DNA methylation refers to methylation of cytosine residues in genomic DNA, which leads to the formation 5-methycytosine (5mC) [1]. This covalently modified nucleotide is found prominently in gene regulatory regions (e.g., promoter elements) and in other regions (e.g., inter- and intra-genic sequences and repetitive elements). Members of the DNA methyltransferase (DNMT) family of enzymes are responsible for promoting de novo methylation and for maintaining methylation. Factors implicated in DNA demethylation have more recently been identified and include DNA excision repair, cytidine deaminase, and Gadd45 proteins. Methyl-CpG-binding domain (MBD) proteins bind specifically to methylated DNA and recruit additional regulatory factors to these loci. This DNA methylation machinery and the corresponding profiles of DNA methylation are dynamically regulated during development, adult life, and aging in a tissue- and cell type-specific manner that is environmentally sensitive (and activity-dependent in the nervous system). DNA methylation is, in concert with other epigenetic mechanisms, involved in mediating a broad range of cellular processes including transcriptional repression (and rarely activation) at individual gene loci and more globally; X chromosome inactivation (XCI) and genomic imprinting; and maintenance of genomic integrity.

DNA methylation plays a role in establishing and maintaining neural cell identity [1]. For example, one recent study showed that in a cellular differentiation paradigm progressing from embryonic stem cells (ESCs) to lineage-committed neural progenitor cells (NPCs), hundreds of promoter regions are methylated in NPCs, including those associated with pluripotency and germline-specific genes, suggesting that DNA methylation is responsible for silencing these genes and promoting neural lineage commitment [3]. Another study showed that, in postnatal neural stem cells (NSCs), DNMT3A methylates intergenic regions and gene bodies flanking proximal promoters regions of a large number of genes, including many regulators of neurogenesis, DNMT3A-dependent non-proximal promoter methylation promotes the expression of these neurogenic genes, and DNMT3A is required for neurogenesis [4]. These observations suggest that DNA methylation mediates neural lineage elaboration by silencing genes associated with pluripotency and activating neuronal lineage-specific genes.

Another modified cytosine residue, 5-hydroxymethylcytosine (5hmC) is also implicated in the epigenetic regulation of cell identity, though its functional roles are not as well understood. 5hmC is likely generated by the oxidation of 5mC, which is catalyzed by the Ten-Eleven Translocation (TET) family of enzymes that are highly expressed in ESCs [5,6]. Mechanistically, 5hmC inhibits the binding of MBD proteins to DNA implying that its role may, in part, be to counterbalance that of 5mC [7,8]. In fact, recent analyses of 5hmC (and TET proteins) in ESCs and induced pluripotent cells suggest that 5hmC is associated with actively transcribed genes in pluripotent cells and, further, that the balance between 5hmC and 5mC levels is important for regulating the switch from a pluripotent to a lineage committed cellular species [6,9,10]. Additional studies have found that 5hmC is widely distributed in brain and is particularly abundant in regions involved in higher cognitive functions, such as the cortex and hippocampus [11-13]. Interestingly, characterizing the distribution of 5hmC in DNA from human brain frontal lobe tissue reveals that 5hmC is selectively targeted to promoter elements and gene bodies rather than intergenic regions and, further, that genes associated with these regions are significantly enriched for those involved in neural development and patterning [13]. These findings suggest that the cell-type specific regulation of this epigenetic modification, like that of 5mC, also plays a role in the establishment and maintenance of neural cell identity.

