Review Article

THE EPIGENETICS OF EARLY DEVELOPMENT: INFERENCES FROM STEM CELLS† Theodore P. Rasmussen1,2,3 1

Department of Pharmaceutical Sciences University of Connecticut Stem Cell Institute 3 Department of Molecular and Cell Biology University of Connecticut, 69 North Eagleville Road, Storrs, Connecticut 06269, USA 2

Key Words: embryo, embryonic stem cell, epigenetics, chromatin Abbreviations: 5-mC, 5-methlcyotosine; EGA, embryonic genome activation; EpiSC, epiblast stem cell; ESC, embryonic stem cell; ICM, inner cell mass; MII, metaphase II of meiosis. Quote: “[Embryonic stem cells] and [epiblast stem cells] can be considered as cell culture models for the behavior of the cells of the [inner cell mass] and the epiblast, respectively.”



This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/mrd.22269]

Received 21 June 2013; Revised 9 October 2013; Accepted 11 October 2013 Molecular Reproduction & Development © 2013 Wiley Periodicals, Inc. DOI 10.1002/mrd.22269

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ABSTRACT Approximately 200 cell types and multiple tissues are established throughout the development of the zygote to an adult mammal. During this process, the cellular genome remains fixed, yet the transcriptome of each of the cell types become widely divergent. This review discusses the epigenetics of preimplantation embryos and the use of embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) as cell-culture models for the inner cell mass (ICM) and epiblast, respectively. Differential patterns of transcription are set up during development by the action of key transcription factors and epigenetics, which are involved in the establishment and maintenance of stable transcriptional states during development. In early embryos, for example, changes in the epigenome consist of alterations to the methylation of CpG dinucleotides and post-translational modification of histones within chromatin. In addition, histone replacement occurs broadly in zygotes. The ICM of the blastocyst, on the other hand, has the amazing ability to contribute to every tissue and cell type present in the adult body. Therefore, ESCs are arguably the most important cell culture model available to developmental biologists. The advantages and risks of using ESCs to model ICM pluripotency are therefore discussed.

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EPIGENETICS AND CHROMATIN

DNA methylation Epigenetics can be broadly defined as any phenomenon or mechanism that affects gene expression without changing the sequence of DNA itself. In recent years, a great effort has been put into research that is aimed to elucidate the biochemical mechanisms that drive gene regulation at the epigenetic level. Through these efforts, it has been found that the molecular basis of epigenetics involves two principle classes of molecular mechanisms. One mechanism has to do with the modification of DNA by cytosine methylation at CpG dinucleotides. The most-studied form of DNA methylation is 5-methyl-cytosine (5mC), but recently, other methylated forms of DNA have been found, including 5-hydroxymethylcytosine (5-hmC). 5-mC marks on genes usually specify transcriptional inactivity. These DNA marks are initially imposed by de novo methyltransferases, such as Dnmt3a, and 5-mC patterns are maintained through subsequent cell division by the methyltransferase Dnmt1. This maintenance mechanism is required because DNA replicates with a semi-conservative mode, wherein the nascent strand of DNA is unmethylated prior to the action of Dnmt1, which converts newly replicated DNA (which is hemimethylated) to symmetrically methylated DNA. In contrast, 5-hmC marks are produced by the oxidation of pre-existing 5-mC marks, an activity that is performed by the TET family of methylcytosine oxidases (Cimmino et al., 2011; Guo et al., 2011; Tan and Shi, 2012). The full impact of 5-hmC marks for development and epigenetics is the focus of intensive current study, and TET-mediated oxidation offers a possible mechanism for the removal of 5-meC marks. Therefore, TET proteins may participate in the reactivation of formerly silenced genes.

Histone modifications

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The other major, molecular-epigenetic regulation system involves the dynamic post-translational modification of histones.

