphorylates and inhibits ERK activity [16]. Therefore, it seems that serum sustains pluripotent ESCs in a metastable state by balancing opposing signaling pathways which increases their heterogeneity and make them prone to undergo spontaneous differentiation [4,17]. In 2008, Smith and coworkers developed a chemically defined medium called ‘2i’ by blocking Mek/Erk and GSK3 pathways using two highly selective chemical inhibitors called PD0325901 and CHIR99021, respectively [7]. They showed that in the absence of serum, feeder cells or LIF, 2i alone is sufficient to maintain pluripotency and clonogenicity of ESCs. Taken together, the net result of developmental cues and signaling pathways is a key determining parameter in controlling cell fate and establishing a final outcome, the so-called ‘cell state’. Indeed, different extrinsic cues redistribute core pluripotency factors via state-specific regulatory partners leading to the rewiring of the regulatory elements, changes in gene expression and in the epigenetic landscape [18,19,20].

Epigenetic landscape in early development and ESCs Both oocyte and sperm are terminally differentiated cells that need to be epigenetically reprogrammed, to extinguish cellular commitment and to establish totipotency in the zygote. Shortly after fertilization, uncommitted cells respond to extrinsic developmental cues and become progressively committed to distinct trajectories to generate different cell states with distinct and characteristic features. These signals trigger a series of downstream cascades and activate a particular set of sequence-specific transcription factors (TFs) or effectors that control gene expression. Once established, gene expression patterns need to be stably maintained during multiple rounds of cell divisions (cell memory) and at the same time should be sufficiently plastic to respond to various external stimuli. In eukaryotes, the genome is packed into a protein-DNA structure called ‘chromatin’. In addition to the passive packaging of eukaryotic DNA, chromatin also serves as a dynamic platform to control DNA accessibility in response to the signaling pathways through downstream www.sciencedirect.com

transcription factors and chromatin regulators [21]. For this purpose, a number of so-called epigenetic mechanisms instigated and controlled by transcription factors are in place that are able to alter the physical and chemical nature of chromatin and govern its accessibility. These series of events play a fundamental role in shaping and establishing the epigenetic landscape which in turn modulates and instructs gene expression. Epigenetic features such as DNA and histone modifications have been extensively used to define and annotate distinct functional regions such as promoters and enhancers. These dynamic processes, that operate during development and are frequently altered in disease, have received and are receiving a great deal of attention as they provide a wealth of information on the factors and mechanisms involved in these processes.

Dynamics of histone modifications in early development and in ESCs So far, our knowledge of the genome-wide landscape of histone marks in pre-implantation development has been limited because of the low availability of the cells and lack of ultra-low input chromatin immunoprecipitation approaches. Recently, several studies have overcome these limitations and have shed light on the dynamics of histone modifications (H3K4me3, H3K27me3) during different phases of gametogenesis and of pre-implantaembryo development (Figure 1) tion [22,23,24,25,26]. H3K4me3: an active or suppressive mark?

H3K4me3 is generally considered to be an active mark; at high signal intensity, it is mainly present at proximal promoters, whereas at low levels it is also observed at ‘active’ enhancers [27]. In mammalian cells, H3K4me3 is deposited by mixed lineage leukemia (MLL) family of SET domain-containing methyltransferases. This family includes Mll1, Mll2, Mll3, Mll4, Setd1a and Setdb1 that are expressed in a stage-specific manner during embryonic development. Mll2 is discontinuously required: firstly throughout oogenesis, again during the first embryonic divisions before zygotic gene activation (ZGA) as well as after gastrulation but is dispensable for pluripotency and self-renewal in ESCs in vitro [28,29]. After the first embryonic divisions and before ZGA, the role of MLL2 as the major H3K4me3 methyltransferase is replaced by SETD1 that is needed for the formation of Oct4 expressing ICM and for maintenance of ESCs [30]. It specifically interacts with OCT4 in a WRD5-independent manner, is required for OCT4 transcriptional circuitry, and activates OCT4 target genes by deposition of H3K4me3 at their promoters [31]. In oocyte, global H3K4me3 is maintained by MLL2 and its absence causes global transcriptional silencing. Initially, this unexpected observation was indirectly linked Current Opinion in Genetics & Development 2017, 46:114–122

116 Cell reprogramming

Figure 1

H3K27me3 Non-Promoter Non-canonical

H3K27me3/H3K4me3 Non-Promoter

Promoter Canonical

M Maternal

H3K4me3

Bivalent

Non-canonical

P Paternal

Promoter Non-canonical

S5P-PolII

Canonical

Protamine-DNA toroid

Mll2

Mature Oocyte

~10kb

~10kb

??

