This chapter will be focused on epigenetic mechanisms known to affect self-renewal and developmental potency of embryonic-like stem cells, and germ cells which mimic similar epigenetic signatures as pluripotent stem cells. Examples of epigenetic regulation have proven crucial for defining the stem cell state. In particular, a wealth of knowledge regarding stem cell-specific epigenetic modifications has occurred over the past decade with discoveries that include describing unique stem cell-specific chromosome structure, DNA and histone modifications and noncoding RNAs. The impact of these findings and the better understanding of epigenetic regulation in pluripotent stem cells provides a foundation for discovering mechanisms which regulate human development and differentiation in addition to those that can facilitate cellular reprogramming.
In eukaryotes, chromosomes consist of repeating chromatin units called nucleosomes, which encompass segments of DNA (~147 bp) wound around a central core of eight histone (
In mammals, heterochromatin is associated with high levels of some methylation marks, including lysine (
2. Epigenetic control in pluripotent stem cells
2.1. Changes in chromatin ultrastructure in pluripotent stem cells
With the discovery of culturing embryonic stem cells (ESCs), several groups have been able to show the progression of global changes in the chromatin architecture of these cells. Through these studies, it has been shown that undifferentiated pluripotent stem cells contain less heterochromatic regions and express less chromatin structural proteins. Moreover, binding of these proteins (i.e. HP1α, lamin B) to heterochromatic regions is weaker compared to lineage-committed cells . Additionally, pluripotent transcription factors and chromatin remodeling proteins are overexpressed in ESCs compared to more differentiated progenitor cells . One study further showed that chromatin remodeler Chd1 knockdown results with heterochromatin accumulation and skewed differentiation in mouse ESCs, which suggests functional relevancy to the ‘open’ chromatin structure . Together, these studies show that pluripotent ESCs has an open and hyperdynamic chromatin structure which transforms into a more compact, repressive-like, chromatin state during differentiation.
As ESC chromatin is more transcriptionally permissive, it is also more sensitive to nuclease activity. This may also be in part due to differences that are seen in the chromatin localization in the nucleus. For instance, one study using the DamID (DNA adenine methyltransferase identification) technique showed that pluripotency genes, including Oct4 and Nanog, move to the nuclear lamina and are silenced while lineage-specific genes disassociate from the lamina and are expressed. This was specifically shown during the differentiation of mouse ESCs into terminal astrocytes which demonstrated cell type-specific relocations of these areas during differentiation . These areas near the nuclear periphery were called Lamina Associated Domains (
Genome-wide ChIP analyses have also described other lamina associated domains with significant overlap with the LADS domains. These regions referred as
Since the discovery of generating induced pluripotent stem (
In embryonic stem cells, lineage-specific gene expression program regulators are repressed, but poised for a rapid response to differentiate . These areas of chromatin, have so called bivalent domains, consist of opposing chromatin marks; i.e. H3K4me for activation and H3K27me for silencing. In ESCs, these domains are believed to be responsible for preventing the transcription responsible for their early differentiation to a specific lineage while priming the area for activation when the appropriate cues are expressed. Consistent with this belief is the findings that the bulk of the protein-encoding genes of human ESCs, including transcriptionally inactive genes have H3K4me, H3K9ac and H3K14ac rich promoter regions in areas of the nucleosome adjacent and downstream of transcription start sites [19, 20]. Moreover, in ESCs, genes with bivalent gene promoters tend to have unmethylated CpG islands . The initial step of active DNA demethylation in mammals occurs by the conversion of 5-methylcytosine of DNA (5mC) to 5-hydroxymethylcytosine (5hmC). A prime example of this in pluripotent stem cells has been shown in regulating the expression of the stem cell transcription factor, Nanog. Here, the demethylated state is critical for the upregulation of Nanog which is an essential regulator of ESC pluripotency and self-renewal, while its downregulation attributed to methylation of its promoter is required for ICM specification . Recent studies demonstrate that demethylation of Nanog is in part contributed to the expression of the Tet methylcytosine dioxygenase 1 (TET1) enzyme which is a TET family member of enzymes that catalyze the conversion of 5mC to 5hmC. This enzyme has been found to demethylate Nanog promoter sites in mouse ES cells [23, 24]. Both TET1 and TET2 expression have also been shown to be regulated by Oct4 expression in mouse ES cells, downregulated following differentiation alongside other stem cell markers, and is induced concomitantly with 5-hmC during fibroblast reprogramming into iPS cells .