DNA is associated with

histone proteins, which are positively charged proteins ranging from 11-21 kDa. Four histones –H2A, H2B, H3, and H4– assemble into a nucleosome, with two units of each histone per octamer, thus forming a nucleosomal octameric core. Around this core are wrapped 146 bp of DNA. The linker histone H1 associates with non-nucleosomal DNA present between nucleosomes, and is involved in chromatin compaction. The amount of DNA present within linker DNA is much less than that found within nucleosomes. The N-terminal tails of each histone protrude from the nucleosome core particle and are rich in lysine and arginine residues. These amino-acid residues are

modified

by

methylation

(mediated

by

the

action

of

histone

methyltransferases) or acetylation (mediated by the action of by histone acetyltransferases). In all, about 40 histone residues can be modified. Each modification seems to have a unique influence on the transcriptional activity of the associated gene. For instance, methylation of lysine 9 of histone H3 (H3K9) generally has a negative effect on transcription. Hereafter, the following widely accepted nomenclature will be used in the review: The histone is first specified, followed by the amino acid involved, followed by the modification. For instance histone H3 trimethylated at lysine 27 is denoted as H3K27me3. Other modifications are denoted as follows: acetylation is ‘Ac’, ubiquitination is ‘Ub’, sumoylation is ‘Su’, and phosphorylation is ‘P’. Histone methylation can have either positive or negative effects on transcription. For instance H3K4me3 is a

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transcriptionally activating mark, while H3K27me3 represses transcription. In some cases, a molecular competition exists between methylation and acetylation. For instance H3K9 can be either methylated or acetylated, but not both. H3K9me3 signals transcriptional repression, whereas H3K9Ac has a generally positive effect on transcriptional activity since histone acetylation weakens DNA-histone interactions.

EPIGENETIC ANALYSES IN INTACT PREIMPLANTATION EMBRYOS The early embryo, from the zygote to the blastocyst, is readily amenable to epigenetic analyses.

Many excellent antibodies are available that are highly

specific for modified histones. One advantage of such studies is that epigenetic analyses can be conducted directly in an intact embryo. An inherent drawback with the use of whole-mount preimplantation embryos, however, is that the action of epigenetic modifications cannot be assessed at the level of specific genes. So far, it has not been possible to perform key procedures such as chromatin immunoprecipitation on single cells, and hence it is not generally easy to correlate immunofluorescence results with effects on specific genes. This said, much has been learned about broad epigenetic reorganizations that occur during the course of preimplantation development using cell biology, as summarized in the following section.

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Early preimplantation development Fertilization triggers a massive reorganization of chromatin in a short amount of time. Male and female genomes are highly dissimilar, especially in the organization of their chromatin and the methods for the packaging of gametic DNA. Both the egg and sperm are transcriptionally silent prior to and shortly after fertilization.

Expansive alterations occur to their haploid genomes soon

after fertilization, even before embryonic transcription begins. The mammalian female egg genome, for example, is packaged into highly specialized maternal chromatin and is arrested at metaphase II of meiosis (MII). The egg cytoplasm contains a large amount of RNA, including protein-coding mRNA to small RNAs that are available for use quickly after fertilization. In contrast, the male sperm genome is highly condensed and is largely (but not completely) devoid of histones; rather, it is associated with protamines that compact the male genome to a very high degree. In short, the egg is a repository (Akiyama et al., 2011) of maternal stores of RNA and proteins that are poised to act upon fertilization, while the sperm head consists mostly of a haploid complement of highly condensed, protamine-laden DNA. Upon fertilization, the final meiotic cell division is completed, the maternal spindle is resolved, and a haploid complement of maternal DNA is extruded as the second polar body. Simultaneously, the male haploid genome is stripped of protamines, and the paternal DNA is packaged with histones. Both the maternal and paternal genomes are then sequestered into separate nuclear envelopes,

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which results in the 2-pronuclear zygote. The sequestered paternal and maternal pronuclei independently re-enter the cell cycle and go through a round of Sphase. They subsequently fuse after nuclear envelope breakdown, and mix when the first mitotic spindle is formed. A diploid complement of DNA of both maternal and paternal sources is drawn to each pole as mitosis proceeds, the nuclear envelope is then re-established, and cytokinesis is completed, resulting in a 2cell embryo. The activation process that occurs soon after fertilization involves a massive reorganization of histones. The male haploid genome is deposited into the egg cytosplasm as highly condensed DNA associated with protamines.

Upon

fertilization, these protamines are rapidly removed and the male haploid genome decondenses and instead associates with histones of oocyte origin. In contrast, the female haploid genome is already associated with histones in the MII oocyte, yet significant replacement of histones also occurs.

