**2. Epigentic mechanisms**

#### **2.1. Post-translational modifications of histones**

Post-translational chemical modifications of histones in chromatin are diverse and include methylation, acetylation, phosphorylation, sumoylation, ubiquitilation, and ADP-ribosyla‐ tion [25, 27]. Here, we will focus on histone methylation and acetylation because these modi‐ fications are considered to be most important and widespread for influencing biological processes during neural differentiation.

Histone methylation occurs mainly at lysine and arginine residues on the tails of the histone H3 and H4 [28]. Histone methylation is reversible. Methylation at these sites has been asso‐ ciated with not only transcriptional activation and repression [27, 29] but also multiple bio‐ logical processes, including heterochromatin formation and genomic imprinting [27, 28]. The differences in the effects of methylation depend on the lysine residues. For example, methylation of histone H3 lysine 9 (H3K9), H3K27, or H4K20 is generally linked to forma‐ tion of gene silencing [30, 31], whereas methylation on H3K4, H3K36, and H3K79 is associat‐ ed with actively transcribed regions and gene activation [29, 32, 33]. In addition, lysine residues can be mono-(me1), di-(me2), or tri-methylated (me3). These differentially methy‐ lated lysine residues lead to different levels of transcriptional activation or repression, re‐ sulting in diverse functional outcomes. For example, H4K20me1 plays an important role in transcriptional repression and X inactivation, while H4K20me2 and H4K20me3 are linked to DNA repair of double-stranded DNA damage [34].

All core histones can be dynamically acetylated on lysine residues in their tails and occa‐ sionally within the globular core. Histone acetylation removes the positive charge on the histones, thereby weakening the interaction with negatively charged DNA [25, 29]. As a con‐ sequence, chromatin is transformed into a more relaxed structure, which is associated with transcriptional activation. Like methylation, histone acetylation is reversible. This reversible acetylation in histones is an important mechanism of controlling gene expression because histone acetylation and deacetylation are linked to transcriptional activation and inactiva‐ tion, respectively [25, 27].

#### **2.2. DNA methylation**

cleosomes [21, 24, 25]. In addition to epigenetic modifications and chromatin remodeling, epigenetic regulators have been recently extended to non-coding RNAs (ncRNAs), because ncRNAs can affect chromatin structure and transcriptional activation by regulating expres‐ sion of key nucleosome modifiers [26]. These epigenetic controls appear to influence gene expression profiles, which are essential for self-renewal and differentiation capacities of ESCs. Thus, a clear understanding of the epigenetic mechanisms underlying gene expres‐ sion patterns will provide significant and novel insights into cell fate specification of ESCs directed to differentiate into neurons. Furthermore, the epigenetic mechanisms are believed to be capable of responding to extrinsic signals such as morphogens and cytokines [8]. Therefore, knowledge of the epigenetic mechanisms is also important for our understanding of neural differentiation by extrinsic factors. In this review, we will describe the major epige‐ netic processes that underlie the acquisition of the NPC fate from ESCs as well as the subse‐ quent neuronal subtype specification. The focus of this review is weighted on neuronal cell

Post-translational chemical modifications of histones in chromatin are diverse and include methylation, acetylation, phosphorylation, sumoylation, ubiquitilation, and ADP-ribosyla‐ tion [25, 27]. Here, we will focus on histone methylation and acetylation because these modi‐ fications are considered to be most important and widespread for influencing biological

Histone methylation occurs mainly at lysine and arginine residues on the tails of the histone H3 and H4 [28]. Histone methylation is reversible. Methylation at these sites has been asso‐ ciated with not only transcriptional activation and repression [27, 29] but also multiple bio‐ logical processes, including heterochromatin formation and genomic imprinting [27, 28]. The differences in the effects of methylation depend on the lysine residues. For example, methylation of histone H3 lysine 9 (H3K9), H3K27, or H4K20 is generally linked to forma‐ tion of gene silencing [30, 31], whereas methylation on H3K4, H3K36, and H3K79 is associat‐ ed with actively transcribed regions and gene activation [29, 32, 33]. In addition, lysine residues can be mono-(me1), di-(me2), or tri-methylated (me3). These differentially methy‐ lated lysine residues lead to different levels of transcriptional activation or repression, re‐ sulting in diverse functional outcomes. For example, H4K20me1 plays an important role in transcriptional repression and X inactivation, while H4K20me2 and H4K20me3 are linked to

All core histones can be dynamically acetylated on lysine residues in their tails and occa‐ sionally within the globular core. Histone acetylation removes the positive charge on the histones, thereby weakening the interaction with negatively charged DNA [25, 29]. As a con‐ sequence, chromatin is transformed into a more relaxed structure, which is associated with transcriptional activation. Like methylation, histone acetylation is reversible. This reversible

lineage specification, and not on glial cell specification.

306 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**2.1. Post-translational modifications of histones**

DNA repair of double-stranded DNA damage [34].

processes during neural differentiation.

**2. Epigentic mechanisms**

DNA methylation is one of the major repressive epigenetic pathways. Methylation occurs at the cytosine residues followed by a guanine (CpG dinucleotides) in the DNA sequence. CpG DNA methylation of gene promoters is a well-known hallmark for transcriptionally inactive genes, and is generally associated with stable gene silencing, such as genomic imprinting and X chromosome inactivation [35, 36]. The DNA methylation state is established during embryogenesis by several DNA methyltransferases (DNMTs) [35]. In mammals, 2 types of DNMTs have been identified. DNMT3A and DNMT3B establish *de novo* DNA methylation, while DNMT1 maintains DNA methylation patterns during DNA replication [37]. These DNA methylation sites then recruit methyl-CpG-binding proteins, including methyl-CpGbinding domain (MBD) proteins [38, 39], which bind the histone deacetylase (HDAC)-con‐ taining repressor complex, and consequently repress transcription [40, 41].