Chromatin refers to the packaging of genomic DNA, along with histone and non-histone proteins and associated factors, within the cell nucleus [1]. The nucleosome is the most essential unit of chromatin. It is formed by 147 base pairs of DNA wrapped around an octamer comprised of two of each of the classical histone proteins (i.e., H2A, H2B, H3, H4) and by linker DNA and histones (i.e., H1). A series of nucleosomes forms the characteristic “beads-on-a-string” configuration that can, in turn, fold into secondary and tertiary structures representing varying degrees of condensation. For example, higher order chromatin conformations include loosely packaged euchromatin and more densely packaged heterochromatin. Chromatin states can be modulated at multiple levels within this hierarchy. Classical histone proteins can be replaced by variant histones (e.g., H2A.Z), with distinct properties. Specific classes of enzymes are responsible for regulating reversible histone post-translational modification levels including acetylation (i.e., histone acetylases and deacetylases [HATs and HDACs]) as well as methylation, phosphorylation, SUMOylation, ADP-ribosylation and others. Additional enzymes and macromolecular complexes promote nucleosome and higher order chromatin (e.g., Polycomb Group [PcG] and Trithorax Group [TrxG] proteins) remodeling. Histone-, nucleosome-, and higher order chromatin-modifying enzymes may act alone; or, because strata within this chromatin hierarchy are interconnected, these factors may be integrated into highly modular complexes, such as the REST and the functionally related CoREST epigenetic regulatory complexes. Rearrangement of chromatin is responsible for the establishment of more or less compacted configurations that play dynamic regulatory, structural, and other functional roles within the cell. These effects can include modulating interactions between DNA sequences with each other and with diverse nuclear factors including ncRNAs, DNA binding proteins, transcriptional co-regulators, transcription factors, and additional nuclear machineries, such as those involved in mediating the processes of transcription and DNA replication and repair. Like DNA methylation, histone and chromatin modifications are involved in mediating a broad range of cellular processes including transcriptional repression and activation at individual gene loci and more globally; XCI and genomic imprinting; and maintenance of genomic integrity. Histone and chromatin modifying factors and the corresponding profiles of chromatin modifications are, like DNA methylation, dynamically regulated during development, adult life, and aging in a tissue- and cell type-specific manner that is environmentally sensitive.

Histone modifications and chromatin remodeling play a role in establishing and maintaining neural cell identity (reviewed in [14,15]). For example, we recently demonstrated that developmental stage-specific deployment of REST and CoREST complexes is responsible for controlling NSC-mediated neural lineage elaboration including neuronal and glial subtype specification, progressive maturation, and terminal differentiation (i.e., oligodendrocyte [OL] myelination) through dynamic and context-specific activation and repression of cell type- and maturational stage-specific gene networks [16-18]. Similarly, recent studies have shown that the histone demethylase, LSD1, regulates the proliferation of NSCs [19]; the TrxG protein, Mll1, is essential for postnatal NSC-mediated neurogenesis [5]; and, PcG proteins play a role in the temporal regulation of NPC fate by modulating neuronal to glial fate switching [20]. Moreover, another study profiled PcG-mediated histone H3 lysine (K) 27 trimethylation (H3K27me3) in ESCs, lineage committed NPCs, and terminally differentiated neurons and found dynamic changes in these marks in promoter regions of many genes including, specifically, loss of H3K27me3 from neuron-specific genes activated upon terminal differentiation in the transition from NPCs to terminally differentiated neurons [3]. Interestingly, this study also reported that promoter regions with H3K27me3 in stem cells are often associated with DNA methylation events during differentiation, highlighting how these developmental processes are likely coordinated by crosstalk between different epigenetic regulatory mechanisms.

The great majority of human genomic DNA does not code for protein; non-coding sequences are nevertheless transcribed, forming various classes of ncRNAs. These ncRNAs are more abundant than protein-coding RNAs in human cells [21]. Certain classes of ncRNAs, such as transfer RNAs and ribosomal RNAs, are well known. Other classes of ncRNAs have more recently been recognized and novel classes are being identified at a rapid pace. These include short ncRNAs such as endogenous short-interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs and small nucleolar RNAs (snoRNAs), and long ncRNAs (lncRNAs) such as long intergenic ncRNAs (lincRNAs). NcRNAs have expression profiles that are cell-, tissue-, and developmental stage-specific (and activity-dependent in the nervous system) [22]. NcRNAs have diverse regulatory, structural, and other operative roles, including many that are still emerging [23]. Known molecular functions for ncRNAs include mediating and/or modulating aspects of DNA methylation, chromatin modification, transcription, and RNA post-transcriptional processing, transport, and translation [24].

MiRNAs, the best-characterized class of ncRNAs, regulate target messenger RNAs (mRNAs) through post-transcriptional mechanisms [25]. Individual miRNAs have the potential to modulate hundreds of complementary mRNA molecules through complete or partial sequence-specific interactions. Single-stranded miRNA molecules bind to mRNAs primarily in 3′ untranslated regions (UTRs) as well as in certain coding regions and 5′ UTRs, preventing the translation of these mRNAs or sequestering them for storage or degradation via the RNA-induced silencing complex. Conversely, a single mRNA may be the target of multiple distinct miRNAs.