For instance, histone

variants of oocyte origin such as linker histone H1FOO, are stripped from the female genome and replaced with canonical linker H1 histones (Gao et al., 2004; Teranishi et al., 2004). The maternal chromatin of the zygote is also modified by the transient loss of the histone variant H3.3, a marker of euchromatin, and H3.2 is incorporated into maternal chromatin upon fertilization (Akiyama et al., 2011). In a final example, the oocyte also contains a cache of macroH2A1, an H2A histone variant, but this is removed from the maternal genome soon after fertilization (Chang et al., 2005). Removal of macroH2A at the pronuclear stage seems to be an active process that does not require cell division because

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macroH2A is removed quickly from transplanted nuclei during somatic cell nuclear transfer (SCNT) experiments (Chang et al., 2010).

The embryonic

genome becomes associated with macroH2A1 of embryonic origin at the 8-cell cleavage stage in SCNT embryos, a timing that is equivalent to that of normal embryos (Chang et al., 2010). Though the dynamics of macroH2A exchanges have been well studied, the role of macroH2A during development is still mysterious. From these examples, it is clear that there is a substantial amount of histone replacement and exchange within the maternal genome that is triggered by fertilization.

DNA methylation in preimplantation development Mammalian DNA is often methylated at cytosine residues in CpG dinucleotides at carbon 5 (5-mCpG). Imprinted genes (both paternal and maternal) often maintain their methylation state through fertilization, but the bulk of genomic DNA undergoes massive changes in CpG methylation upon fertilization and during cleavage-stage embryogenesis (please see excellent, recent reviews on imprinting by Amor and Halliday, 2008; Barlow, 2011; Ferguson-Smith and Surani, 2001).

An interesting study compared bovine

oocytes and 8-cell embryos using microarrays to define pools of stored and expressed mRNAs; genes encoding DNA methylation enzymes were found to be upregulated in mature oocytes (Misirlioglu et al., 2006). Yet, DNA methylation genes and other chromatin modifiers are also expressed in cleavage-stage

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embryos, and likely participate in epigenetic remodeling of the genome during early embryogenesis. Sperm and eggs are imbued with remarkably different configurations of CpG DNA methylation. Egg genomes surprisingly show a positive correlation between DNA methylation and gene bodies, whereas sperm genomes are nearly completely methylated except at CpG islands (Kobayashi et al., 2012). Eggs and sperm also differ greatly in their overall content of 5-mCpG. Overall, sperm DNA is more methylated than egg DNA, but the male haploid genome undergoes rapid demethylation soon after fertilization. Recently this has been shown to be an active process: After years of searching, the identification of a possibly biologically relevant 5-meCpG demethylases has finally been documented in the form of TET family of proteins (Cimmino et al., 2011; Guo et al., 2011; Tan and Shi, 2012). During cleavage-stage embryogenesis, levels of 5-me-CpG decline, and then de-novo methylation occurs by the action of Dnmt3a and Dnmt3b. Thereafter, symmetric methylation on both strands is maintained by the CpG methyltransferase Dnmt1. genome

to

Accessibility of the early preimplantation embryo

methyltransferase

activity

may

be

restricted

to

de

novo

methyltransferases, however, since Dnmt1 is excluded from the nuclei of embryos up to the 8-cell stage (Ratnam et al., 2002). Enhancer of zeste 2 (Ezh2) is present as a maternal store in mature oocytes. Ezh2 is a polycomb group 2 (PcG2) histone methyltransferase that produces H3K27me3. Knockout of Ezh2 causes early embryonic lethality (O'Carroll et al., 2001). Depletion of the maternal store of Ezh2 leads to delayed development,

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even though Ezh2 of embryonic origin is supplied following embryonic genome activation (EGA) (Erhardt et al., 2003), suggesting that Ezh2 is involved in epigenetic histone methylation at H3K27 patterning the early embryo during preimplantation development. This is supported by the finding that Ezh2 is most highly expressed in very early bovine preimplantation embryos (Ross et al., 2008). In contrast, JmJD3 (an H3K27me3 demethylase), is highly expresses in porcine 4-cell embryos, but decreased in hatched blastocysts (Gao et al., 2010). Though these observations were made in two different species, it is tempting to consider that a window exists for the establishment of H3K27me3 between the 1and 4-cell stages. In mouse development, the action of PcG1 is required during oocyte maturation in order to form an embryo after fertilization. Thus, histone methylation is quite important for establishing zygotic totipotency (Posfai et al., 2012).

Indeed, both egg and sperm chromatin seem to be epigentically

predisposed to totipotency based on epigenetic marks present in mature germ cells (Rasmussen and Corry, 2010). Upon fertilization, sperm DNA is rapidly denuded of protamines, and then becomes associated with acetylated histones. (Rousseaux et al., 2008), contributing to the zygotic asymmetry between paternal and maternal pronuclei.