#### **2.3. Chromatin remodeling**

Chromatin structure is not static, but subject to change in response to internal and external developmental signals [23]. Dynamic changes in the chromatin structure are regulated by ATP-dependent chromatin remodelers, which allow the transcriptional machinery to access its targets more or less effectively [42-44]. Using energy derived from ATP hydrolysis, ATPdependent chromatin remodelers relocate nucleosomes either by mobilizing or restructuring nucleosomes [45, 46]. Thus, ATP-dependent chromatin remodelers can function both in tran‐ scriptional activation and repression via their nucleosome remodeling activity. Nearly all ATP-dependent chromatin remodelers are multi-protein complexes that contain an ATPase subunit, which belongs to the sucrose non-fermenting 2 (SNF2) family of ATPases. Based on the homology between their ATPase domains, ATP-dependent chromatin remodeling com‐ plexes are divided into 4 groups: switch/sucrose non-fermenting (SWI/SNF), imitation switch (ISWI), chromo helicase DNA binding (CHD), and inositol auxotroph 80 (INO80) [42-44].

Genetic and biochemical studies indicate that some ATP-dependent chromatin remodeling complexes contain epigenetic factors such as HDAC and MBD proteins. For example, nucle‐ osome-remodeling deacetylase (NuRD) is a multi-subunit complex that includes a SWI2/ SNF2 helicase/ATPase domain-containing Mi2 protein, HDAC1, HDAC2, and MBD3 [47]. The NuRD complexes promote the establishment of a specific chromatin structure at rRNA genes that are transcriptionally inactive but are poised for transcriptional activation and control transcription of these genes [48]. Thus, ATP-dependent chromatin complexes play essential roles in epigenetic regulation of transcription along with several histone-modifying enzymes and/or modified histone codes.

#### **2.4. Non-coding RNAs**

Apart from the role of histone modifications and DNA methylation, another form of epige‐ netic regulation involves non-coding RNAs (ncRNAs). A large variety of ncRNAs can be classified into 2 major classes based on their transcript size: small ncRNAs (less than 200 nu‐ cleotides) and long ncRNAs (greater than 200 nucleotides) [49]. Each of these classes can be further divided into subclasses. Micro RNA (miRNA) is a subgroup of small ncRNA mole‐ cules ~22 nucleotides in length. miRNAs post-transcriptionally regulate gene expression by binding to complimentary or uncomplimentary sequences on target mRNA transcripts, which results in either mRNA degradation or inhibition of translation [50]. In animal cells, miRNA genes tend to be clustered in the genome, and are widely distributed [51]. Approxi‐ mately half of the human miRNA genes are located in the introns of protein-coding genes. Expression studies have revealed that the clustered miRNA genes are often co-expressed, suggesting that they are jointly transcribed as a polycistron [52, 53].

mediated by PcG and TrxG proteins plays a key epigenetic role in maintenance of the undif‐

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**Figure 1. Schematic diagram of transcriptional states during neuronal fate acquisition from ESCs.** In ESCs, pluri‐ potency genes are transcriptionally activated (green "ON"). Key developmental genes are transcriptionally silent, yet competent for expression (orange "OFF"). This silencing ensures rapid reactions to extracellular inductive signals. Upon neural lineage choice, the transcriptional states of various developmental genes are altered. Pluripotency genes and genes associated with other lineages become repressed during the transition from ESCs to neurogenic NPCs (red "OFF"). Such a state of transcriptional repression is maintained over a long period. Gene activation and repression cor‐ relates with the presence of H3K4 tri-methylation (green flags) and H3K27 tri-methylation (red flags). DNA methyla‐

Genome-wide mapping by chromatin immunoprecipitation has revealed that differentiation of ESCs is generally accompanied by global changes in histone methylation [55, 56]. In the course of neural lineage commitment, the promoters of many neural lineage genes have been shown to lose H3K27me3 from the bivalent domain, retain H3K4me3, and become acti‐ vated. At the same time, the promoter loci of non-neural lineage genes maintain H3K27me3 in the bivalent domain, while removing H3K4me3, which results in a stable silent state. The promoters of some pluripotency genes such as *SOX2*, *POU5F1*, and *NANOG* shift from modification by H3K4me3 alone to neither H3K4 nor H3K27 methylation as they are re‐ pressed during differentiation. As ESCs differentiate into NPCs, H3K27 specific demethy‐

tion (shocking pink stars) contributes to repression in combination with H3K27 tri-methylation.

ferentiated state and suppress neural differentiation.

### **3. Transition from ESCS to NPCS**

#### **3.1. Histone modifications**

Expression of pluripotency genes, such as *OCT4* or *NANOG*, is a hallmark of undifferentiated ESCs. The promoters and/or enhancers of these pluripotency genes are marked by H3K4me3, which is strongly associated with transcriptionally active genes. In contrast, the majority of genes, whose upregulation leads to differentiation, are inactivated or expressed at very low levels [54, 55]. These genes loci are maintained in a transcriptionally competent but inactive state characterized by both active (H3-K4me3) and repressive (H3-K27me3) histone marks, a configuration described as a ''bivalent domain'' (Figure 1) [54, 55]. Bivalent histone methyla‐ tion in the promoters of proneural genes, such as Neurogenins (*Ngns*), *Pax6*, and *Mash1*, has been reported in undifferentiated ESCs [56]. H3K27 and H3K4 methylation is catalyzed by Polycomb-group (PcG) and Trithorax-group (TrxG) proteins, respectively [57]. PcG proteins form a complex referred to as the Polycomb Repressor Complex (PRC). PRCs can be biochem‐ ically subdivided into 2 groups: PRC1 and PRC2. PRC1 and PRC2 are essential for the repres‐ sion of key developmental genes and maintenance of pluripotency in ESCs [58, 59]. The PRC2 complexes, which contain Enhancer of Zeste Homolog (EZH), a histone methyltransferase (HMTase), catalyze tri-methylation of H3K27 [60]. This histone mark leads to the recruitment of PRC1, thereby contributing to a repressive chromatin state [61, 62]. Consistent with this, ESCs deficient in a PRC2 component display de-repression of tissue-specific genes, including neural-associated genes [59]. TrxG proteins also act as large multimeric complexes [57]. TrxG complexes possess methyltransferase activity directed specifically towards H3K4, thereby leading to increased levels of H3K4me3 [57]. *Dpy-30* is a mammalian homolog of the *Drosophi‐ la* TrxG protein, and a core component of myeloid/lymphoid or mixed-lineage leukemia (MLL) histone methyltransferase complexes. Depletion of *Dpy-30* in ESCs results in a defect in their neural lineage specification through reduced H3K4 methylation at bivalent domains of key developmental loci [63]. Collectively, these findings suggest that the bivalent domain mediated by PcG and TrxG proteins plays a key epigenetic role in maintenance of the undif‐ ferentiated state and suppress neural differentiation.