In addition to promoter regulation, methylC-Seq genome-wide analysis has also discovered novel types of DNA methylation regulation at non-CG sites (CHG and CHH sites where H = A, C, or T residues). These analyses showed that non-CG methylation accounted for 25% of the total ESC methylome and that these sites were more commonly found within gene bodies than within promoter sites . Furthermore, the methylation of these sites was lost when differentiation was induced in ESCs, and restored during the generation of in induced pluripotent stem cells. This included many differentially methylated regions associated with genes involved in pluripotency and differentiation.
2.3. Polycomb and trithorax group proteins in pluripotent stem cells
Recent studies have established that developmental gene priming and bivalency are crucial for pluripotency whereby the chromatin of pluripotent stem cells are transcriptionally permissive, with normally silent DNA repeat regions, transcriptionally related histone modifications such as H3K9ac, H3K4me3, H3K36me3 and low stochastic transcription of lineage-restricted genes [8, 9]. The poised state is believed to inhibit the activity of RNA Polymerase II (
PcG, TrxG, and BAF complex associated genes are conserved from fly to man and are important in the regulation of organogenesis and development. PcG proteins were initially discovered as repressors of the Hox or homoeotic genes in
PcG proteins produce two distinct protein complexes that act sequentially to regulate gene expression – the “Bmi-1 complex” also known as Polycomb Repressive Complexes (
Polycomb repressive complexes have been shown associated with many developmental regulator regions in ESCs, and many of the PcG repressed targets of ESCs are also ‘bivalent’ [30, 39]. For instance, PRC2 target genes have been shown to be preferentially turned on during ESC differentiation and that the pluripotent stem cell regulating genes Oct4, Sox2, and Nanog co-occupy a significant subset of these genes. Therefore, it has been suggested that the PRC2 complex represses a distinct group of developmental genes that have to be repressed to maintain pluripotency. This would promote a poised or primed state which could be readily activated during early differentiation . For example, the histone methyltransferase Ezh2 is known to catalyze H3K27me3. In fact, bivalency domains at PRC2 regulated promoters are roughly five times more likely to become DNA methylated during differentiation than those with non-PRC2 regulated promoters  suggesting that the PRC2 complex plays a pivotal role in the switch for early lineage commitment . Jarid2, a member of the Jumonji family of histone demethylases, has also been shown to play an important role in properly recruiting PRC1 and PRC2 and initiating the RNA Polymerase II activating form (Ser5P-RNAPII)  to bivalent loci to promote differentiation [43-45]. While Jarid2 is enzymatically inactive in ESCs, recent evidence has shown that Jarid2 is regulated by pluripotency factors in ESCs . In null ESCs lacking Jarid2 expression were able to self-renew but unable to differentiate despite expressing appropriate PRC2 target genes demonstrating that transcriptional priming of bivalent genes in ESCs was dependent on Jarid2 expression.
In addition to the bivalent marks associated with PRC2 associated H3 modifications in pluripotent stem cells, bivalent genes in stem cells also exhibit repressive marks of H2AK119Ub1 at their promoter and throughout the coding region. For this reason, members of the PRC1 complex Ring1A and Ring1B proteins which catalyze H2AK119ub1 have also reveal a role in regulating pluripotency [30, 46]. For instance, in ESCs, double mutants of Ring1A/B demonstrate reduced levels of H2AK119Ub1, repression of known stemness genes (including Oct4 targets), increased expression of developmental regulator targets, and spontaneous differentiation. Furthermore, upon differentiation, Ring1A/B lost binding to their target loci suggesting that a Ring 1/B mediated complex functions downstream of the stem cell core transcriptional machinery to maintain the ESC state .