?? Sperm

Fertilization

Mll2

PN5 zygotes

M

& P

KDM5B

KDM5B

Early two-stage

ZGA

Setd1 M

Late two-stage P

Setd1 M ICM P

Serum/LIF

Setd1 M

ESC

& P

2i/LIF Current Opinion in Genetics & Development

Dynamic Landscapes of histone modifications H3K4me3 and H3K27me3 during early stages of mouse development. In contrast to the canonical H3K4me3 modification in late-two cell embryos and onwards, H3K4me3 in zygotes and early two-stage embryos is mainly enriched in nonpromoter regions with low-CpG density (non-canonical H3K4me3). MLL2 functions as the major H3K4me3 methyltransferase during oogenesis and before zygotic gene activation (ZGA). After ZGA MLL2 function is replaced by SETD1. After ZGA, ncH3K4me3 is replaced by canonical H3K4me3. In the mature sperm, most of the histones 85–90% are replaced by protamines and protamine-DNA form a toroid like structure. Only 10–15% of histones are preserved at some loci, for example, developmental genes. Pattern of H3K27me3 in MII oocyte and in pre-implantation embryos including the PN5 zygote, 2-cell and ICM is different from sperm and ESCs. In oocyte and pre-implantation embryos, H3K27me3 domains are much larger and mainly enriched in non-promoter regions. It is absent at many developmental genes during pre-implantation but its reestablishment starts at the ICM stage onward. The demethylase KDM5B is responsible for the shortening of broad H3K4me3 domains from late two-cell onwards. During pre-implantation development, bivalent marks are mostly absent from many developmental genes. The global pattern of H3K27me3 in early embryo is quite different from that in in vitro-grown ESCs.

to the lack of MLL2 to mediate the expression of factors required for global silencing. However, recent studies revealed an unexpected role of H3K4me3 in gene silencing [23,25,26]. In contrast to the generic H3K4me3 makeup observed in late-two cell embryos and onwards, MII stage oocytes, zygotes and early two-stage embryos show strong enrichment of broad H3K4me3 domains in Current Opinion in Genetics & Development 2017, 46:114–122

distal (non-promoter) regions with low-CpG density socalled ‘non-canonical (nc) H3K4me3’. Additionally, not only distal regions but also promoters show a non-canonical pattern in MII oocytes. Strikingly, establishment of ncH3K4me3 coincides with genome silencing during oogenesis to the early two-cell embryo. In the late twocell embryos, coincidental with ZGA, ncH3K4me3 is www.sciencedirect.com

Transcriptional and epigenetic control in pluripotency Habibi and Stunnenberg 117

replaced by canonical H3K4me3 and zygotic transcription is essential for this switch. This observation was supported by overexpression of histone demethylase KDM5B, but not KDM5A, which results in reduced H3K4me3 and reactivation of transcription in oocytes. These studies further show that the histone demethylase KDM5B is the enzyme responsible for the shortening of broad H3K4me3 domains from late two-cell onwards. Interestingly, these events coincide with the expression of LIF and its receptor LIF-R/gp130 [31]. It has been shown that KDM5B directly functions as a downstream chromatin regulator of LIF/gp130/NANOG underpinning how developmental cues and downstream TFs shape the epigenetic landscape. H3K27me3: a suppressive mark dispensable for pluripotency

Polycomb Group (PcG) proteins are a group of chromatin modifiers that repress transcription and play a critical role during normal development and differentiation [32]. PcG proteins are classified into two major complexes with different enzymatic activities: Firstly PRC1, an E3 ubiquitin ligase containing complex that ubiquitinates H2A at lysine 119 causing chromatin compaction. Secondly, the PRC2 complex that includes EZH2 the catalytic subunit of the methyltransferase that deposit H3K27me3 [32]. Over the past decade, many studies have reported that knockout of PRC2 and PRC1 components including Ezh2 / , Eed / , Suz12 / and Ring1b / die around 7.5-9.5 days post coitum (dpc). In contrast, Ring1a / mice are viable and fertile showing only minor abnormalities [33–37]. The respective ESCs knockout lines, either generated in vitro or derived from blastocysts of knockout mice, are viable although developmental genes are deregulated and de-repressed [38–40]. Collectively, PRC1 and PRC2 complexes are essential for post-implantation development and differentiation but not for maintenance and self-renewal of pluripotent cells. Recently, it has also been shown that in the mouse, the pattern of H3K27me3 in MII oocyte and in pre-implantation embryos including the PN5 zygote, 2-cell and ICM is quite different from those in sperm, ESCs and postimplantation epiblast (E6.5) [22,25]. In contrast to latter stages in which H3K27me3 is mostly present at the promoters of canonical polycomb target genes, H3K27me3 domains in the former stages are much larger in length and mainly enriched in distal regions (nonpromoter) with low CpG density. Although H3K27me3 at the canonical polycomb promoters is absent at many developmental genes during pre-implantation, its reestablishment starts at the ICM stage onwards [22,25]. Bivalency: a debate on the functional significance