For instance: H3 lysine methylation in the male

pronucleus is negative for H3K9me3 whereas the female pronucleus is positive (Santos et al., 2005); PcG proteins are found in the male pronucleus soon after fertilization, but H3K27 methylation does not occur until after the first mitosis; and

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a store of heterochromatin protein 1 (HP1) is also observed in the male pronucleus, even before H3K9me3 is completed. For years, the opinion in the field was that EGA occurs at the 2-cell stage in mice and the 8-cell stage in human embryos. As it has recently become possible to collect RNA from single blastomeres, however, it is apparent that the human embryo also undergoes waves of EGA beginning at the 2-cell stage (Vassena et al., 2011). In 2-cell embryos each of the blastomere cells are totipotent, as they can give rise to cells of the embryo proper as well as extraembryonic cells of the placenta and trophectoderm.

During subsequent cleavage stages of

preimplantation development, each of the blastomeres retains totipotency. Shortly after syngamy is completed, the preimplantation embryo undergoes a rapid set of cleavage-stage mitotic divisions; when it reaches the morula stage, there may be patterning that assists in the establishment of the first distinct cellular lineages that appear in the blastocyst.

A recent, alternative model

proposes that asymmetry exists at the first embryonic cleavage, and that a single blastomere may be primed to differentiate into the embryo proper while the other gives rise to extra-embryonic lineages (Plusa et al., 2005). Yet, it is not known if the mechanisms responsible for this extraordinary method of lineage allocation exist and/or are epigenetic in nature. Parthenogenetic development to live offspring does not occur in mammals. This is likely due to the barrier posed by imprinted genes, and is supported by somatic-cell nuclear transfer experiments. For example, parthenogenetic embryos can be made that contain genetic material acquired from non-growing

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oocytes, and develop to embryonic day 13.5, much farther into development than occurs during parthenogenetic activation of mature oocytes (Kono et al., 1996). In addition, these results suggest that maternal imprinting of genes may occur, at least in part, during oocyte maturation.

THE

BLASTOCYST

AND

BEYOND:

MODELING

EPIGENETICS

AND

DEVELOPMENT USING EMBRYONIC STEM CELLS AND EPIBLAST STEM CELLS The morula is poised to undergo the first cell divisions that yield developmentally asymmetric cells that can be discerned in the blastocyst. The blastocyst contains three primary cell types: the inner cell mass (ICM), which gives rise to the embryo proper; trophectodermal cells, which give rise to extraembryonic portions of the placenta; and the extraembryonic endoderm, which gives rise to other placental tissues. Embryonic stem cells (ESCs) are derived from the ICM of blastocysts (Fig. 1). Two principle lines of evidence suggest that ESCs may provide a functional developmental model of the ICM. First, ESCs can be injected into blastocyst stage embryos and contribute to chimeras in the mouse. This suggests that the configuration of ESC chromatin and the potential for cell-lineage specification (i.e. pluripotency) are retained in ESCs. Secondly, it is possible to make liveborn mice that are entirely derived from ESCs using tetraploid embryo complementation.

In this procedure a host tetrapoid blastocyst is used.

Tetraploid blastocysts cannot yield live mice upon implantation, but can support

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the development of diploid ESCs to birth via the formation of a supporting tetraploid placenta. Another type of pluripotent stem cells is the epiblast stem cell (EpiSC). In vivo, blastocysts undergo implantation into the uterine wall where subsequent differentiation of the ICM occurs as early invasive stages of placentation occur. In vitro, however, it is possible to culture preimplantation embryos to the egg cylinder stage, in which epiblast cells are well-formed (Fig. 1); EpiSCs can be derived from these epiblast-stage embryos (Brons et al., 2007; Tesar et al., 2007). EpiSCs seem to be developmentally “primed”, meaning that they are especially prone to differentiation into ectoderm, endoderm, mesoderm, and their derivatives. Recent reports have shown that EpiSCs can be reverted to ESCs by exposing them to reprogramming conditions. For instance, mouse EpiSCs were reverted to ESC-like cells by manipulating their response to leukemia inhibitory factor (LIF)-Stat3 signaling (Bao et al., 2009). In another report, reprogramming transgenes (Oct4, Klf4, and Klf2) were used to revert EpiSCs to ESCs (Hanna et al., 2010). While further studies are warranted to investigate the full range of epigenetic changes that occur in these reprogramming experiments, these initial findings that reprogramming from EpiSCs to ESCs can be achieved suggests that distinct epigenetic states exist that distinguish EpiSCs from ESCs (and likely embryonic epiblast cells from ICM cells, respectively).

