Utrecht University
From stem cells to neurons: epigenetic mechanisms governing
neurogenesis
Master Thesis
Corina Maria Markodimitraki
Student Number 3349241
Under the supervision of Prof. Michiel Vermeulen
Radboud University Nijmegen
Utrecht, June 2014
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Abbreviations
PTM
Post translational modification
DNMT
DNA methyltransferase
HP1
Heterochromatin protein 1
mC
Methyl cytosine
hmC
Hydroxymethyl cytosine
ESC
Embryonic stem cell
hESC
Human embryonic stem cell
mESC
Mouse embryonic stem cell
NSC
Neural stem cell
NPC
Neural progenitor cell
NuRD
Nucleosome remodeling and histone deacetylation
CHD1
Chromodomain-helicase-DNA-binding protein 1
MLL
Mixed lineage leukemia
RARE
Retinoic acid response elements
REST
Repressor element 1 (RE1) silencing transcription factor
TSS
Transcription start sites
BDNF
Brain-derived neurotrophic factor
UHRF2
Ubiquitin-like with PHD and rind finger domains 2
ZHX1/2
Transcriptional repressors zinc fingers and homeoboxes ½
VZ
Ventricular zone
SVZ
Sub ventricular zone
NGN
Neurogenin
PAX6
Paired box gene 6
SOX1
SRY-Box 1
SOX3
SRY-Box 3
MASH1
Achaete-scute complex homolog 1 or Ascl1
NKX2.2
NK2 transcription factor related locus 2
REST
RE1 silencing transcription factor
PRC1
Polycomb-recruiting complex 1
PRC2
Polycomb-recruiting complex 2
TET
Ten-eleven translocation
TGF-β
Transforming Growth Factor-β
1. Introduction
The development of one of the most important systems of the human body, the nervous system, is
definitely a very complicated and less understood process. The nervous system contains a wide range of
different cell types, all of which originated from the ectoderm of the early embryo during development.
The neural stem cells or else called neural progenitor cells are controlled by both intrinsic and extrinsic
signals in order to produce a stunning diversity of neural cells at the right time and place. The fate of
each stem cell can be defined among others, by the epigenetic landscape of its DNA. Epigenetics control
gene expression from the early stages of development. From the embryonic stem cell stage, to the
neural stem cell stage to the terminally differentiated neuron. In this review, we will take a closer look at
the complicated and precise epigenetic mechanisms governing neural development.
2. Epigenetics
Epigenetic mechanisms establish mitotically heritable changes in gene expression potential without
modifying the DNA sequence. The process of marking the chromatin is achieved by proteins that read,
write and erase these marks, both on the DNA itself (5mC, 5hmC) but also on the histone tails of the
nucleosomes. This way, the chromatin can be either active, inactive or bimodal, balancing in between
expression and repression [1]. All the different epigenetic mechanisms don’t act independently but can
often be seen to work cooperatively with other epigenetic mechanisms underlining the complexity of
gene expression and repression pathways and development. The result of one such cooperating event
are the poised promoters, very often seen during development. They possess both inhibitory and
activating marks resulting in a bivalent chromatin mark that prepares the cell for upcoming gene
activation while at the same time preventing aggressive differentiation. Upon differentiation one of the
histone markers composing the bivalent mark will be enhanced by either the Trithorax or the Polycomb
complex.
The term epigenetics can be defined by either one of the following mechanisms:
2.1 Histone post translational modifications (PTMs)
The dynamic N tails of the histones can be modified by the covalent binding of a small chemical
compound (for example an acetyl group) whose addition is catalyzed by histone acetyltransferases
(HATs) and removed by histone deacetylases (HDACs) and takes place on multiple lysine residues. Other
possible modifications are phosphorylation, methylation, ubiquitination, sumoylation, ADP-ribosylation,
proline isomerization, citrullination, butyrylation, propionylation and glycosylation. Depending on the
modification and the cross-talk between multiple PTMs, their outcome on the chromatin state can be
different. A modification that corresponds with open chromatin state is acetylation. It adds a positive
charge to the lysine molecule: H3K9ac, H3K14ac and H4K16ac [2]. Another PTM that causes chromatin
de-condensation is the phosphorylation of serine residues, for example H3S10p. On the other hand
there are modifications whose outcome differs depending on the degree of modification and the
residue. Examples are the H3K4me3 gene associated with activation, the H3K9me3 which marks
heterochromatin and the H3K27me3 linked to facultative heterochromatin [2].