Bivalent (poised) promoters are characterized by the cooccurrence of the opposing histone modifications, www.sciencedirect.com

H3K27me3 and H3K4me3. Bivalency was first reported in mouse ESCs and is a signature of developmental and lineage-specifying genes [41,42]. Although bivalent promoters are stably transcriptionally poised, they are plastic for a response to developmental cues to become either stably active or silent [43]. The MEK/ERK signaling pathway has been shown to play a key role in establishing the bivalent state of developmental genes. Activation of ERK2 and its binding to the promoter results in the PRC2-mediated trimethylation of H3K27 and conversion of RNAPII to a poised state, a form of RNAPII harboring phosphorylated serine 5 [44,45]. Several lines of evidence suggest that bivalency might be functionally dispensable during early stages of development and in ESCs [28,46]. Remarkably, inhibition of Erk signaling pathway in 2i ESCs and the subsequent decrease in the PRC2-mediated trimethylation of H3K27 causes reduced numbers of bivalent loci as compared to serum (to 1/3 or less depending on the applied threshold). However, associated genes do not get activated and remain in a silent state [46]. Additionally, recent studies in early embryos report that bivalent marks are largely missing from many developmental genes during pre-implantation development to appear in post-implantation embryos [22,25]. A comparison between early embryos and mouse ESCs showed that the global pattern of H3K27me3 in early embryo is quite different from that in in vitro serum-grown ESCs [22]. Collectively, these results raise the question whether signaling cues induced by culture conditions place ESCs in a developmentally more advanced state as compared to ICM. Macfarlan et al. found a subpopulation in serum/ LIF that resembles in vivo 2-cell (2C) blastomers [47]. It will be interesting to see whether, and to what extent the pattern of H3K27me3 in this subpopulation is similar to its in vivo counterpart in embryo.

Dynamics of DNA methylation in pluripotent cells Methylation of DNA at the 5’ position of cytosine, mainly in the context of a palindromic CpG dinucleotide (CpG dyad), is mediated by DNA methyltransferases (Dnmt’s) consisting of the so-called maintenance DNMT1 and de novo enzymes DNMT3A and DNMT3B. DNA methylation plays an essential role during embryogenesis [48]. During replication, the UHRF1 protein (also known as NP95) [49–51] binds to the replication fork via two different domains, the tandem Tudor binding H3K9me2/3 and SRA binding hemimethylated CpG. Uhrf1 recruits Dnmt1 to the newly generated hemimethylated CpGs and ensures high fidelity symmetric DNA methylation of the newly synthesized DNA strand. Once established, DNA methylation can be removed either passively through successive rounds of replication with impaired maintenance or enzymatically through subsequent oxidation steps Current Opinion in Genetics & Development 2017, 46:114–122