The functions of lncRNAs, the most abundant class of ncRNAs, are not as well understood. Only a few lncRNAs have been studied in detail, such as the X inactivation-specific transcript, which mediates X chromosome inactivation. More recently, however, lncRNA biogenesis and function have been examined more systematically. LncRNAs are defined as transcripts longer than 200 nucleotides that can be, but are not necessarily, 5′ capped, polyadenylated, and spliced like mRNAs [26-29]. Some lncRNAs are derived from intergenic regions, whereas others are transcribed in antisense, bi-directional, or overlapping orientations relative to protein-coding genes—genes that may be specifically regulated by their associated lncRNAs. LncRNAs can form a range of functional secondary structures, including particular protein interaction domains [30-32]. LncRNAs can be cleaved into shorter ncRNAs (e.g., siRNAs, snoRNAs and miRNAs) [33]. Thus, lncRNAs represent highly versatile molecules. Correspondingly, these factors have a broad spectrum of functions, including the ability to recruit transcriptional and epigenetic (e.g., chromatin remodeling complexes) regulatory factors to specific genomic sites, to form of nuclear subdomains (e.g., paraspeckles), to mediate nuclear-cytoplasmic transport of proteins, and to control translation (e.g., local protein synthesis in synaptic compartments) [34,35]. Moreover, lncRNAs are implicated in mediating a wide range of normal and pathological processes, from development, plasticity, and homeostasis to cancer metastasis [36].

Many studies have begun to elucidate the expression and function of miRNAs in NSC self-renewal, maintenance and lineage restriction, neuronal and glial lineage commitment, neuronal and glial subtype specification, progressive maturation, and plasticity [37]. For example, one important study showed that miR-124, the most abundant miRNA in the central nervous system (CNS), regulates the progression of NSCs into neurons [38]. Specifically, in NSCs, knocking down miR-124 derepresses its target, Sox9, and results in disruption of neuronal lineage elaboration with arrest in the proliferative precursor state. Two recent studies demonstrated that miRNAs also regulate OL development [39,40]. Both studies showed that eliminating the presence of mature miRNAs via targeted disruption of the miRNA biogenesis factor, Dicer1, leads to profound impairments in the progressive maturation of specified OL lineage species. Further, the introduction of specific miRNAs (e.g., miR-219 and miR-338) rescues the abnormal OL developmental phenotypes by modulating the expression of proteins with important roles in regulating the fidelity of OL maturation.

In addition, we, and others, have shown that lncRNAs play a role in establishing and maintaining neural cell identity. For example, one key study showed that lncRNAs are specifically expressed in the developing brain and preferentially associated with protein-coding genes expressed in the brain or involved in transcriptional regulation of CNS development. Moreover, we found 169 lncRNAs that are significantly differentially expressed during neuronal-glial fate specification and OL lineage maturation including those associated with key neural developmental genes, such as AK053922, a lncRNA transcribed from the Gli3 locus, and Sox8OT, a lncRNA transcribed from the Sox8 locus [41]. Others have similarly identified lncRNAs with roles in OL lineage elaboration including Nkx2.2AS, which is transcribed in an antisense orientation relative to Nkx2.2 [42]. Another study found more than 1,000 lincRNAs in various cell types, including neural precursor cells and identified a “brain cluster” of lncRNAs associated with hippocampal development, OL maturation, and GABAergic neuronal function [43].

Intriguingly, REST and CoREST regulate the expression levels of particular ncRNAs (i.e., miRNAs and lncRNAs) [32]. In turn, specific ncRNAs can modulate the expression levels of REST and CoREST [32]. Moreover, lncRNAs are implicated in mediating the function of these and other chromatin remodeling complexes, by recruiting them to their genomic sites of actions. These observations, once again, highlight the complex crosstalk that occurs between different epigenetic regulatory mechanisms that modulate neural cell fate decisions.