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2.2 DNA methylation
The methylation of a 5’cytosine next to a guanine (CpG dinucleotides) is an epigenetic modification of
the DNA, hallmarking transcriptional repression and is established by the DNMT family of DNA
methyltransferases. While DNMT1 is a maintenance enzyme, preferentially methylating hemimethylated
DNA during replication, DNMT3a and DNMT3b are de novo methyltransferases important in
development. Of all the CpGs found in the genome, 60-80% are methylated and 5mCs are more often
found in CpG-dense regions called CpG islands (CGIs) [3]. Although 50-60% of CGIs are located in
promoter regions, methylation also occurs in intergenic regions and gene bodies. This suggests distalpromoter methylation is important and plays a role in tissue-specific gene expression [4, 5].
As the role of the DNMT enzymes in neural development is concerned, the DNMT3b protein is important
for neural development in early NPCs but its levels go down during development. On the other hand,
DNMT3a’s protein levels are low in early development but later increase and it continues being
expressed during adulthood in the SVZ and the hippocampal dentate gyrus [6]. For mice lacking the
DNMT3a protein, postnatal neurogenesis is severely misregulated [6].
The establishment of mC by itself causes a cascade of chromatin-remodeling events, with the binding of
methyl CpG-binding proteins that will either recruit chromatin remodeling complexes or block
transcription factors from binding to their target sequences and initiating transcription [7]. Such methyl
CpG-binding proteins are the MBD (methyl-CpG binding domain) family, the Kaiso family, the Kaiso-like
family and the SRA domain proteins. The binding of MBD proteins for example prevents initiation of
transcription by either recruiting chromatin remodeling complexes like HDACs or blocking the binding of
transcription factors. The MBD family member MeCP2 in particular is associated with the maturation of
the central nervous system (CNS) and stimulates neuronal development and dendrogenesis.
2.3 DNA hydroxymethylation
DNA hydroxymethylation is another epigenetic mark directly coating the DNA. It results from Tet
enzymes converting mC to 5-hydroxymethylcytosine (hmC). This modification is particularly abundant in
the brain and in embryonic stem cells [8]. During mammalian development active demethylation
converts methyl groups to hydroxymethylgroups by ten-eleven translocation (Tet) proteins TET1 and
TET2, forming a unique distribution pattern [9, 10]. The hydroxymethylgroups can in turn be removed by
enzymes AID and APOBEC1 resulting in deamination [11]. This all happens in primordial germ cells
(PGCs), and hydroxymethylation levels have been reported to be higher in pluripotent stem cells
contributing to the pluripotency of the cells, as well as in multipotent stem cells and the CNS, namely in
the Purkinje cells of the cerebellum [2].
2.4 Regulatory ncRNAs
Only 3% of the transcribed genome codes for proteins. However, more than three quarters of the
genome is transcribed giving rise to non-coding RNAs (ncRNAs) which won’t be translated into protein
[12, 13]. The ncRNAs can be sorted into long and short, taking a length threshold of 200 nucleotides into
account: they can range from 21 (mature miRNAs) to 100000 (AIR ncRNA) nucleotides. These non-coding
transcripts can specifically guide enzymatic activities to targets thus repressing gene expression.
Different ncRNAs have been found to be highly expressed in the nervous system: microRNAs (miRNAs),
long non coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) [6].
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MiRNAs are single strands of 18-24 nucleotides. They post-transcriptionally repress by binding to specific
mRNAs with anti-sense sequence homology resulting in degradation. miRNAs can also recruit chromatin
remodeling complexes to the DNA and thus alter the expression pattern of the chromatin. They have
also been shown to help establishing de novo DNA methylation in mouse ESC [2]. Stem cell proliferation
and differentiation are also in part regulated by miRNAs [2]. One miRNA can regulate more than one
gene due to imperfect base-pairs with their targets.
Some lncRNAs, namely the large intergenic noncoding RNAs (lincRNAs) can also change the expression
of specific loci by recruiting chromatin modifying complexes [14]. The first lincRNA to be found
influencing the expression of a locus on another chromosome was the lincRNA HOTAIR that binds the
PRC2 complex and controls the expression of the HOXD locus during development [15].