118 Cell reprogramming

mediated by Ten-eleven translocation 1–3 (TET1–3) proteins [52,53]. The TET proteins are 2-oxoglutarate and Fe(II)-dependent dioxygenases that use a-ketoglutarate as a co-substrate [54]. During mouse pre-implantation development, the genome undergoes global DNA demethylation. After fertilization, the paternal pronucleus is actively demethylated, whereas the maternal pronucleus passively loses methylation in a replication-dependent manner [55]. From 2-cell to blastula stage, 5mC is passively diluted and gradually reaches a steady state with the lowest 5mC level in the blastocyst (21% CpG methylation) [55]. Recent studies have shown that compared to the hypermethylated (on average 70%) state of the DNA derived from serum-grown ESCs, the genome in ground state 2i cells is globally hypomethylated (20%), illustrating the strong impact of culture conditions on the methylome state [56–58]. This striking difference in methylome state was surprising as both serum and 2i cells have the same embryonic origin [56–59]. A comparison between these in vitro states (serum and 2i) and that of early embryos showed that ESCs in serum are more reminiscent of epiblast at around E6.5 representing a post-implantation embryonic state, while the 2i condition catches ESCs in a hypomethylated state resembling ICM at E4.5 [56,57,59,60]. The 2i culturing condition promotes expression of Prdm14 which in turn strongly downregulates expression of the de novo methyltransferases (Dnmt3a/b) [61]. Therefore, it appeared that a reduced de novo methylation deposition could be the main cause of the gradual loss of methylation during serum-to-2i reprogramming, a process that takes around 2 weeks in cell culture. However, this assumption was not fully in line with the observation that global loss of methylation in Dnmt3 knock-out line is a very slow process that takes several months [62,63]. Recently, a comprehensive study by von Meyenn, Iurlaro and Habibi et al. systematically dissected the contribution of the different pathways and factors to obtain mechanistic insights into how DNA methylation is globally erased upon transition from serum-to-2i [60]. Assessment of DNA methylation in various knock-out lines and the use of mathematical modeling showed that neither DNMT3’s nor the components of active enzymatic demethylation pathways including TET1, TET2, TET3, AICDA, and TDG could explain the observed rate of global demethylation. Instead, the data strongly pointed to impaired DNA methylation maintenance as the cause of global demethylation in naive ground state ESCs. It was shown that reduced maintenance occurs because of a reduction in UHRF1 protein level and global loss of H3K9me2 that together result in a less efficient recruitment of DNMT1 to replication forks and consequently in a global loss of DNA methylation. Current Opinion in Genetics & Development 2017, 46:114–122

Co-factors and metabolites: new players in epigenetic regulation and pluripotency There is a growing body of evidence unveiling the profound impact of culture conditions on pluripotency and cell fate [60,64,65,66,67,68,69]. Recently, it was shown that addition of vitamin C (VitC) during serum to 2i transition accelerates the loss of DNA methylation through activation of the TET enzymes [60]. VitC interacts with the C-terminal catalytic domain of TET enzymes and serves as an antioxidant to recycle iron from an oxidized (Fe3+) to a reduced form (Fe2+) [70,71]. Supplementing serum with different levels of VitC and L-Proline (L-Pro) shifts ESCs towards naı¨ve (low L-Pro/ high VitC) or early primed (high L-Pro/low VitC) states, respectively [67]. VitC and L-Pro have opposing effects on DNA methylation: high levels of VitC causes DNA hypomethylation, whereas high levels of L-proline induces hypermethylation [67]. Moreover, it is shown that also retinol/retinoic acid (vitamin A) reduces DNA methylation levels by increasing cellular levels of TET proteins and enhances the generation of naı¨ve ESCs [72]. In a recent study, it was shown that higher levels of cellular aKG/succinate ratio in 2i medium support selfrenewal and pluripotency in naı¨ve ESCs [65]. High levels of aKG changed the epigenetic landscape by changing H3K27me3 and TET-mediated DNA demethylation. Systematic investigations are needed to assess whether and how culture supplements affect the trajectory between cell state transitions and where the resulting cell state should be placed in the developmental panel of in vivo counterparts. Such findings will provide more insights into how alterations in metabolites and co-factors modulate the epigenetic landscape of pluripotency and cell state transitions during lineage commitment.

Transcriptional heterogeneity in pluripotent cells revealed by single cell analysis Most of our knowledge regarding gene expression in pluripotent cells has been obtained from analysis of cell populations. Such assays average the behavior of lowoccurrent to high-occurrent phenotypes in the population and hence rare and transient phenotypes escape detection or are discarded as experimental noise. Advances in measuring RNA expression at single-cell level provide valuable information with respect to cell-to-cell variation and diversity/heterogeneity of pluripotent stem cells, these variations might be caused by the stochastic nature of gene transcription or the existence of diverse extrinsic cues in the microenvironment. Recently, the transcriptional profile of in vitro-grown pluripotent stem cells has been determined at single cell level providing new insight into the heterogeneity of cell populations in different growth conditions. Compared to different stages of mouse early development from zygote to blastocyst, three distinct cultured ESCs including serum, 2i and www.sciencedirect.com