Editing is a key mechanism for generating diversity in the information content of ncRNA molecules (as well as protein-coding RNAs and even DNA) [44,45]. RNA editing refers to nucleoside modifications, including adenosine-to-inosine (A-to-I) and cytidine-to-uridine (C-to-U) deaminations in RNA molecules, catalyzed by the adenosine deaminase that act on RNA (ADAR) and the apolipoprotein B editing catalytic subunit (APOBEC) families of enzymes, respectively. ADARs are differentially expressed during development with ADAR1 and 2 preferentially found in the CNS and ADAR3 restricted to brain [46,47]. The targets of A-to-I RNA editing were initially thought to encompass protein-coding transcripts, particularly those involved in the establishment and maintenance of neuronal cell identity and synaptic transmission such as voltage-gated ion channels, ligand-gated receptors, and signal transduction molecules [44,48]. More recent studies have recognized that A-to-I RNA editing occurs primarily in ncRNA transcripts derived from Alu elements and in UTRs of protein-coding transcripts [49-52] and that these editing events occur at significant levels within the human brain [53].

RNA editing plays a role in establishing and maintaining neural cell identity. For example, one study showed that human ESC differentiation is generally associated with an ADAR1-mediated global decrease in the editing levels of Alu elements, and these changes are particularly significant during neural lineage restriction [54]. Further, knocking down ADAR1 in ESCs leads to the deregulation of various genes including, particularly, those that are involved in neuronal development. Another recent study found that, as human NPCs differentiate into neuronal and glial lineages, the levels of ADAR2 and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit pre-mRNA editing increase [55]. This change in editing levels is associated with a transition from AMPA receptor calcium-permeability in NPCs to impermeability in more mature cells. In turn, overexpression of ADAR2 impairs NPC differentiation.

Nuclear organization refers to the three dimensional arrangement of the genome, from entire chromosomes to individual gene loci, within the context of the nuclear envelope and matrix and other factors. The execution of cellular programs including DNA replication and repair, gene transcription, and RNA post-transcriptional processing and transport is associated with dynamic nuclear reorganization such as the formation and disassembly of functional nuclear domains [1,24,56-60]. The genome can undergo controlled local and long-range movements, more global reorganization, as well as other shifts (e.g., chromatin states) that have functional consequences for nuclear processes. Further, the nucleus contains a number of specialized sub-organelles comprised of particular proteins and RNA species, including some whose roles have been characterized and others that are less well understood. These heterogeneous nuclear domains can dynamically assemble and associate with specific genomic loci, interact with chromatin, move, and undergo other changes. These three-dimensional features of nuclear structure and function do not arise randomly. Rather, they are associated with particular physiological cues and cellular processes, such as cell-cycle progression and differentiation. Although the regulation and function of many nuclear features has not yet been elucidated, recent advances have highlighted the interconnected nature of nuclear organization and dynamics with epigenetic regulation, in general and in the CNS [61], both in health and in disease [62].

Nuclear reorganization plays a role in establishing and maintaining neural cell identity. For example, one important study showed how the expression of Mash1, a pro-neural gene, is mediated by the repositioning of the Mash1 locus from the nuclear periphery to the interior of the nucleus, which occurs during neural lineage commitment of ESCs [63]. At the nuclear periphery, the Mash1 locus is associated with profiles of histone modifications indicative of transcriptional repression, whereas in the interior of the nucleus, it is associated with profiles of histone modifications linked to transcriptional activation. By contrast, another study demonstrated that the expression of PLP, a myelin gene, is up regulated during progressive stages of OL lineage maturation, while the PLP locus remains at the nuclear periphery [64]. Interestingly, the increased transcription of PLP is associated with the local assembly of SC35 domains (a.k.a., nuclear speckles) proximal to the PLP gene locus. These nuclear domains are enriched in NEAT1/2, lncRNAs that we showed are upregulated during the progressive maturation of OLs [28], and are implicated in modulating transcription and RNA processing. Other ncRNAs have been linked unequivocally to gene regulation, nuclear organization, and OL development. The miRNA, miR-23, negatively regulates the expression of lamin B1 (LMNB1), a nuclear lamina protein with diverse roles in epigenetic phenomena including chromatin formation and euchromatin and heterochromatin transitions. The LMN1B gene is duplicated in adult-onset autosomal dominant leukodystrophy resulting in increased levels of LMNB1 that lead to defects in OL maturation and myelination accompanied by abnormalities of the nuclear envelope and myelin gene expression and localization [65,66].

Along with inherited and acquired genetic abnormalities, epigenetic mechanisms are also implicated in promoting cellular transformation via mechanisms that include silencing tumor suppressor genes and activating oncogenes; deregulation of genes involved in cell growth and proliferation, stress responses, and survival and apoptosis; loss of genomic imprinting, derepression of parasitic repetitive elements, and producing genomic instability.