**2.4. Non-coding RNAs**

308 Trends in Cell Signaling Pathways in Neuronal Fate Decision

Apart from the role of histone modifications and DNA methylation, another form of epige‐ netic regulation involves non-coding RNAs (ncRNAs). A large variety of ncRNAs can be classified into 2 major classes based on their transcript size: small ncRNAs (less than 200 nu‐ cleotides) and long ncRNAs (greater than 200 nucleotides) [49]. Each of these classes can be further divided into subclasses. Micro RNA (miRNA) is a subgroup of small ncRNA mole‐ cules ~22 nucleotides in length. miRNAs post-transcriptionally regulate gene expression by binding to complimentary or uncomplimentary sequences on target mRNA transcripts, which results in either mRNA degradation or inhibition of translation [50]. In animal cells, miRNA genes tend to be clustered in the genome, and are widely distributed [51]. Approxi‐ mately half of the human miRNA genes are located in the introns of protein-coding genes. Expression studies have revealed that the clustered miRNA genes are often co-expressed,

Expression of pluripotency genes, such as *OCT4* or *NANOG*, is a hallmark of undifferentiated ESCs. The promoters and/or enhancers of these pluripotency genes are marked by H3K4me3, which is strongly associated with transcriptionally active genes. In contrast, the majority of genes, whose upregulation leads to differentiation, are inactivated or expressed at very low levels [54, 55]. These genes loci are maintained in a transcriptionally competent but inactive state characterized by both active (H3-K4me3) and repressive (H3-K27me3) histone marks, a configuration described as a ''bivalent domain'' (Figure 1) [54, 55]. Bivalent histone methyla‐ tion in the promoters of proneural genes, such as Neurogenins (*Ngns*), *Pax6*, and *Mash1*, has been reported in undifferentiated ESCs [56]. H3K27 and H3K4 methylation is catalyzed by Polycomb-group (PcG) and Trithorax-group (TrxG) proteins, respectively [57]. PcG proteins form a complex referred to as the Polycomb Repressor Complex (PRC). PRCs can be biochem‐ ically subdivided into 2 groups: PRC1 and PRC2. PRC1 and PRC2 are essential for the repres‐ sion of key developmental genes and maintenance of pluripotency in ESCs [58, 59]. The PRC2 complexes, which contain Enhancer of Zeste Homolog (EZH), a histone methyltransferase (HMTase), catalyze tri-methylation of H3K27 [60]. This histone mark leads to the recruitment of PRC1, thereby contributing to a repressive chromatin state [61, 62]. Consistent with this, ESCs deficient in a PRC2 component display de-repression of tissue-specific genes, including neural-associated genes [59]. TrxG proteins also act as large multimeric complexes [57]. TrxG complexes possess methyltransferase activity directed specifically towards H3K4, thereby leading to increased levels of H3K4me3 [57]. *Dpy-30* is a mammalian homolog of the *Drosophi‐ la* TrxG protein, and a core component of myeloid/lymphoid or mixed-lineage leukemia (MLL) histone methyltransferase complexes. Depletion of *Dpy-30* in ESCs results in a defect in their neural lineage specification through reduced H3K4 methylation at bivalent domains of key developmental loci [63]. Collectively, these findings suggest that the bivalent domain

suggesting that they are jointly transcribed as a polycistron [52, 53].

**3. Transition from ESCS to NPCS**

**3.1. Histone modifications**

**Figure 1. Schematic diagram of transcriptional states during neuronal fate acquisition from ESCs.** In ESCs, pluri‐ potency genes are transcriptionally activated (green "ON"). Key developmental genes are transcriptionally silent, yet competent for expression (orange "OFF"). This silencing ensures rapid reactions to extracellular inductive signals. Upon neural lineage choice, the transcriptional states of various developmental genes are altered. Pluripotency genes and genes associated with other lineages become repressed during the transition from ESCs to neurogenic NPCs (red "OFF"). Such a state of transcriptional repression is maintained over a long period. Gene activation and repression cor‐ relates with the presence of H3K4 tri-methylation (green flags) and H3K27 tri-methylation (red flags). DNA methyla‐ tion (shocking pink stars) contributes to repression in combination with H3K27 tri-methylation.

Genome-wide mapping by chromatin immunoprecipitation has revealed that differentiation of ESCs is generally accompanied by global changes in histone methylation [55, 56]. In the course of neural lineage commitment, the promoters of many neural lineage genes have been shown to lose H3K27me3 from the bivalent domain, retain H3K4me3, and become acti‐ vated. At the same time, the promoter loci of non-neural lineage genes maintain H3K27me3 in the bivalent domain, while removing H3K4me3, which results in a stable silent state. The promoters of some pluripotency genes such as *SOX2*, *POU5F1*, and *NANOG* shift from modification by H3K4me3 alone to neither H3K4 nor H3K27 methylation as they are re‐ pressed during differentiation. As ESCs differentiate into NPCs, H3K27 specific demethy‐ lase JMJD3 is recruited to and resolves the bivalent domain at the promoters of neuronspecific genes such as nestin [64]. The majority of the JMJD3 target genes are key inducers of neurogenesis, including Pax6 and Sox1. Knockdown of the H3K4me2/3 demethylase *JAR‐ ID1B* results in upregulation of stem cell-specific genes in ESC-derived NPCs [65]. Further‐ more, *JARID1B* knockdown ESCs fail to progress beyond the NPC stage [65]. These results suggest that JARID1B promotes ESCs to differentiate towards a neural lineage by silencing genes associated with pluripotency. Thus, when ESCs are committed to a neural lineage, bi‐ valent domains appear to be resolved in a lineage-specific fashion by leaving methyl marks either being activated or repressed. This mechanism is believed to allow rapid transcription of developmental genes in response to a variety of extrinsic cues.

neural lineage commitment and loss of pluripotency than with terminal neural differentia‐ tion. The promoters of highly expressed housekeeping and pluripotency genes in ESCs ex‐ hibit low methylation levels [75]. By contrast, most key developmental and tissue-specific genes exhibit high methylation levels, and are transcriptionally repressed [75]. As ESCs un‐ dergo differentiation, significant changes in DNA methylation patterns are observed. *De no‐ vo* methylation occurs on the promoter regions of pluripotency-associated factors [74].