2.5 Reader, writer and eraser proteins
Epigenetic marks on the DNA and histones can be altered by writer, reader and eraser proteins, altering
the epigenetic landscape and thus the expression pattern of genes. An example of a reader protein is
the Polycomb Repressive Complex 2 (PRC2) which trimethylates H3K27, leading to PRC1 recruitment and
a sequential cascade of gene silencing [6]. Especially during neurogenesis starting from undifferentiated
ESCs, the gene targeting of the PcG protein family is dynamic.
An example PRC2 regulation is the X chromosome inactivation in the female embryo after fertilization,
when the lncRNA XIST represses one of the two copies of the X chromosome in mammalian females.
This is achieved by the coating of the to-be silenced X chromosome by the XIST transcript and the
recruitment of the PRC2 complex alongside with the addition of the repressive mark H3K27me3, H3 and
H4 hypoacetylation and methylation of CpG-rich promoters [16].
2.6 Epigenetic cross-talk
Histone PTMs and mC can interact, creating pivotal crosstalk such as the overlap of mC and H3K9me3.
Histone PTMs can also crosstalk with each other resulting in a poised state of chromatin where
expression can be either repressed or promoted by the recruitment of different factors. The poised state
is especially important during development where the modifications in a bimodal state can follow
different pathways and will lead to specification faster [11]. Example of epigenetic crosstalk is H3K4
methylation that is strongly enriched at unmethylated active promoters, but also the histone variant
H2A.Z whose presence anti-correlates with the DNA methylation mark during lymphomagenesis [17].
2.7 Histone variants
Histone variants can alter the chromatin state structurally and functionally. Some histone variant
examples are H3.1 and H2A.1 which are expressed during replication in S phase, H2AZ and H3.3 which
are expressed throughout the whole cell cycle [2].
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3. Epigenetics in ESCs
3.1 Open chromatin
The epigenetic landscape differs widely between ESCs and differentiated cells [18]. According to
biochemical and imaging studies the chromatin in ESCs has an open structure, which is characterized by
active H3 and H4 acetylation and H4K36me2 and H3K4me3 marks whereas heterochromatin
characterized by H3K9me3 is limited [19]. This serves the purpose of the cell having a full potential to
differentiate into any cell type. During the differentiation process the open chromatin marks are
reduced and repressive marks increase, leading to lineage-committed gene expression [2].
The architectural proteins heterochromatin protein 1 (HP1) and the linker histone H1 are dynamic in
ESCs, resulting in an open chromatin state. As soon as the differentiation process starts, the
abovementioned proteins will bind the DNA in a hyperdynamic manner, causing the chromatin to adopt
a more closed state [20, 21]. which gets more compact as the cells commit to a cellular lineage fate and
start expressing lineage-specific genes [22]. H1 exchange rate is regulated by the chromodomainhelicase-DNA-binding protein 1 (CHD1) and when it is depleted, the number of differentiated cells that
are produced increases showing that open chromatin is a characteristic of stem cells [22].
Another finding that confirms the open chromatin state of ESCs, is the high transcription rates of ESCs
which undergo high-scale silencing during differentiation [21]. A proposed model for similar regulation
in NPCs includes decondensation of chromatin at regions with neural specific genes and glial specific
which allows expression and thus promotes neurogenesis. Later toward the gliogenic phase, the HMGA
protein levels would decrease resulting in loss of neurogenic potential and increase in gliogenic potential
[22].
The biggest changes in methylation levels occur during development when CpGs in promoters of
housekeeping or developmental genes are kept constitutively hypomethylated. These methylation
changes can determine the transition between developmental stages [3].
3.2 Global demethylation
After fertilization, epigenetic reprogramming occurs in the form of complete erasure of the methylation
patterns followed by de novo methylation by the DNMT enzymes. First the paternal nucleus undergoes
fast demethylation as maternally derived histones replace the “condensing” proteins, the protamines.
Soon after, the maternal nucleus also demethylates with a result of both nuclei having the same levels
of methylation at the 4-cell stage [23]. The CpGs of promoters of housekeeping or developmental genes
are kept constitutively hypomethylated. All these methylation changes can determine the transition
between developmental stages [3].
The re-establishment of de novo methylation is crucial for development. This is demonstrated by the
fact that mESCs (mouse embryonic stem cells) have a tolerance to global demethylation when
maintenance and de novo DNMTs are depleted, having no effect on pluripotency. However as soon as
differentiation has to take place in Dnmt (3a-/-, 3b-/-) ES cells, it is completely inhibited, confirming that
DNA methylation promotes differentiation [24].