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Epigenetics in preimplantation embryos In some cases, epigenetic phenomena can be modeled in ESCs that have also been directly studied in preimplantation embryos.

For instance, proviral

sequences are usually silenced by DNA methylation and other chromatin modifications. Interestingly, rare ESCs can be found that exhibit active proviral transcripts reminiscent of that observed in 2-cell embryos. These long-terminal repeat (LTR)-driven transcripts come from a subpopulation of ESCs that lack the expression of pluripotency factors such as Oct4 and Sox2, suggesting that these cells that may have an increased propensity to be developmentally committed. Surprisingly, these Oct4- and Sox2-negative cells can contribute extraembryonic components to embryos (unlike normal ESCs), suggesting that the 2-cell embryo may reside in a state of relative epigenetic plasticity (Macfarlan et al., 2012). In short, this study shows that a subpopulation of ESCs resembles 2-cell blastomeres. X-chromosome inactivation occurs in an imprinted fashion in female trophectoderm, whereas two transiently active X chromosomes reside in morula in cells whose lineages are bound for ICM (Okamoto et al., 2004). This is largely dictated by the methylation status of the Xist gene (whose product is involved in the initiation of X-chromosome inactivation), which is differentially regulated in placenta versus embryo proper. Interestingly, two transiently active X chromosomes are also found in mouse ESCs (Panning et al., 1997; Penny et al., 1996). Therefore, female ESCs serve as a reasonable model of the epigenetic

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process of X chromosome inactivation, which normally occurs shortly after implantation. But X chromosome inactivation can also be reversed in pluripotent stem cells. Like all somatic cells, EpiSCs contain one inactive X chromosome, yet conversion of EpiSCs to developmentally more-primitive ESCs causes reactivation of the inactive X chromosome, thus demonstrating that this reprogramming event culminates in the establishment of an earlier epigenetic state in which the choice of paternal or maternal X chromosome for inactivation has not yet been made (Hanna et al., 2010). The histone variant macroH2A plays a role in early development, and somatic cells with reduced levels of macroH2A exhibit higher efficiencies of reprogramming into induced pluripotent stem cells (Pasque et al., 2012).

MacroH2A is also associated with the inactive X

chromosomes after mouse ESCs differentiate (Costanzi et al., 2000). Yet, knockdown of the two closely related genes encoding MacroH2A in female mouse ESCs failed to prevent X chromosome inactivation (Tanasijevic and Rasmussen, 2011). The first developmentally distinct cell lineages that differentiate during embryogenesis are the trophectoderm, the extraembryonic endoderm, and the ICM, which are all present at the blastocyst stage. In a recent study, pluripotent cells derived from all three of these lineages –embryonic stem cells (ESCs), trophectoderm stem cells (TSCs), and extraembryonic endoderm cells (XENCs)– were compared using epigenetic assays, including the measurement of the replication timing of lineage-specific genes for each of the cell types (Santos et al., 2010).

Since replication timing is thought to be associated with open

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chromatin, this study suggests that the chromatin of all three cell lineages is already epigenetically accessible at the blastocyst stage – a point further supported by the similar differences observed in modified histones among the three cell types. The transcriptome is presumed to be the functional output of the underlying epigenome.

Another recent study followed transcriptome-wide

changes in the derivation of ESCs from the ICM at the single-cell level (Tang et al., 2010), under the assumption that the transcriptome represents the functional output of the underlying epigenome. This study showed that many transcript variants can be detected during the ICM-to-ESC derivation process, even for house-keeping genes. In addition, genes encoding negative epigenetic regulators increased during ESC derivation from the ICM. Thus, caution must be used when inferring developmental information when using stem cell lines as models of development since the derivation process itself induces changes in gene expression.

Establishment of tissue-specific gene expression Developmentally important Hox genes are assembled into bivalent chromatin in ESCs and presumably in the developing epiblast. Bivalent domains consist of lysine methylation of H3K4me3 and H3K27me3 (Atkinson et al., 2008), where H3K4me3 is ordinarily a transcription-activating modification while H3K27me3 is normally a repressive modification.