Indeed, epigenetic profiling in cancer has revealed a spectrum of abnormalities. Cancer cells exhibit changes in normal patterns of DNA methylation including global hypomethylation in intergenic and heterochromatic regions, which may lead to genomic instability, reactivation of transposable elements, and loss of imprinting, and local changes in regulatory regions that can modulate specific genes, including the repression of tumor suppressor genes and the activation of oncogenes. Further, hypermethylation may lead to long term gene silencing of tumor suppressor genes by promoting the establishment of “locked” repressive chromatin states. Cancer cells also exhibit abnormal profiles of histone, nucleosome, and chromatin regulation. Like changes in DNA methylation, these patterns can be deregulated globally or more locally. In fact, global H3K4me2 and H3K18ac patterns correlate with tumor phenotype and predict prognosis, treatment response, and outcome for different cancers [67-69]. By contrast, combinations of histone modifications at particular gene loci, such as repressive PcG-mediated H3K27me3 and H3K9me2 at tumor suppressor genes, are associated with deregulation of these specific genes [70]. Various cancers are further associated with mutations and other alterations in the expression and function of epigenetic factors including, for example, DNMTs, histone and chromatin modifying enzymes, as well as ncRNAs.

Cancer cells have deregulated miRNA expression profiles that are implicated in initiating and maintaining the abnormal cellular phenotype. These miRNAs target a broad range of mRNAs including those that control cell growth and proliferation, stress responses, and survival and apoptosis as well as those encoding factors involved in reading and writing the epigenetic modifications. Cancer cells also display changes in the methylation and chromatin states associated with miRNA genes. In turn, these abnormalities can lead to deregulation of miRNAs that act as tumor suppressor genes and oncogenes through effects on their target mRNAs and those that have more global effects on genomic stability [71]. Other ncRNAs, such as lncRNAs, may similarly have roles in the molecular mechanisms underlying cancer. For example, a number of lncRNAs are associated with the establishment of cell identity, including pluripotency [72-74] and fate restriction, lineage specification and maturation; with p53-mediated cell stress responses, survival, and apoptosis [75,76]; with genomic imprinting; XCI; and with cancer metastasis [36]. Cumulatively, these signatures highlight the emerging roles played by epigenetic deregulation in promoting the cancer phenotype.

In contrast to the canonical stochastic theory that attributes the molecular pathogenesis of cancer to clonal evolution, the hierarchical CSC hypothesis suggests that tumor maintenance, progression, treatment resistance, and recurrence are largely driven by cells with stem cell-like characteristics, including the capacity to undergo self-renewal and differentiate into the potentially heterogeneous subpopulations of cells present within a particular tumor. Studies on acute myeloid leukemia supplied preliminary data supporting the CSC hypothesis [139]. Subsequent studies have provided evidence for the existence of CSCs in a broad range of solid tumors including those that manifest in the CNS, such as gliomas, medulloblastomas, neuroblastomas, and ependymomas [140,141]. Cells derived from nervous system tumors are operationally defined as CSCs if they express certain cell surface markers (i.e., CD133, CD15/SSEA-1, L1CAM, A2B5, or integrin a6) and exhibit particular functional properties (i.e., the ability to initiate and propagate tumors in various assays). Our understanding of these so-called CSCs, their in situ interactions with the tumor microenvironment, and corresponding methodologies used to identify and study them ex vivo are still evolving. Furthermore, the relevance of the CSC model to specific cancers remains the subject of ongoing controversies [142], though stochastic and hierarchical models are not mutually exclusive [143,144].