In addition to PcG complexes, core members of the TrxG and BAF chromatin remodeling complexes have also been shown to contribute to the bivalent mark in stem cells by acting in concert to establish and preserve H3K4me3 [48, 49]. Another core member, WD repeat domain 5 (
3. Epigenetic control in primordial germ cells and pluripotent embryonic germ cells
Primordial germ cells (
Key initiators of PGC induction in the epiblast include the Blimp1 (
In addition to pluripotent associated genes, early germ-cell development and imprinted genes also undergo demethylation during this time. These include well-established postmigratory germ cell genes Mvh (also known as Ddx4), Sycp3 (synaptonemal complex protein 3) and Dazl (deleted in azoospermia-like). These genes are demethylated in germ cells and repressed in somatic cells. This demethylation occurs during the migration of PGCs into the gonad at CpG islands of their promoters as well as at differentially methylated regions (
Another important epigenetic process required for germ cell development and cellular reprogramming to the pluripotent state involves the X chromosome. In female adult cells, one of the two X chromosomes is inactivated to compensate for the differences in gene expression between sexes. For this purpose, X chromosome inactivation is initiated in early embryos by noncoding X-inactive specific transcript (Xist) RNA followed by chromatin modifications on the inactive X chromosome which leads to stable gene repression in somatic cells. Likewise, reactivation of the X chromosome is required for the totipotency of the female blastocyst and germ cell development. Reactivation of the X chromosome also occurs to establish pluripotency in iPS cells. During development, epigenetic reprogramming or re-activation of the inactive X-chromosome commences in PGCs during their migration through the hindgut along their route to the developing gonads where imprint erasure is completed . In mouse PGCs, decreased Xist expression, and the displacement of PcG repressor proteins EED and SUZ12, results in the loss of the inactive X associated histone modification, H3K27me3 . In humans, PcG proteins YY1, EZH2, and EED have also been found in the ovarian follicles, oocytes and preimplantation embryos. YY1 and EZH2 transcripts were additionally detected in human metaphase II oocytes suggesting they may be play a similar role in human germ cell reprogramming .
4. MicroRNAs and stem cells
Studies in mice have shown that induction of neural differentiation in mouse ESCs with retinoic acid results in increased miR-134, miR-296, and miR-470 which in turn interact with the coding sequences of the pluripotency transcription factors Oct4, Sox2, and Nanog. These results suggest that through interaction of the miRNAs these pluripotent stem cells genes are downregulated thereby permitting differentiation to proceed . Additionally, the ESC-specific miR-290 cluster has also been shown to regulate Oct4 methylation in differentiating ESCs . Other studies have shown that mouse ESCs deficient in proteins of the miRNA processing apparatus such as Dicer, Drosha, DGCR8, and Ddx5 exhibit differentiation and developmental defects [95-97].
Interestingly, PcG proteins have been shown to be both regulators of miRNA expression as well as their targets. For instance, miRNA-101 has been shown to directly regulate the expression of the PRC protein EZH2 in highly aggressive cancers [98, 99].
5. Epigenetic regulation in progenitor and adult stem cells
Progenitor cells and adult stem cells are thought to be predecessors of pluripotent or multipotent stem cells that are generated during early differentiation. During their transition in development, bivalently marked stem cell genes can become either active, or inactive, or remain bivalent, dependent in part, on the activity of key enzymes which drive these chromatin modifications such as lysine demethylases (
Specific differences occur in the chromatin states between pluripotent stem cells, progenitor cells and more differentiated cell types which include active, repressed and poised states of chromatin. Several lines of evidence suggest that priming in the poised state enables genes to respond rapidly when differentiation cues are presented . For example, during neural induction, several hundred genes including those required to maintain stem cell-ness become de novo mCG and therefore transcriptionally silenced. Furthermore, the observation was made that neural precursors that are derived from ESCs acquired more mCG than terminal neurons, suggesting that the transition from pluripotent to lineage-committed cells is associated with these changes [17, 21, 101, 102].
Polycomb group proteins also appear to play a unique role in defining the progenitor or adult stem cell state. It has been shown that the PRC1 complex protein Bmi-1 activates multiple pathways that are important for regulating the stem cell-like state. For example, it has been shown that Bmi-1 is potentially upregulated via the pluripotent stem cell marker SALL4 signaling and has been shown to regulate stem cell self-renewal by repressing Hox genes, as well as INK4a locus genes, p16INK4a and p19ARF. BMI1 has also been shown to facilitate stem cell-like features in adult stem cells such as increased telomerase activity, transcriptional factor GATA3, and NF-kB pathways. These pathways are associated with the prevention of senescence, differentiation and apoptosis, while promoting immortalization and proliferation (for review see ).