Thus, bivalently marked genes have

“opposing” chromatin marks. In ESCs (and presumably also in blastomeres of the ICM), however, it has been shown that the net effect of bivalency is

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transcriptional silence – although this silencing is not locked, but rather in a developmentally poised state (Fig. 2). Upon differentiation, bivalent domains are resolved in two ways, depending upon which methyl mark is removed: H3K4me3 is the remaining mark (of the original bivalent domain mark) found at active genes, whereas H3K27me3 is the sole remaining mark at silent genes (unmodified histones are not shown in conventional nomenclature).

Other

chromatin modifications are also likely established that serve to stabilize the gene expression status of resolved bivalent domains. For instance, 5-mC marks can become associated with silenced bivalent domain genes to lock in the transcriptionally inactive state. The ability to perform single-cell chromatin immunoprecipitation is needed to determine if findings about bivalency in ESCs hold true in preimplantation embryos.

SUMMARY AND FUTURE DIRECTIONS Based on the above writing, it should be clear that ESCs and EpiESCs can be considered as cell culture models for the behavior of the cells of the ICM and the epiblast, respectively. Arguably, the ICM is one of the most important primordial cell types to model in the embryo because this population can give rise to all the cell types present in the embryo proper. Much of this developmental potency is retained in ESCs, which can readily differentiate into a multitude of derivative cell types spanning all three principle germ layers. Development itself makes heavy use of epigenetics, because all ~200 cell types established once embryogenesis is complete share the same genetic complement of DNA.

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Therefore, the

changes that occur over the course of development from the ICM to the adult organism likely involve the coordinated action of key transcription factors, combined with the action of epigenetic mechanisms that stabilize specific transcriptional states. The study of epigenetic mechanisms in ESCs, especially those induced by differentiation in vitro, provides a good cell-culture model for initial stages of development. A prudent note of caution should be emphasized, however. In epigenetic studies of ESCs, it is assumed that similar mechanisms exist in the ICM, but comparative RNA-seq analysis of single ICM cells and ESCs demonstrated tractable changes that occur during the derivation of ESCs from the ICM (Tang et al., 2010). This result is not surprising since the ICM is an important, albeit transient, structure of development, likely existing for a period of hours.

In

contrast, ESCs are immortal in cell culture, and exist in a state of “suspended animation” with regards to their developmental potential, until differentiation is intentionally induced. Therefore, while ESCs can serve as a good model of the ICM for many aspects of developmental biology, some key alterations occur during the derivation, establishment, and propagation of ESC lines. The technical ability to assess the entire transcriptome in single cells has outpaced the ability to assess the epigenome.

It is only a matter of time,

however, until the ascertainment of the single-cell epigenome will be possible; at that time in the near future, we will be able to determine how well the ESC epigenome models the ICM epigenome.

Based on the present comparative

transcriptome studies, we can predict that the ESC epigenome will resemble that

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of the ICM, but that specific differences will be found that may be attributed to the cell-line derivation process.

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Figure Legends Figure 1. Derivation of pluripotent stem cells. The ICM is a structure contained within the blastocyst. Prior to the blastocyst, all cells are totipotent; at the blastocyst stage, ICM cells are developmentally restricted to contribution to the embryo proper. ESCs are derived from the ICM of preimplantation embryos. Like ICM cells, ESCs can differentiate into endoderm, ectoderm, and mesoderm lineages; in contrast, ESCs proliferate indefinitely. EpiSCs, on the other hand, are derived from epiblast cells that are present in embryos at the egg-cylinder stage.

Figure 2. Configuration of bivalent domains in embryonic chromatin and in differentiated cells. Bivalent domains are physically composed of nucleosomes that contain histone H3 that is trimethylated at both lysines 4 and 27. H3K4me3 (green) is normally permissive of transcription while H3K27me3 (red), is normally repressive of transcription.

In ESCs, genes that are associated with both

H3K4me3 and H3K27me3 are said to contain bivalent domains. These genes are silenced, but poised for either activation or permanent silencing upon differentiation. If the gene is allocated to a cell lineage in which it is silenced, the bivalent domain is resolved leaving only the repressive histone mark H3K27me3. If the gene is allocated to a cell lineage in which it is expressed, then only the H3K4me3 mark is retained. 24

Figure 1

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Figure 2

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The epigenetics of early development: inferences from stem cells.

Approximately 200 cell types and multiple tissues are established throughout the development of the zygote to an adult mammal. During this process, th...
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