However thorough the initial demethylation might be, it has been shown that there is a small
percentage of the genome that retains its initial methylation. Specific genomic regions like LINEs (Long
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Interspersed Elements), minor satellites and imprints are more prone to escape silencing than for
example intracisternal A-type particles [25]. LINE elements are also hydroxymethylated in ESCs [26, 27].
3.3 Other kinds of DNA methylation
Although the primary sites of DNA methylation are CpGs, methylation of CpAs is common in ESCs and
shows high de novo methylase activity by DNMT3L [28-30]. However, non-CpG methylation gradually
disappears as the cells undergo differentiation. Co-occurring1 with non-CpG methylation are high levels
of CpG methylation, both on global levels [31].
“Poised” regions in ESCs and genes activated during differentiation often have a high coverage of 5hmC,
which leads to the assumption that hydroxymethylation primes regions for transcription [2].
3.4 Pluripotency factors
On a global level in ESCs, pluripotency factor binding (e.g. OCT4 and NANOG) and hypomethylation
characterizes nucleosome depleted regions-which can be found at transcription start sites and
enhancers-linked to cell-type-specific regulation [32]. What’s more, the pluripotency factor Oct4
controls the expression of H3K9me2/3 demethylases thus sustaining the epigenetic landscape [2]. These
key transcription factors as well as TAF3, E-cadherin, KLF4 and PBX1 also regulate the expression of
target genes maintaining pluripotency.
Pluripotency factors have also been shown to regulate non-coding RNAs. Some lncRNAs are regulated by
the pluripotent transcription factors Oct4 and Nanog and are involved in regulation of lineage-specific
gene expression contributing to mESCs pluripotency [33].
3.5 Histone PTMs, readers and writers
3.5.1 Histone acetylation
The balance between proliferation and differentiation lies in the fine tuning of the acetylation mark at
promoters, regulated by the HATs and HDACs [19]. An example are the H3K9ac levels that are higher in
ESCs than in differentiated cells and promote expression of pluripotency genes [34]. Genome-wide
analysis of HDAC1 occupancy in mESCs showed HDAC1 sometimes binding with the MBD3 protein -a
subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex- at actively
transcribed genes, including genes involved in self-renewal like Nanog, Sox, Oct4 and Klf4 [35]. What’s
more, inhibition of HDACs class I and II with trichostatin A (TSA) in mESCs, promotes lineage-specific
gene expression and down-regulates pluripotency genes thus stimulating differentiation [35, 36].
Together these results suggest that HDACs limit the expression levels of lineage-specific genes and help
maintain the proliferating and pluripotent state of ESCs. In hESCs, high levels of the class III HDAC family
member SirT1, coincides with deacetylated H3K9 residues at promoters of repressed genes. Such genes
are DLL4, TBX3, and PAX6 and get activated as soon as SirT1 protein levels decrease [37]. SirT1 which is
known to sense redox cellular stress and respond to it, also represses pluripotency genes Oct4 and
Nanog through a possible indirect mechanism [37].
3.5.2 Histone methylation
Histone methylation is established by the SET1/MLL histone methyltransferases, which form complexes
with catalytic subunits. Arginine and lysine residues can be methylated in a variety of ways, leading to a
variety of outcomes on gene expression. An example are the bivalent domains that are formed when
histones are marked with both H3K27me3 and H3K4me3, marking gene promoters, as shown in mESCs
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[38]. It has been proposed that bivalent domains serve the purpose of keeping developmental genes
under repression in ESC state until differentiation has to initiate quickly [38]. What’s more, it’s shown
from the oscillated epigenetic state of genes that DNA and H3K9me3 cooperate in ESCs concurrently
[39]. In mESCs the Wdr5 core subunit maintains H3K4me3 levels and associates with the OCT4
transcriptional factor establishing the H3K4me3 mark on pluripotency-related genes, preserving the
pluripotency condition [40]. H3K4me3 regulation is complex as indicated by contradicting results: while
the deletion of one core subunit (Wdr5) in ESCs marks the loss of pluripotency, the knockout of another
(Dpy-30) prolongs the pluripotency state and considerably changes the differentiation potential of ESCs,
especially along the neural lineage [40, 41]. Although the role of the methyltransferases is controversial
in ESCs, the deletion of the components of the Polycomb Repressive Complex (PRC2) -which establishes
the H3K27 methylation mark- leads to impaired lineage commitment upon external signals in mESCs
[42].