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DNA methylation potentially accompanies histone modifications during neural differentia‐ tion from ESCs. Cross-referencing DNA methylation patterns with mapping of histone H3K27me3 in ESCs and ESC-derived neurons has revealed that, upon differentiation, the re‐ gions marked by H3K27me3 acquire DNA methylation in a sequence-independent manner [74, 75]. H3K27me3 and DNA methylation are compatible throughout most of the genome [76]. Furthermore, deficiency of *DNMT* in ESCs causes widespread H3K27me3 genomic changes [76]. Taken together, these data suggest that DNA methylation, in cooperation with histone modifications, may function as a protective gear by repressing pluripotency and oth‐

In addition to histone modifications and DNA methylation, the ATP-dependent chromatin remodeling complexes also play a pivotal role in the maintenance of ESC pluripotency. As previously described, Tip60-p400 chromatin remodeling complexes appear to be necessary for the maintenance of ESCs, including pluripotency [71]. Subunits of the NuRD complexes have also been shown to be important for ESC pluripotency and differentiation [77, 78]. Fur‐ thermore, ESCs contain another specialized ATP-dependent chromatin remodeling complex: the Brahma-associated factor (BAF) complex. The BAF complexes are characterized by 2 SWI2/SNF2-like ATPases, BRG1 and BRM. The BAF complexes in ESCs have a unique subu‐ nit composition (termed esBAF) that is not seen in other tissues, such as NPCs and post-mi‐ totic neurons [79]. This specialized subunit composition is essential for establishment and maintenance of ESCs. The esBAF complexes contain BRG1 but not BRM, and BAF155 but not BAF170 (Figure 2). As ESCs differentiate into NPCs, these complexes undergo several subunit exchanges. The esBAF complexes incorporate BRM and excludes BAF60B, thereby forming the neural progenitor-specific BAF (npBAF) complexes in NPCs. The npBAF com‐

plex-specific subunit is necessary and sufficient for amplification of NPCs [80].

Increasing evidence demonstrates contributions of specific miRNAs in establishing ESC properties and their transitioning to NPCs. The miR-290-295 cluster codes for miRNAs are the most abundant in mouse ESCs and constitute over 70% of their entire miRNA popula‐ tion [81]. Consistent with their high expression levels, miR-290-295 miRNAs are involved in many functions in ESCs. For example, miR-290-295 miRNAs promote the transition from mitosis to S phase by targeting G1/S transition inhibitors such as Cdkn1a [82]. Furthermore, miR-290-295 miRNAs have been shown to target the *Rbl2* gene, which controls the expres‐ sion of the DNA methyltransferases, DNMT3A and DNMT3B, thereby establishing *de novo*

er lineage-specific genes during differentiation.

**3.3. Chromatin remodeling**

**3.4. Non-coding RNAs**

Other repressive histone marks have also been reported to play important roles during dif‐ ferentiation. A sustained increase in silent chromatin marked by H3K9 methylation is ob‐ served in ESC-derived cells undergoing differentiation [66], suggesting that repressive histone marks H3K9me2/3 is essential for promoting differentiation. Additionally, it is pos‐ sible that H3K9me2/3 marks play an important role in the establishment of an expression profile of neuron specific genes in response to extracellular signals [67]. Ciliary neurotrophic factor (CNTF), an astrocyte differentiation factor, is incapable of inducing expression of glial fibrillary acidic protein (*GFAP*), an astrocytes-specific marker gene, in NPCs, because the *GFAP* promoter is marked by H3K9 methylation. However, fibroblast growth factor 2 (FGF2) confer NPCs with responsiveness to CNTF by adding active H3K4 methylation and removing H3K9 methylation at the GFAP promoter. Thus, H3K9 methylation controls the timing of astrogliogenesis through regulation of CNTF-mediated signaling.

In addition to histone methylation, histone acetylation also appears to be an important epi‐ genetic modification during neural differentiation from ESCs, as ESCs generally undergo striking changes in the global pattern of histone acetylation during neural differentiation [68]. Histone acetylation is catalyzed by histone acetyltransferase (HAT), while histone de‐ acetylation is catalyzed by histone deacetylase (HDAC) [69]. Generally, HATs induce the transcriptional activation of their target genes. However, the 60-kDa HIV-Tat interactive protein (Tip60) histone acetyltransferase has been implicated in both transcriptional repres‐ sion and activation [70]. Tip60-p400 chromatin remodeling complexes containing Tip60 are necessary to maintain characteristic features of ESCs [71]. Tip60-p400 complexes acetylate histone H4 on the promoters of both activated and repressed genes. Additionally, distribu‐ tion patterns of p400 in ESCs strongly correlate with H3K4me3 marks on the promoters of both active and inactive genes. These results suggest that Tip60-p400 complex-mediated his‐ tone acetylation functions as an active mark in the bivalent domain together with the active H3K4me3 mark in ESCs.

#### **3.2. DNA methylation**

Genome-wide mapping of DNA methylation patterns has revealed dynamic DNA methyla‐ tion states at gene promoters during ESC differentiation (Figure 1) [72, 73]. The most pro‐ nounced changes in the DNA methylation state occur during neural lineage commitment [74], suggesting that alterations in the DNA methylation state more strongly correlate with neural lineage commitment and loss of pluripotency than with terminal neural differentia‐ tion. The promoters of highly expressed housekeeping and pluripotency genes in ESCs ex‐ hibit low methylation levels [75]. By contrast, most key developmental and tissue-specific genes exhibit high methylation levels, and are transcriptionally repressed [75]. As ESCs un‐ dergo differentiation, significant changes in DNA methylation patterns are observed. *De no‐ vo* methylation occurs on the promoter regions of pluripotency-associated factors [74].

DNA methylation potentially accompanies histone modifications during neural differentia‐ tion from ESCs. Cross-referencing DNA methylation patterns with mapping of histone H3K27me3 in ESCs and ESC-derived neurons has revealed that, upon differentiation, the re‐ gions marked by H3K27me3 acquire DNA methylation in a sequence-independent manner [74, 75]. H3K27me3 and DNA methylation are compatible throughout most of the genome [76]. Furthermore, deficiency of *DNMT* in ESCs causes widespread H3K27me3 genomic changes [76]. Taken together, these data suggest that DNA methylation, in cooperation with histone modifications, may function as a protective gear by repressing pluripotency and oth‐ er lineage-specific genes during differentiation.