Preliminary studies of tumor initiating and propagating cells, if not CSCs, derived from nervous system tumors have demonstrated that these cells are epigenetically deregulated. In fact, the PcG protein, BMI1, is highly enriched in GBM-derived CD133+ cells and is required for sustaining stem cell self-renewal. Further, examining GBMs and associated cell lines reveals that the CD133 gene, itself, is subject to regulation by DNA methylation, though, conspicuously, this only seems to be the case in the cancer state [145]. The methylation status of the CD133 gene promoter is generally inversely correlated with the expression of this cell surface marker in GBM-derived CD133+ and CD133- cell populations [145,146]. Also, the CASP8 gene promoter is hypermethylated in GBM-derived CD133+ cells, leading to low levels of CASP8 expression in these cells [147]. Notably, this protein is required for mediating the sensitivity of the tumor to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); and not surprisingly, these cells are resistant to TRAIL-induced cell death. The BMP receptor 1B gene, which typically inhibits proliferation and promotes NSC differentiation, is also epigenetically silenced in GBM-derived tumor initiating and propagating cells. It is subject to promoter hypermethylation and is also the target of the H3K27 histone methyltransferase, enhancer of zeste 2 (EZH2), a PcG protein [148]. In addition, comparative profiling of miRNAs reveals that many miRNAs are differentially expressed between GBM-derived CD133+ and CD133- cells [149]. Specifically, miRNAs that may have roles in promoting brain development and neural cellular differentiation, such as miR-451, miR-486 and miR-425 [149] and miR-29b, miR-125a and miR-149 [150], are upregulated in CD133- cells. Moreover, the introduction of miR-124 or miR-137 inhibits proliferation and induces differentiation in GBM-derived (and oligodendroglioma-derived) tumor initiating and propagating cells [151]. Neuroblastoma- and medulloblastoma-derived tumor initiating and propagating cells are also subject to epigenetic regulation, though, these processes are less well characterized than those operating in GBM-derived tumor initiating and propagating cells. For example, the CD133 gene is similarly subject to regulation by DNA methylation in neuroblastoma cell lines [152]. And, in medulloblastoma, the introduction of miR-199b-5p depletes the CD133+ population of cells and reduces the expression of genes linked to the stem cell state, such as HES1 [153].

Given the recognition of the increasingly roles of epigenetic mechanisms in mediating all aspects of neural cellular development, these observations suggest that epigenetic deregulation is a critical factor for determining tumor initiating and propagating cell functions.

By definition, CSCs have the ability to undergo self-renewal and to differentiate into potentially heterogeneous subpopulations of cells that comprise an individual tumor. CSCs are, thus, responsible for maintaining and propagating tumors. However, our understanding of the processes by which CSCs are generated and maintained in nervous system (and other) tumors that conform to the CSC model remains limited. Many studies have focused on identifying the cells of origin—cells that acquire genetic or other lesions and are truly responsible for initiating nervous system tumors in situ [154]. Indeed, it has been shown that GBMs, medulloblastomas, neuroblastomas, and other nervous system tumors can arise from cells of origin including neural stem and progenitor cells that escape from normal proliferation and differentiation programs and even from more mature cell types that undergo de-differentiation. For example, both NSCs or cerebellar granule neuronal precursor cells can form medulloblastomas [155], and mature macroglial cells, such as astrocytes, can form gliomas. These cells of origin are distinct from CSCs, but they may be closely related to and are either directly of indirectly responsible for generating CSCs [154]. Because epigenetic mechanisms are implicated in mediating neural cell identity and function and also because they are deregulated in cancer generally and in populations of tumor initiating and propagating cells derived from nervous system tumors, epigenetic deregulation is likely to be a very important pathogenic mechanism in the cancer cell of origin and in the establishment and maintenance of the CSC state [156].