Transcriptional and epigenetic control in pluripotency Habibi and Stunnenberg 119

alternative 2i (containing to small molecule inhibitors for GSK3 and SRC pathways [8]) showed the highest similarity to the blastocyst stage, the stage ESCs are derived from [10]. The results also confirm previous findings indicating that 2i cells are more close to blastocyst as compared to serum cells [73,74]. Pathway analysis based on dynamically expressed genes in 2i-grown mouse ESCs versus pre-implantation epiblast showed a significant enrichment for metabolic processes indicating the influence of environmental factors and nutrition on gene expression [46,74]. Self-renewal is a characteristic feature of ESCs and is not present in vivo [17]. However, this feature can be observed during diapause, a transient embryonic arrest that occurs due to delayed implantation accompanied with decreased c-Myc expression [75]. Recently, Scognamiglio et al. found that c-Myc knock-out cells grown in 2i have similar transcriptome profile to embryonic diapause. c-Myc is needed to retain the pluripotent state in serumgrown ESCs, however, its expression is strongly suppressed in 2i (by 10 fold). Scognamiglio et al. observed that although c-Myc knock-out causes cell cycle arrest and forces 2i-grown ESCs into a ‘dormant state’, pluripotency is preserved [76]. In the absence of cMyc, pathways involved in biosynthesis, metabolism and proliferation are compromised, a situation called ‘biosynthetic quiescence’. These studies shows that pluripotency can be uncoupled from proliferation and biosynthetic pathways. Previous studies considered serum-grown ESCs as a heterogeneous and metastable cell state, while 2i-grown ESCs was regarded as a more homogenous state. However, single cell studies reveal that serum, 2i and a2i ESCs are equally heterogeneous, although in each condition different subsets of genes cause the heterogeneity [10]. Subpopulations in both serum and 2i exist in a dynamic equilibrium and their subcellular states are readily interconverted. Additionally, recent studies have addressed the underlying regulatory networks controlling the heterogeneity and substate transitions suggesting that microRNAs might be major players of the regulatory circuit causing heterogeneity and controlling cell state transitions in ES populations [77,78,79].

and optimizing in vitro culture conditions that preserve the (often suspected but not known) characteristics of the cells or embryo states they originated from and to differentiating them into the different lineages. Recent ground breaking advances in single-cell — omics enable deconvolution of the cellular heterogeneity in cell populations. A main challenge is to link the cellular state of the cell with its position in the embryo or tissue to assess how physical or chemical cues from its niche finetunes the fate of the cell. By unplugging cells from their 3D space, valuable information is lost. The knowledge gained by the integration of positional information from barcoding/labeling, cell tracing and time lapse high-resolution imaging with single cell analysis of regulatory features combined with advanced computational approaches will revolutionize biology and medicine.

Conflict of interest statement Nothing declared.

Acknowledgements E.H. and HGS are supported by CEU-ERC Advanced Grant SysStemCell (No. 339431).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Frum T, Ralston A: Cell signaling and transcription factors regulating cell fate during formation of the mouse blastocyst. Trends Genet 2015, 31:402-410.

2.

Guo G, Huss M, Tong GQ, Wang C, Li Sun L, Clarke ND, Robson P: Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev Cell 2010, 18:675-685.

3.

Morris SA, Graham SJ, Jedrusik A, Zernicka-Goetz M: The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos. Open Biol 2013, 3:130104.

4.

Hackett JA, Surani MA: Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014, 15:416-430.

5.

Bradley A, Evans M, Kaufman MH, Robertson E: Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 1984, 309:255-256.

6.

Yang Y, Liu B, Xu J, Wang J, Wu J, Shi C, Xu Y, Dong J, Wang C, Lai W et al.: Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 2017, 169 243– 257 e225.

7.

Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A: The ground state of embryonic stem cell self-renewal. Nature 2008, 453:519-523.

8.

Shimizu T, Ueda J, Ho JC, Iwasaki K, Poellinger L, Harada I, Sawada Y: Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling. Stem Cells 2012, 30:1394-1404.

9.

Li X, Zhu L, Yang A, Lin J, Tang F, Jin S, Wei Z, Li J, Jin Y: Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell 2011, 8:46-58.

Conclusion and outlook Over the past few decades, many efforts have been made to address one of the most fundamental questions in biology: how genetically identical descents of a totipotent single cell can initiate different spatiotemporally controlled programs to engender a whole organism? Due to technical barriers, in vivo monitoring and measuring of regulatory features in embryos during early developmental stages has been challenging. To gain more insight into the molecular mechanism regulating pluripotency the focus has been on isolating these cells, establishing www.sciencedirect.com