#### **3.3. Chromatin remodeling**

lase JMJD3 is recruited to and resolves the bivalent domain at the promoters of neuronspecific genes such as nestin [64]. The majority of the JMJD3 target genes are key inducers of neurogenesis, including Pax6 and Sox1. Knockdown of the H3K4me2/3 demethylase *JAR‐ ID1B* results in upregulation of stem cell-specific genes in ESC-derived NPCs [65]. Further‐ more, *JARID1B* knockdown ESCs fail to progress beyond the NPC stage [65]. These results suggest that JARID1B promotes ESCs to differentiate towards a neural lineage by silencing genes associated with pluripotency. Thus, when ESCs are committed to a neural lineage, bi‐ valent domains appear to be resolved in a lineage-specific fashion by leaving methyl marks either being activated or repressed. This mechanism is believed to allow rapid transcription

Other repressive histone marks have also been reported to play important roles during dif‐ ferentiation. A sustained increase in silent chromatin marked by H3K9 methylation is ob‐ served in ESC-derived cells undergoing differentiation [66], suggesting that repressive histone marks H3K9me2/3 is essential for promoting differentiation. Additionally, it is pos‐ sible that H3K9me2/3 marks play an important role in the establishment of an expression profile of neuron specific genes in response to extracellular signals [67]. Ciliary neurotrophic factor (CNTF), an astrocyte differentiation factor, is incapable of inducing expression of glial fibrillary acidic protein (*GFAP*), an astrocytes-specific marker gene, in NPCs, because the *GFAP* promoter is marked by H3K9 methylation. However, fibroblast growth factor 2 (FGF2) confer NPCs with responsiveness to CNTF by adding active H3K4 methylation and removing H3K9 methylation at the GFAP promoter. Thus, H3K9 methylation controls the

In addition to histone methylation, histone acetylation also appears to be an important epi‐ genetic modification during neural differentiation from ESCs, as ESCs generally undergo striking changes in the global pattern of histone acetylation during neural differentiation [68]. Histone acetylation is catalyzed by histone acetyltransferase (HAT), while histone de‐ acetylation is catalyzed by histone deacetylase (HDAC) [69]. Generally, HATs induce the transcriptional activation of their target genes. However, the 60-kDa HIV-Tat interactive protein (Tip60) histone acetyltransferase has been implicated in both transcriptional repres‐ sion and activation [70]. Tip60-p400 chromatin remodeling complexes containing Tip60 are necessary to maintain characteristic features of ESCs [71]. Tip60-p400 complexes acetylate histone H4 on the promoters of both activated and repressed genes. Additionally, distribu‐ tion patterns of p400 in ESCs strongly correlate with H3K4me3 marks on the promoters of both active and inactive genes. These results suggest that Tip60-p400 complex-mediated his‐ tone acetylation functions as an active mark in the bivalent domain together with the active

Genome-wide mapping of DNA methylation patterns has revealed dynamic DNA methyla‐ tion states at gene promoters during ESC differentiation (Figure 1) [72, 73]. The most pro‐ nounced changes in the DNA methylation state occur during neural lineage commitment [74], suggesting that alterations in the DNA methylation state more strongly correlate with

of developmental genes in response to a variety of extrinsic cues.

310 Trends in Cell Signaling Pathways in Neuronal Fate Decision

timing of astrogliogenesis through regulation of CNTF-mediated signaling.

H3K4me3 mark in ESCs.

**3.2. DNA methylation**

In addition to histone modifications and DNA methylation, the ATP-dependent chromatin remodeling complexes also play a pivotal role in the maintenance of ESC pluripotency. As previously described, Tip60-p400 chromatin remodeling complexes appear to be necessary for the maintenance of ESCs, including pluripotency [71]. Subunits of the NuRD complexes have also been shown to be important for ESC pluripotency and differentiation [77, 78]. Fur‐ thermore, ESCs contain another specialized ATP-dependent chromatin remodeling complex: the Brahma-associated factor (BAF) complex. The BAF complexes are characterized by 2 SWI2/SNF2-like ATPases, BRG1 and BRM. The BAF complexes in ESCs have a unique subu‐ nit composition (termed esBAF) that is not seen in other tissues, such as NPCs and post-mi‐ totic neurons [79]. This specialized subunit composition is essential for establishment and maintenance of ESCs. The esBAF complexes contain BRG1 but not BRM, and BAF155 but not BAF170 (Figure 2). As ESCs differentiate into NPCs, these complexes undergo several subunit exchanges. The esBAF complexes incorporate BRM and excludes BAF60B, thereby forming the neural progenitor-specific BAF (npBAF) complexes in NPCs. The npBAF com‐ plex-specific subunit is necessary and sufficient for amplification of NPCs [80].

#### **3.4. Non-coding RNAs**

Increasing evidence demonstrates contributions of specific miRNAs in establishing ESC properties and their transitioning to NPCs. The miR-290-295 cluster codes for miRNAs are the most abundant in mouse ESCs and constitute over 70% of their entire miRNA popula‐ tion [81]. Consistent with their high expression levels, miR-290-295 miRNAs are involved in many functions in ESCs. For example, miR-290-295 miRNAs promote the transition from mitosis to S phase by targeting G1/S transition inhibitors such as Cdkn1a [82]. Furthermore, miR-290-295 miRNAs have been shown to target the *Rbl2* gene, which controls the expres‐ sion of the DNA methyltransferases, DNMT3A and DNMT3B, thereby establishing *de novo* DNA methylation in ESCs [83]. miR-9 is gradually upregulated during neural differentiation from ESCs [84, 85]. Furthermore, miR-9 performs diverse functions in different aspects of neuronal differentiation [86]. For example, during neural differentiation of human ESCs, miR-9 expression is not detectable in embryoid bodies, but is turned on in NPCs [84]. Inhibi‐ tion of miR-9 activity has been found to suppress proliferation and simultaneously promote migration of NPCs [84]. Collectively, miRNAs could regulate multiple developmental proc‐ esses at the post-transcriptional level.

promotes neurogenesis and inhibits astrocytic differentiation [87]. During the transition from neurogenic NPCs to astrogenic NPCs, the level of repressive H3K27me3 mark at the promoter of the *Ngn1* gene gradually increases, leading to gene silencing [88]. Therefore, ac‐ tivation of the Wnt signaling does not lead to transactivation of *Ngn1* at later developmental stages. This illustrates how histone methylation can modulate responsiveness of NPCs to ex‐ tracellular cues, thereby rendering NPCs to switch from neurogenesis to astrogenesis.