Indeed, emerging evidence has demonstrated that epigenetic mechanisms play roles in mediating all of the major acquired abilities that are the hallmarks of cancer and the characteristics that enable them (recently reviewed by Hanahan and Weinberg [157]). These properties include the capacity to sustain proliferative signaling, evade growth suppressors, resist cell death, achieve replicative immortality, induce angiogenesis, activate invasion and metastasis, reprogram energy metabolism and evade immune destruction and are underpinned by genome instability and inflammation and through bidirectional interactions with the tumor microenvironment. Here we provide leading examples of how diverse epigenetic mechanisms are involved in the molecular circuitry underlying all of these pathological processes. Proliferative signaling pathways include those that are mediated by growth factors and related developmental cues. REST, an epigenetic regulatory factor deregulated in nervous system tumors, modulates Shh signaling [158], and in turn, the activity of REST is modulated by the Wnt signaling pathway [159,160]. Growth suppressors, such as the pRb and p53 tumor suppressors, themselves, participate in epigenetic pathways. For example, the mechanism of action of pRb includes modulation of local and more global chromatin environments by recruitment of enzymes that modify histones, remodel nucleosomes, and promote the formation of heterochromatin [161]. Similarly, p53 binds to its response element, where it recruits coregulatory factors such as histone modifying enzymes, chromatin remodeling factors, and subunits of the mediator complex. This stimulus-specific p53 activity is also important for sensing cell stress and promoting apoptosis. Replicative immortality is mediated by telomeres, which are, by definition, chromosomal structures. The organization of telomeric chromatin influences telomere maintenance, and telomere dysfunction affects telomeric and subtelomeric chromatin formation and activates alternative telomere maintenance mechanisms [162]. Further, treatment of nervous system tumor cells with HDAC inhibitors has been shown to have antiproliferative and proapoptotic effects, mediated by alterations in chromatin at the telomerase reverse transcriptase (TERT) promoter [163]. Moreover, telomeres are transcribed into ncRNAs, telomere repeat-containing RNA (TERRA), that may have roles in modulating telomeric heterochromatin formation, the accessibility of telomeres to DNA repair mechanisms, and the activity of telomerase [164]. Angiogenesis is regulated by the actions of various pro- and anti-angiogenic factors including many, such as VEGF, with expression levels that are modulated by DNA methylation, histone modifications, and microRNAs [165]. In turn, VEGF influences expression of the PcG protein, EZH2, in the tumor vasculature. EZH2 promotes tumor angiogenesis by repressing the anti-angiogenic factor, Vash1 [166]. Invasion and metastasis are also mediated by epigenetic factors such as ncRNAs. The expression of certain miRNAs can correlate positively or negatively with the invasive and metastatic phenotype of cancer. For example, miR-10b is a miRNA that promotes invasion and metastasis in gliomas [167]. In contrast, miR-124a is generally downregulated in GBMs, and ectopic expression of miR-124a in GBM cells inhibits tumor invasion and migration [168]. In addition, expression of certain lncRNAs is also correlated with the phenotype of particular cancers. Higher levels of HOTAIR, a lincRNA transcribed from the HOX locus, are implicated in promoting metastasis [36] and recurrence [169] and indicate a poor clinical prognosis for particular cancers. Many aspects of energy metabolism are also modulated by epigenetic factors. In fact, HDACs play a pivotal role in glucose homeostasis by regulating Forkhead box O transcription factors, which have critical roles in modulating metabolism [170,171]. The immune response and its evasion by cancer cells are also mediated by epigenetic factors, including HDACs. For example, HDACs are implicated in transcriptional regulation of both pro- and anti-inflammatory cytokines, in immune cell differentiation, in immunological signaling pathways (i.e., STAT and NF-κB) and inflammatory responses, in forming the antigen-presenting cell/T-cell immunological synapse, and specifically in modulating the immune response to cancer [172].

Tumor microenvironments (or niches) include the extracellular matrix (ECM) and various stromal, endothelial, and other cell types that create the local milieu permitting tumor initiation and subsequently supporting tumor maintenance and progression. The bidirectional communication that exists between CSCs and tumor microenvironments located in perivascular and, putatively, in hypoxic and invasive edge regions is modulated, at least in part, by epigenetic factors. For example, GBM CSCs secrete VEGF that promotes endothelial cell growth [173], and we have already noted how VEGF signaling is under epigenetic control. In addition, Notch signaling maintains CSCs [174], and many epigenetic factors such as histone modifying enzymes and corepressor complexes and associated epigenetic marks are important for regulating Notch target gene expression [175]. Furthermore, it is likely that many other bidirectional communications are mediated by epigenetic factors. Indeed, integrins and ECM interactions are important for CSC maintenance [176], and emerging evidence suggests an epigenetic level of control for these factors, mediated particularly by REST and CoREST [16-18,177]. Diffusible cerebrospinal fluid-borne signals that impact NSC proliferation, including specifically those derived from patients with GBMs, are partly dependent on insulin-like growth factor 2 (IGF2) levels [178], and IGF2 is regulated by intricate epigenetic mechanisms including imprinting and lncRNAs [179,180]. The release of exosomes containing regulatory and functional ncRNAs, including those derived from transposable elements, also represents an intriguing mechanism for bidirectional intercellular communication in the tumor microenvironment, and more general evidence already exists for such neural-endothelial cell communication promoting an angiogenic phenotype [96,181].