Current Opinion in Genetics & Development 2017, 46:114–122

120 Cell reprogramming

10. Kolodziejczyk AA, Kim JK, Tsang JC, Ilicic T, Henriksson J,  Natarajan KN, Tuck AC, Gao X, Buhler M, Liu P et al.: Single cell RNA-sequencing of pluripotent states unlocks modular transcriptional variation. Cell Stem Cell 2015, 17:471-485. This study by applying single cell transcriptome profiling describes the population structure and cellular substrates of ESCs in three different conditions. 11. Nakai-Futatsugi Y, Niwa H: Transcription factor network in embryonic stem cells: heterogeneity under the stringency. Biol Pharm Bull 2013, 36:166-170. 12. Burdon T, Chambers I, Stracey C, Niwa H, Smith A: Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs 1999, 165:131-143. 13. Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A: FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 2007, 134:2895-2902. 14. Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998, 12:2048-2060. 15. Stavridis MP, Lunn JS, Collins BJ, Storey KG: A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development 2007, 134:2889-2894. 16. Li Z, Fei T, Zhang J, Zhu G, Wang L, Lu D, Chi X, Teng Y, Hou N, Yang X et al.: BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells. Cell Stem Cell 2012, 10:171-182. 17. Martello G, Smith A: The nature of embryonic stem cells. Annu Rev Cell Dev Biol 2014, 30:647-675. 18. Buecker C, Srinivasan R, Wu Z, Calo E, Acampora D, Faial T,  Simeone A, Tan M, Swigut T, Wysocka J: Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 2014, 14:838-853. This study along with reference [19] rewiring of the regulatory element landscape during pluripotent state transitions. 19. Factor DC, Corradin O, Zentner GE, Saiakhova A, Song L,  Chenoweth JG, McKay RD, Crawford GE, Scacheri PC, Tesar PJ: Epigenomic comparison reveals activation of ‘seed’ enhancers during transition from naive to primed pluripotency. Cell Stem Cell 2014, 14:854-863. See annotation to Ref. [18].

chromatin domains in pre-implantation embryos. Nature 2016, 537:558-562. See annotation to Ref. [22]. 26. Dahl JA, Jung I, Aanes H, Greggains GD, Manaf A, Lerdrup M, Li G,  Kuan S, Li B, Lee AY et al.: Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition. Nature 2016, 537:548-552. See annotation to Ref. [22]. 27. Calo E, Wysocka J: Modification of enhancer chromatin: what, how, and why? Mol Cell 2013, 49:825-837. 28. Denissov S, Hofemeister H, Marks H, Kranz A, Ciotta G, Singh S, Anastassiadis K, Stunnenberg HG, Stewart AF: Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant. Development 2014, 141:526-537. 29. Lubitz S, Glaser S, Schaft J, Stewart AF, Anastassiadis K: Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2. Mol Biol Cell 2007, 18:2356-2366. 30. Fang L, Zhang J, Zhang H, Yang X, Jin X, Zhang L, Skalnik DG, Jin Y, Zhang Y, Huang X et al.: H3K4 methyltransferase Set1a is a key Oct4 coactivator essential for generation of Oct4 positive inner cell mass. Stem Cells 2016, 34:565-580. 31. Nichols J, Davidson D, Taga T, Yoshida K, Chambers I, Smith A: Complementary tissue-specific expression of LIF and LIFreceptor mRNAs in early mouse embryogenesis. Mech Dev 1996, 57:123-131. 32. Simon JA, Kingston RE: Occupying chromatin: polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 2013, 49:808824. 33. Faust C, Lawson KA, Schork NJ, Thiel B, Magnuson T: The polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 1998, 125:4495-4506. 34. Faust C, Schumacher A, Holdener B, Magnuson T: The eed mutation disrupts anterior mesoderm production in mice. Development 1995, 121:273-285. 35. Morin-Kensicki EM, Faust C, LaMantia C, Magnuson T: Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis. Genesis 2001, 31:142-146. 36. O’Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T: The polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 2001, 21:4330-4336.

20. Galonska C, Ziller MJ, Karnik R, Meissner A: Ground state  conditions induce rapid reorganization of core pluripotency factor binding before global epigenetic reprogramming. Cell Stem Cell 2015, 17:462-470. This study characterizes epigenomic changes and reconfiguration of regulatory circuits during serum-2i reprogramming.

37. Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K: Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J 2004, 23:40614071.

21. Badeaux AI, Shi Y: Emerging roles for chromatin as a signal integration and storage platform. Nat Rev Mol Cell Biol 2013, 14:211-224.

38. Chamberlain SJ, Yee D, Magnuson T: Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency. Stem Cells 2008, 26:1496-1505.

22. Zheng H, Huang B, Zhang B, Xiang Y, Du Z, Xu Q, Li Y, Wang Q,  Ma J, Peng X et al.: Resetting epigenetic memory by reprogramming of histone modifications in mammals. Mol Cell 2016, 63:1066-1079. This study along with references [23,24,25,26] is the first study describing dynamics of histone modifications after fertilization and early stages of development by developing ultra-low input ChIP-seq profiling.