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CREB-binding protein (CBP), one of the most extensively studied HAT proteins, has been found to play an essential role in motor neuron differentiation by interacting with retinoic acid (RA) signaling [89]. Retinoic acid receptors (RARs) are DNA-binding proteins and form a complex with Neurogenin2 (Ngn2) on the promoter of a motor neuron enhancer gene. Binding of RA to RARs triggers recruitment of CBP on the promoter, which leads to acetyla‐ tion of core histone proteins and activation of the motor neuron enhancer gene. These re‐ sults indicate that neuronal subtype specification is regulated, at least in part, by the

Histone deacetylation by HDACs also plays essential roles in neuronal differentiation, as evidenced by studies using histone deacetylase inhibitors, such as valproic (VP) and trichos‐ tatin A (TSA) [90-92]. VPA promotes neuronal differentiation from adult hippocampal NPCs by inducing the proneural genes, *Ngn1*, *Math1*, and *NeuroD*, and histone H4 acetylation, while inhibiting glial differentiation [92]. A combination of TSA with sonic hedgehog (Shh), fibroblast growth factor 8 (FGF8) and Wnt instructs non-mesencephalic NPCs to give rise to dopaminergic neurons [93]. Inhibiting the activity of all HDAC1, 2, and 3 in NPCs leads to suppression of oligodendrocyte differentiation, while HDAC2 activity alone inhibits astro‐ cyte differentiation. On the other hand, the HDAC1 activity is required for neural differen‐ tiation [94]. HDACs are generally present within large multi-subunit protein complexes in the nucleus [69]. Among them in association with neuronal differentiation is the HDAC/ CoREST/REST repressor complex. The repressor element 1-silencing transcription factor (REST) is a vertebrate zinc finger transcriptional repressor protein, which plays a fundamen‐ tal role in neurogenesis [95-97]. REST is expressed in both ESCs and NSCs, but is not ex‐ pressed in ES-derived neurons [98]. REST binds to an evolutionally conserved DNA motif known as the repressor element 1 (RE1) [96, 97]. REST represses the expression of RE1-con‐ taining neuronal genes via recruitment of HDAC/CoREST complexes containing HDAC1 and HDAC 2 [97]. Thus, RE1 motif-associated neuronal genes in ESCs and NPCs appear to be suppressed by REST. This repression could block premature expression of genes associat‐ ed with terminal differentiation at earlier stages than needed. Recently, a genome-wide binding site analysis revealed the target genes of REST during cholinergic, GABAergic, glu‐ tamatergic, and medium spiny projection neuronal specification from NPCs [99]. A large number of the identified REST target genes are unique for each neuronal subtype, strongly suggesting that histone deacetylase plays essential roles in epigenetic control of neuronal

DNA methylation has been shown to suppress astrocyte-specific genes in NPCs during ear‐ ly stages of development. Promoters of many astrocyte-specific genes contain the signal

interplay between intrinsic epigenetic mechanisms and extrinsic cues.

subtype specification as well as neuronal lineage commitment.

**4.2. DNA methylation**

**Figure 2. A switch in subunit composition of the BAF complexes during neuronal differentiation from ESCs.** The exchange of the components is essential for the transition from ESCs to post-mitotic neurons. The exchangeable subu‐ nits are colored as follows: esBAF complex-specific subunit, ocher; npBAF complex-specific subunit, red; and nBAF complex-specific subunit, yellow. Cell type-specific BAF complexes have distinct functions that are indispensable for their properties. The BAF complex in ESCs, NPCs, and neurons are defined as esBAF, npBAF, and nBAF, respectively. The microRNA miR-124, binds to *BAF53A* mRNA transcripts to suppress its expression, thereby facilitating the replace‐ ment of BAF53 in the npBAF complexes.

## **4. Transition from NPCs to neurons**

#### **4.1. Histone modifications**

After being committed to a neural lineage, NPCs exit the cell cycle and sequentially undergo neural and glial differentiation. Wnt signaling, which plays important roles in the mainte‐ nance of embryonic and adult stem cells, promotes differentiation of neurogenic ESCs to neurons, but does not promote differentiation of astrogenic NPCs into neurons [8]. In neuro‐ genic NPCs, one of the target genes for Wnt signaling is the proneural gene *Ngn1*, which promotes neurogenesis and inhibits astrocytic differentiation [87]. During the transition from neurogenic NPCs to astrogenic NPCs, the level of repressive H3K27me3 mark at the promoter of the *Ngn1* gene gradually increases, leading to gene silencing [88]. Therefore, ac‐ tivation of the Wnt signaling does not lead to transactivation of *Ngn1* at later developmental stages. This illustrates how histone methylation can modulate responsiveness of NPCs to ex‐ tracellular cues, thereby rendering NPCs to switch from neurogenesis to astrogenesis.

CREB-binding protein (CBP), one of the most extensively studied HAT proteins, has been found to play an essential role in motor neuron differentiation by interacting with retinoic acid (RA) signaling [89]. Retinoic acid receptors (RARs) are DNA-binding proteins and form a complex with Neurogenin2 (Ngn2) on the promoter of a motor neuron enhancer gene. Binding of RA to RARs triggers recruitment of CBP on the promoter, which leads to acetyla‐ tion of core histone proteins and activation of the motor neuron enhancer gene. These re‐ sults indicate that neuronal subtype specification is regulated, at least in part, by the interplay between intrinsic epigenetic mechanisms and extrinsic cues.