6. Epigenetic dysregulation in cancer stem cells
Cancer stem/initiating cells (CSC) have been defined as a subset of cancer cells that have clonal ability or self-renewal and are resilient against cancer therapies [104, 105]. As such CSCs are implicated in cancer initiation, metastasis, and recurrence of some cancers . Although the most well established pluripotent stem cell genes OCT4, NANOG, cMYC and SOX2 are implicated in many poorly differentiated or metastatic cancers [107-109], they are not expressed in all and they are not all elevated concordantly. In addition, targets of NANOG, OCT4, SOX2, and c-MYC are often overexpressed in tumors that are poorly differentiated, more so than in those that are well differentiated . These genes also play a significant role in the induction of pluripotency into iPS cells from differentiated cell types and are thus involved in regulating epigenetic reprogramming [111-113]. More specifically, it is found that c-MYC, which is also an oncogene is sufficient for the reactivation of ESC-like transcriptional program in both, normal and cancer cells . Additionally, studies have shown that one of the inherent issues with generating iPS cells is their propensity to become cancer stem cell-like [115, 116]. Taken together, these results indicate that aberrant activation of an ESC or iPS-like transcriptional program might cause induction of pathological self-renewal in adult differentiated cells, characteristic of cancer stem cells.
Aberrant function of PcG proteins has also been established in the malignancy of various cancers . This is not surprising as it is well known that polycomb complexes contribute to the epigenetic regulation of key networks associated with self-renewal , differentiation, and proliferation [92, 119-123]. These roles for polycombs have been demonstrated in cancer cells and normal stem cells  and more recently studied for their targeted function in CSCs . For instance, there is much evidence that overexpression of the EZH2 polycomb gene occurs in multiple human malignancies (see [117, 126]). One study showed that this may in part be atributed to a genomic loss of miR-101 which has been shown to lead to increased EZH2 levels [99, 127]. Although how EZH2 contributes to carcinogenesis remains poorly defined, recent evidence suggests that overexpression of EZH2 can contribute to improper silencing of tumor suppressor genes . In this case, EZH2 was shown to target a pro-differentiation tumor suppressor gene, retinoic acid receptor β2 (
The pluripotent stem cells have a chromatin that is hyperdynamic, with a preponderance of modified histones and chromatin remodelers that ensures low-level transcription and tight regulation. Losing pluripotency is accompanied with a more compact, repressive, chromatin structure, which leads to cellular differentiation. Chromatin architecture is regulated at multiple levels in conjunction with known pluripotent genes to constitute an interwoven pluripotency network. Although there are many gaps in our knowledge of how epigenetic modifications regulate the pluripotent state, it is known that PcG repressor proteins prevent the precocious expression of lineage-restricted gene expression in pluripotent stem cells and germ cells by contributing to a unique ‘primed’ bivalent state of the chromatin. Future studies will provide mechanistic insights into the signaling cues required to maintain this state and inhibit differentiation while iPS cells and adult stem cells provide a renewed opportunity to study the role of chromatin architecture for controlling the pluripotent state. This will include understanding the mechanisms that interplay between pluripotent transcription factors, epigenetic regulators, and miRNAs to balance self-renewal and differentiation, properties which regulate reprogramming and carcinogenesis.
cdk, cyclin-dependent kinase; H2A-K119-Ub, ubiquitinylated histone H2A lysine 119; H3K27me3, tri-methylated histone H3 lysine K27; PcG, Polycomb group genes; ESC, embryonic stem cells; EGC, embryonic germ cells; PGC, primordial germ cells; iPS, induced pluripotent stem cells; CSC, cancer stem cells; RARβ2, retinoic acid receptor β2; Hh, Hedghog; KDMs, lysine demethylases; DUBs, histone deubiquitylases; DNMT, DNA methyltransferases; YY1, Ying Yang 1; EZH2 Enhancer of Zeste-2; EED, embryonic ectoderm development; GDF3, growth differentiation factor 3; DMR, differentially methylated regions; DAZL. deleted in azoospermia-like; Mvh, deadhead box 4; Sycp3, synaptonemal complex protein 3; E, embryonic day; RNAP II, RNA Polymerase II; trxG, trithorax group proteins; AID, activation-induced cytidine deaminase;
This work was supported by NIH grants R01 AR053851 and R01 CA131074 to R Eckert and R21HD057487 awarded to C Kerr as well as by the State of Maryland Stem Cell Research Fund 2010-MSCRFI-0110-00 to R Eckert and 2007-MSCRFII-0159-00 awarded to C Kerr.