39. Montgomery ND, Yee D, Chen A, Kalantry S, Chamberlain SJ, Otte AP, Magnuson T: The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr Biol 2005, 15:942-947.

23. Zhang B, Zheng H, Huang B, Li W, Xiang Y, Peng X, Ming J, Wu X,  Zhang Y, Xu Q et al.: Allelic reprogramming of the histone modification H3K4me3 in early mammalian development. Nature 2016, 537:553-557. See annotation to Ref. [22].

40. Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, Yuan GC, Orkin SH: EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 2008, 32:491-502. 41. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M, Warnes G, Merkenschlager M et al.: Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006, 8:532-538.

24. Wu J, Huang B, Chen H, Yin Q, Liu Y, Xiang Y, Zhang B, Liu B,  Wang Q, Xia W et al.: The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 2016, 534:652657. See annotation to Ref. [22].

42. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K et al.: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006, 125:315-326.

25. Liu X, Wang C, Liu W, Li J, Li C, Kou X, Chen J, Zhao Y, Gao H,  Wang H et al.: Distinct features of H3K4me3 and H3K27me3

43. Voigt P, Tee WW, Reinberg D: A double take on bivalent promoters. Genes Dev 2013, 27:1318-1338.

Current Opinion in Genetics & Development 2017, 46:114–122

www.sciencedirect.com

Transcriptional and epigenetic control in pluripotency Habibi and Stunnenberg 121

44. Stock JK, Giadrossi S, Casanova M, Brookes E, Vidal M, Koseki H, Brockdorff N, Fisher AG, Pombo A: Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol 2007, 9:14281435. 45. Tee WW, Shen SS, Oksuz O, Narendra V, Reinberg D: Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 2014, 156:678-690. 46. Marks H, Kalkan T, Menafra R, Denissov S, Jones K, Hofemeister H, Nichols J, Kranz A, Stewart AF, Smith A et al.: The transcriptional and epigenomic foundations of ground state pluripotency. Cell 2012, 149:590-604. 47. Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff SL: Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 2012, 487:57-63. 48. Meissner A: Epigenetic modifications in pluripotent and differentiated cells. Nat Biotechnol 2010, 28:1079-1088. 49. Sharif J, Muto M, Takebayashi S, Suetake I, Iwamatsu A, Endo TA, Shinga J, Mizutani-Koseki Y, Toyoda T, Okamura K et al.: The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 2007, 450:908-912. 50. Sharif J, Koseki H: Recruitment of Dnmt1 roles of the SRA protein Np95 (Uhrf1) and other factors. Prog Mol Biol Transl Sci 2011, 101:289-310. 51. Bostick M, Kim JK, Esteve PO, Clark A, Pradhan S, Jacobsen SE: UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 2007, 317:1760-1764. 52. Kriaucionis S, Heintz N: The nuclear DNA base 5hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 2009, 324:929-930. 53. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L et al.: Conversion of 5methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009, 324:930-935. 54. Loenarz C, Schofield CJ: Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends Biochem Sci 2011, 36:7-18. 55. Wu H, Zhang Y: Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 2014, 156:45-68. 56. Ficz G, Hore TA, Santos F, Lee HJ, Dean W, Arand J, Krueger F, Oxley D, Paul YL, Walter J et al.: FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 2013, 13:351-359. 57. Habibi E, Brinkman AB, Arand J, Kroeze LI, Kerstens HH, Matarese F, Lepikhov K, Gut M, Brun-Heath I, Hubner NC et al.: Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 2013, 13:360-369. 58. Leitch HG, McEwen KR, Turp A, Encheva V, Carroll T, Grabole N, Mansfield W, Nashun B, Knezovich JG, Smith A et al.: Naive pluripotency is associated with global DNA hypomethylation. Nat Struct Mol Biol 2013, 20:311-316. 59. Smith ZD, Chan MM, Mikkelsen TS, Gu H, Gnirke A, Regev A, Meissner A: A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 2012, 484:339-344. 60. von Meyenn F, Iurlaro M, Habibi E, Liu NQ, Salehzadeh-Yazdi A,  Santos F, Petrini E, Milagre I, Yu M, Xie Z et al.: Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells. Mol Cell 2016, 62:983. This study shows the underlying molecular mechanism causes loss of DNA methylation during transition between two distinct states of pluripotency (serum and 2i). 61. Yamaji M, Ueda J, Hayashi K, Ohta H, Yabuta Y, Kurimoto K, Nakato R, Yamada Y, Shirahige K, Saitou M: PRDM14 ensures naive pluripotency through dual regulation of signaling and www.sciencedirect.com