Histone deacetylation by HDACs also plays essential roles in neuronal differentiation, as evidenced by studies using histone deacetylase inhibitors, such as valproic (VP) and trichos‐ tatin A (TSA) [90-92]. VPA promotes neuronal differentiation from adult hippocampal NPCs by inducing the proneural genes, *Ngn1*, *Math1*, and *NeuroD*, and histone H4 acetylation, while inhibiting glial differentiation [92]. A combination of TSA with sonic hedgehog (Shh), fibroblast growth factor 8 (FGF8) and Wnt instructs non-mesencephalic NPCs to give rise to dopaminergic neurons [93]. Inhibiting the activity of all HDAC1, 2, and 3 in NPCs leads to suppression of oligodendrocyte differentiation, while HDAC2 activity alone inhibits astro‐ cyte differentiation. On the other hand, the HDAC1 activity is required for neural differen‐ tiation [94]. HDACs are generally present within large multi-subunit protein complexes in the nucleus [69]. Among them in association with neuronal differentiation is the HDAC/ CoREST/REST repressor complex. The repressor element 1-silencing transcription factor (REST) is a vertebrate zinc finger transcriptional repressor protein, which plays a fundamen‐ tal role in neurogenesis [95-97]. REST is expressed in both ESCs and NSCs, but is not ex‐ pressed in ES-derived neurons [98]. REST binds to an evolutionally conserved DNA motif known as the repressor element 1 (RE1) [96, 97]. REST represses the expression of RE1-con‐ taining neuronal genes via recruitment of HDAC/CoREST complexes containing HDAC1 and HDAC 2 [97]. Thus, RE1 motif-associated neuronal genes in ESCs and NPCs appear to be suppressed by REST. This repression could block premature expression of genes associat‐ ed with terminal differentiation at earlier stages than needed. Recently, a genome-wide binding site analysis revealed the target genes of REST during cholinergic, GABAergic, glu‐ tamatergic, and medium spiny projection neuronal specification from NPCs [99]. A large number of the identified REST target genes are unique for each neuronal subtype, strongly suggesting that histone deacetylase plays essential roles in epigenetic control of neuronal subtype specification as well as neuronal lineage commitment.

#### **4.2. DNA methylation**

DNA methylation in ESCs [83]. miR-9 is gradually upregulated during neural differentiation from ESCs [84, 85]. Furthermore, miR-9 performs diverse functions in different aspects of neuronal differentiation [86]. For example, during neural differentiation of human ESCs, miR-9 expression is not detectable in embryoid bodies, but is turned on in NPCs [84]. Inhibi‐ tion of miR-9 activity has been found to suppress proliferation and simultaneously promote migration of NPCs [84]. Collectively, miRNAs could regulate multiple developmental proc‐

**Figure 2. A switch in subunit composition of the BAF complexes during neuronal differentiation from ESCs.** The exchange of the components is essential for the transition from ESCs to post-mitotic neurons. The exchangeable subu‐ nits are colored as follows: esBAF complex-specific subunit, ocher; npBAF complex-specific subunit, red; and nBAF complex-specific subunit, yellow. Cell type-specific BAF complexes have distinct functions that are indispensable for their properties. The BAF complex in ESCs, NPCs, and neurons are defined as esBAF, npBAF, and nBAF, respectively. The microRNA miR-124, binds to *BAF53A* mRNA transcripts to suppress its expression, thereby facilitating the replace‐

After being committed to a neural lineage, NPCs exit the cell cycle and sequentially undergo neural and glial differentiation. Wnt signaling, which plays important roles in the mainte‐ nance of embryonic and adult stem cells, promotes differentiation of neurogenic ESCs to neurons, but does not promote differentiation of astrogenic NPCs into neurons [8]. In neuro‐ genic NPCs, one of the target genes for Wnt signaling is the proneural gene *Ngn1*, which

esses at the post-transcriptional level.

312 Trends in Cell Signaling Pathways in Neuronal Fate Decision

ment of BAF53 in the npBAF complexes.

**4.1. Histone modifications**

**4. Transition from NPCs to neurons**

DNA methylation has been shown to suppress astrocyte-specific genes in NPCs during ear‐ ly stages of development. Promoters of many astrocyte-specific genes contain the signal transducer and activator of transcription (STAT) binding elements. With this, astrocyte-spe‐ cific genes are transcriptionally activated through the Janus kinase (JAK)-STAT pathway, one of whose ligands is the cytokine leukemia inhibitory factor (LIF). However, neurogenic NPCs are not competent to differentiate into astrocytes even when they are grown with LIF, because the STAT-binding elements within astrocyte-specific genes promoters are methylat‐ ed [100, 101]. This DNA methylation inhibits the association of activated STATs with the promoter of astrocyte-specific genes, thereby repressing their transcription. Conditional de‐ letion of *DNMT1* in embryonic NPCs results in DNA hypomethylation and alteration of the timing of astrocytogenesis [101]. In addition, knockdown of *DNMT3B* in ESCs alters the tim‐ ing of their neural differentiation [102]. These findings suggest that DNA methylation con‐ trols the timing and developmental switch from neurogenesis to astrogliogenesis of NPCs by altering responsiveness to their extracellular developmental cues.

miR-9 has been shown to negatively regulate NPC proliferation and accelerate differentia‐ tion through targeting *TLX* transcription [111]. These findings suggest that miR-9 can switch from undifferentiated to differentiated state of NPCs by downregulating *TLX* expression. Another miRNA, miR-124, has been reported to promote neuronal differentiation by target‐ ing several genes involved in selection of non-neuronal cell fates. For example, miR-124 reg‐ ulates the BAF complex subunit composition during differentiation from NPCs to neurons (Figure 2) by binding and thus suppressing *BAF53A* mRNA transcripts, which allows facili‐ tation of a switch between BAF53 subunits [112]. These studies indicate that miRNA expres‐ sion should be strictly controlled to ensure proper differentiation of ESCs into neurons. Consistent with this assumption, miR-124 is expressed in neurons, but not in astrocytes [113], and the miR-124 level increases during neural differentiation [84]. Remarkably, the control of miR-124 expression itself is mediated by an epigenetic mechanism. The promoter of the *miR-124* gene contains a functional RE1 site. In ESCs and NPCs, REST occupies the miR-124 gene locus and represses its expression [114], allowing persistent expression of nonneuronal mRNAs. However, once NPCs start to differentiate into neurons, REST is downre‐ gulated, thereby disinhibiting miR-124 expression. miR-124 then triggers degradation of non-neuronal mRNA transcripts, which promotes differentiation towards a neuronal line‐ age. One of the known target genes for miR-124 is C-terminal domain phosphatase 1 (*SCP1*), which represses transcription of RE1-containing neuronal genes by REST, thereby prevent‐ ing cells from adopting a neuronal lineage and producing non-neural tissues [115]. Togeth‐ er, *miR-124* is a target for REST, but at the same token also targets the REST co-repressor. This represents the presence of a negative feedback loop between miRNA and a REST si‐ lencing complex, and such a mechanism may be broadly used to ensure proper cell fate