3.5.3 Bivalent marks
ESC are characterized by the simultaneous presence of both active and inactive histone marks on
developmental genes, such as the Hox clusters called, “bivalent marks”. Depending on the direction of
the differentiation these marks would either loose or gain impact by silencing or activating the gene in
question as the cell permanently leaves its’ pluripotency properties behind [2]. In ESC cells poised
promoters can be found with both the H3K27me3 inhibitory mark and the H3K4ac activation mark
which can have different outcomes depending on the path the cell is going to take.
3.5.4 Readers/Writers
One of the most important protein complexes involved in epigenetic control is the Polycomb repressive
complex 2 (PRC2). One of its many roles is to inhibit neuronal genes in ESCs. What’s more, the
Polycomb-like 3 (Pcl3) protein component of the PRC2 complex is involved in ESC proliferation by
facilitating the binding of PRC2 to CpG islands and promoting H3K27me3 [43].
Early in differentiation the germ-line specific genes are repressed, possibly by continuous methylation at
downstream sequences of transcriptional repressors [44]. The E2F6 factor is a member of the E2F family
whose members play a role in cellular proliferation. Forming a complex with PRC2, E2F6 represses
promoters leading to tissue-specific gene expression [45]. E2F6 can also recruit DNMT3b directly and
silence germ-line genes in murine somatic tissue during early mouse development [46]. The knockouts
of E2F6 and DNMT3b deregulate a comparable group of genes whose promoters in somatic cells lack
silencing epigenetic marks like H3K9me [47].
On the other hand, the transcriptionally activating Trithorax protein group (TrxG) counteracts the
Polycomb complexes’ repressive marks with its’ component protein mixed lineage leukemia (MLL) that
establishes the H3K4me3 mark [2].
3.5.5 Transcription factors and other proteins
Homeobox (Hox) genes play a vital role in the development of the ectoderm of the blastocyst into the
CNS, as different waves of signaling Hox transcription factors define the time, place and type of
differentiation that neural stem cells (NSC) will undergo. This complex signaling is regulated by retinoic
acid (RA) whose effect is mediated by the retinoic acid response elements (RARE) [2].
Another factor keeping ESCs under control by specifically inhibiting premature neuronal differentiation
is the repressor element 1 (RE1) silencing transcription factor (REST) or else known as neuron restrictive
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silencing factor (NRSF). It silences neuronal genes not only in ESCs but also in NSCs and non-neuronal
tissues. Silencing is achieved by recruiting the HDAC complexes, PRC1, PRC2, coREST and mSin3 which
will modify chromatin in a repressive way [2]. REST also seems to control microRNAs mir-9, mir-124a
and mir-132 [48].
Other proteins modifying the chromatin state are the HMGA1 and HMGA2 proteins of the HMG
subfamily that bind to AT enriched DNA regions, can bind to transcription factors and their expression
levels are high early in development [22]. They have been found to bind and regulate the IMP2 gene
whose protein product helps maintain mRNAs and which can partially rescue neurogenic potential in
absence of HMGA [22].
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4. Epigenetics during cell line commitment (from ESCs to NPC/NSC)
Neural progenitor cells (NPCs) or neural stem cells (NSCs) are lineage committed cells destined to
differentiate into astrocytes, neurons or oligodendrocytes or any other neural cell type. The two terms
are often used interchangeably, however some studies place NSCs at an earlier developmental time
point with more differentiating and self-renewing potential than NPCs [2]. NSCs first undergo the
neurogenic state during which various types of neurons are produced and subsequently the astrogenic
phase follows during which glia (astrocytes) are being produced. When a neural progenitor produces a
neuron, it first undergoes neuronal fate commitment and afterwards acquires the features of the
specific cell type, for instance axons, dendrites, synapses and polarized membrane potential [22]. During
the commitment of ESC to the neural lineage, changes in the genome-wide regulation of chromatin
structure occur [22]. It is worth mentioning that neurogenesis is not limited during the differentiation
stages of embryonic development but also occurs during adult life in certain regions of the brain such as
the sub ventricular zone (SVZ) of the lateral ventricles and the sub granular zone of the dentate gyrus of
the hippocampus.