epigenetic pathways in mouse embryonic stem cells. Cell Stem Cell 2013, 12:368-382. 62. Chen T, Ueda Y, Dodge JE, Wang Z, Li E: Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 2003, 23:5594-5605. 63. Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW, Jones PA: Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol 2002, 22:480-491. 64. Blaschke K, Ebata KT, Karimi MM, Zepeda-Martinez JA, Goyal P, Mahapatra S, Tam A, Laird DJ, Hirst M, Rao A et al.: Vitamin C induces Tet-dependent DNA demethylation and a blastocystlike state in ES cells. Nature 2013, 500:222-226. 65. Carey BW, Finley LW, Cross JR, Allis CD, Thompson CB:  Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 2015, 518:413-416. This study describes the effect of various metabolites in particular Krebs cycle intermediates on the maintenance of cellular identity and epigenetic/transcriptional regulation in mouse ESCs. 66. Chen J, Guo L, Zhang L, Wu H, Yang J, Liu H, Wang X, Hu X, Gu T, Zhou Z et al.: Vitamin C modulates TET1 function during somatic cell reprogramming. Nat Genet 2013, 45:1504-1509. 67. D’Aniello C, Habibi E, Cermola F, Paris D, Russo F, Fiorenzano A,  Di Napoli G, Melck DJ, Cobellis G, Angelini C et al.: Vitamin C and L-proline antagonistic effects capture alternative states in the pluripotency continuum. Stem Cell Rep 2017, 8:1-10. In this study describes methylome, transcriptome and metabolome reprograming upon changing the relative levels of ascorbic acid and Lproline as two physiological metabolites. 68. Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, Li W, Weng Z, Chen J, Ni S et al.: Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 2010, 6:71-79. 69. Stadtfeld M, Apostolou E, Ferrari F, Choi J, Walsh RM, Chen T, Ooi SS, Kim SY, Bestor TH, Shioda T et al.: Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat Genet 2012, 44:398-405 S391–S392. 70. Minor EA, Court BL, Young JI, Wang G: Ascorbate induces teneleven translocation (Tet) methylcytosine dioxygenasemediated generation of 5-hydroxymethylcytosine. J Biol Chem 2013, 288:13669-13674. 71. Yin R, Mao SQ, Zhao B, Chong Z, Yang Y, Zhao C, Zhang D, Huang H, Gao J, Li Z et al.: Ascorbic acid enhances Tetmediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc 2013, 135:1039610403. 72. Hore TA, von Meyenn F, Ravichandran M, Bachman M, Ficz G, Oxley D, Santos F, Balasubramanian S, Jurkowski TP, Reik W:  Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naive pluripotency by complementary mechanisms. Proc Natl Acad Sci U S A 2016, 113:12202-12207. In this study the effect of retinol and ascorbate during naı¨ve pluripotent stem cell reprogramming has been investigated. 73. Boroviak T, Loos R, Bertone P, Smith A, Nichols J: The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat Cell Biol 2014, 16:516-528. 74. Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, Nichols J, Smith A, Bertone P: Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev Cell 2015, 35:366-382. 75. Fenelon JC, Banerjee A, Murphy BD: Embryonic diapause: development on hold. Int J Dev Biol 2014, 58:163-174. 76. Scognamiglio R, Cabezas-Wallscheid N, Thier MC, Altamura S, Reyes A, Prendergast AM, Baumgartner D, Carnevalli LS, Atzberger A, Haas S et al.: Myc depletion induces a pluripotent dormant state mimicking diapause. Cell 2016, 164:668-680. Current Opinion in Genetics & Development 2017, 46:114–122

122 Cell reprogramming

77. Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V,  Peshkin L, Weitz DA, Kirschner MW: Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 2015, 161:1187-1201. This study along with references [78,79] provides new insights into the underlying regulatory circuit that control and generate heterogeneity in ESCs.

Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature 2014, 516:56-61. See annotation to Ref. [77]. 79. Schmiedel JM, Klemm SL, Zheng Y, Sahay A, Bluthgen N,  Marks DS, van Oudenaarden A: Gene expression. MicroRNA control of protein expression noise. Science 2015, 348:128-132. See annotation to Ref. [77].

78. Kumar RM, Cahan P, Shalek AK, Satija R, DaleyKeyser AJ, Li H,  Zhang J, Pardee K, Gennert D, Trombetta JJ et al.:

Current Opinion in Genetics & Development 2017, 46:114–122

www.sciencedirect.com