Epigenetic Regulation of Neural Differentiation from Embryonic Stem Cells

http://dx.doi.org/10.5772/53650

315

Neurons derived from different human ESC lines exhibit distinctive cellular properties due to the fact that human ESC lines were established under diverse conditions and from em‐ bryos with different genetic backgrounds. Comparison of neurons derived from HSF1 and HSF6 ESC human lines has revealed that the HSF1 line produces forebrain neurons with GABAergic and dopaminergic neurotransmitter phenotypes, while HSF6-derived neurons produce midbrain/hindbrain neurons bearing dopaminergic, cholinergic, serotonergic, nonforebrain GABAergic, and glutamatergic phenotypes [116]. Significant differences in the miRNA expression profile was noted between these 2 human ESC lines [116], suggesting that miRNA expression patterns might dictate in defining various neuronal subtypes arisen

Epigenetic mechanisms are regulatory processes that control gene expression via changes in chromatin structure without alterations in the DNA sequence. Changes in chromatin struc‐ ture alter the accessibility of transcription factors and RNA polymerase to genes packed into chromatin, thereby modulating the efficiency of gene transcription. Epigenetic mechanisms act to control this accessibility through histone modifications, DNA methylation, chromatin

transitions during development.

from ESCs.

**5. Conclusions**

#### **4.3. Chromatin remodeling**

As NPCs exit the cell cycle and differentiate into mature neurons, BAF60C is incorporated into the npBAF complexes [80, 103]. BAF45A and BAF53A in the complexes give way to BAF45B/C and BAF53B, respectively (Figure 2) [80, 103], establishing the post-mitotic neu‐ ron-specific nBAF complexes. Preventing the exchange of npBAF and nBAF components im‐ pairs neuronal differentiation, indicating that a switch in subunit composition of the BAF complexes is required for the transition from pluripotent ESCs to post-mitotic neurons [80, 103]. The nBAF complexes, along with Ca2+-responsive dendritic regulator CREST, also play a role in regulating the activation of genes essential for activity-dependent dendritic out‐ growth, suggesting that the nBAF complexes are required for morphological/synaptic devel‐ opment of neurons [103, 104].

The BAF complexes incorporate the BAF57 subunit containing DNA-binding HMG-box do‐ mains [105]. In addition, the BAF complex subunits contain motifs known to bind to modi‐ fied histones, including chromo-, bromo-, and PHD domains [103]. The bromodomain can bind acetylated histones [106]. The chromo- and PHD domains function as lysine-methylat‐ ed histone-binding domains [106]. The esBAF and npBAF complexes contain different chro‐ modomain proteins (BAF155 or BAF170) [103], whereas the npBAF and nBAF complexes contain different PHD domain proteins (BAF45a or BAF45b) [80, 103]. Thus, changes in sub‐ unit composition could alter targets of the BAF complexes, thereby causing changes in gene expression patterns during neuronal differentiation.

#### **4.4. Non-coding RNAs**

A number of miRNAs involved in cell fate decision during stem cell differentiation is also highly expressed in the nervous systems. Among these is miR-9, which is expressed specifi‐ cally in neurogenic areas of the embryonic and adult brains [107, 108]. TLX, an orphan nu‐ clear receptor, is essential for maintaining a self-renewable and undifferentiated state [109], as well as cell cycle progression [110] of NPCs in the developing brain. TLX is highly ex‐ pressed in NPCs, but its expression is down-regulated upon neural differentiation [111]. Conversely, miR-9 expression increases during neural differentiation [111]. Furthermore, miR-9 has been shown to negatively regulate NPC proliferation and accelerate differentia‐ tion through targeting *TLX* transcription [111]. These findings suggest that miR-9 can switch from undifferentiated to differentiated state of NPCs by downregulating *TLX* expression. Another miRNA, miR-124, has been reported to promote neuronal differentiation by target‐ ing several genes involved in selection of non-neuronal cell fates. For example, miR-124 reg‐ ulates the BAF complex subunit composition during differentiation from NPCs to neurons (Figure 2) by binding and thus suppressing *BAF53A* mRNA transcripts, which allows facili‐ tation of a switch between BAF53 subunits [112]. These studies indicate that miRNA expres‐ sion should be strictly controlled to ensure proper differentiation of ESCs into neurons. Consistent with this assumption, miR-124 is expressed in neurons, but not in astrocytes [113], and the miR-124 level increases during neural differentiation [84]. Remarkably, the control of miR-124 expression itself is mediated by an epigenetic mechanism. The promoter of the *miR-124* gene contains a functional RE1 site. In ESCs and NPCs, REST occupies the miR-124 gene locus and represses its expression [114], allowing persistent expression of nonneuronal mRNAs. However, once NPCs start to differentiate into neurons, REST is downre‐ gulated, thereby disinhibiting miR-124 expression. miR-124 then triggers degradation of non-neuronal mRNA transcripts, which promotes differentiation towards a neuronal line‐ age. One of the known target genes for miR-124 is C-terminal domain phosphatase 1 (*SCP1*), which represses transcription of RE1-containing neuronal genes by REST, thereby prevent‐ ing cells from adopting a neuronal lineage and producing non-neural tissues [115]. Togeth‐ er, *miR-124* is a target for REST, but at the same token also targets the REST co-repressor. This represents the presence of a negative feedback loop between miRNA and a REST si‐ lencing complex, and such a mechanism may be broadly used to ensure proper cell fate transitions during development.

Neurons derived from different human ESC lines exhibit distinctive cellular properties due to the fact that human ESC lines were established under diverse conditions and from em‐ bryos with different genetic backgrounds. Comparison of neurons derived from HSF1 and HSF6 ESC human lines has revealed that the HSF1 line produces forebrain neurons with GABAergic and dopaminergic neurotransmitter phenotypes, while HSF6-derived neurons produce midbrain/hindbrain neurons bearing dopaminergic, cholinergic, serotonergic, nonforebrain GABAergic, and glutamatergic phenotypes [116]. Significant differences in the miRNA expression profile was noted between these 2 human ESC lines [116], suggesting that miRNA expression patterns might dictate in defining various neuronal subtypes arisen from ESCs.