4.1 More compact chromatin
In general, as ESCs move towards a particular lineage, non-lineage genes as well as genes associated
with proliferation are silenced and inaccessible to transcription factors, whereas the promoters and
enhancers of lineage-specific genes become accessible [49]. Upon depletion of the H1 protein,
differentiation of ESCs is impaired and this has led to the assumption that through H1 and chromatin
compaction, the pluripotent genes are repressed whereas in ESCs chromatin maintains a more plastic
nature [50]. What’s more, the need for the sequential activation of the developmental Hox genes has
led to the suggestion of unidirectional chromatin opening along each chromosome thus allowing the
formation of a precise expression pattern, which has been shown for the Hoxd4 locus [2]. The HOX
genes’ expression is regulated by a variety mechanisms such as PTMs, ncRNAs, chromatin condensation
and other transcription factors [2].
4.2 Hydroxymethylation
During development and differentiation, hmC and TET oxidase levels go down and reestablish in certain
terminally differentiated cells. In NPCs and neurons for example, it was found that the hydroxyl mark is
more abundant on gene loci expressed in brain and that the levels increase during differentiation. In
general the brain has the highest amount of hmC than any other tissue [22, 26]. In ESCs, hmC and TET1
are enriched at bivalently marked genes, and assists the PRC2 complex in binding [5].
Hydroxymethylation occurs in early development in NPCs at intragenic regions and is associated with
high levels of transcription, especially for genes involved in neuronal differentiation, migration and axon
guidance. On the other hand, whenever the hydroxyl mark was found around promoter or TSS
(transcription start sites) in early stages in the brain, the genes were not transcribed [22]. This leads to a
possible model in which 5hmC can serve as a “poised” mark, just like PTMs in ESCs [22]. In the brain
cortex, TET2 and 3 have the highest expression of all the TET enzymes and their levels go up during
differentiation possibly explaining the increasing abundance of 5hmC. When the TET2, TET3 and EZH2
enzymes are depleted, neural differentiation is abnormal suggesting that both H3K27me3 and 5hmC are
essential for brain development [51]. Although it is still unclear if hydroxymethylation itself has a
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function or if it is just an intermediate between methylated and unmethylated state, the recent
discovery of hydroxymethyl-readers in NPCs differentiated from mESCs and from adult mouse brain,
indicates that there is more to the hydroxyl mark [8]. Some of the hydroxyl readers identified were
UHRF2, ZHX1/2 as well as MeCP2 which was also found earlier [22]. The same study identifies
methylation mark readers in the different stages of NPC differentiation and confirmation of these data
in vivo need to be provided.
4.3 Methylation
As far as methylation is concerned, its global levels go down during differentiation and is established de
novo on promoter regions of genes responsible for early development, ensuring a transition from ESC to
lineage committed cells [26, 52]. The methylation mark is maintained through the DNMT1 and DNMT1o
enzymes as well as the NLPR2, NLPR7, and ZFP57 genes, whose role however is not yet fully understood
[12, 53]. The ZFP57 protein is known to maintain both maternal and paternal imprints, as well as adding
de novo imprints on the oocyte DNA and helps maintain global methylation levels [54].
Upon differentiation, promoters of pluripotency genes like Oct4 and Nanog get hypermethylated. This is
the result of a signaling pathway involving the binding of repressor proteins on the gene, the G9Amediated H3K9 methylation, and the recruitment of HP1 causing de novo methylation and
differentiation [55]. This process from early to terminal silencing is problematic in LSH-deficient ESCs [3].
Demethylated regions upon differentiation include the Notch-signaling responsive genes like Hes5. This
way, Notch signaling can generate NSCs [6]. Demethylation is also important for adult neurogenesis in
the hippocampus of the brain and is promoted by the GADD45b gene which is able to detect
environmental changes and remove the methylation mark accordingly. This way genes like FGF2 and
BDNF (brain-derived neurotrophic factor) are activated and initiate adult neurogenesis [6].
What’s more, upon differentiation DNA methylation coincides with nucleosome assembly on
nucleosome-depleted regions of pluripotency genes like Oct4 and Nanog, thus inhibiting transcription
factor binding [32].
Finally, robust and coordinated promoter methylation occurs, and has been shown for the sets of genes
that are deregulated by both the E2F6 and the DNMT3b knockouts in mice, as well as for certain
repetitive elements [46, 56].
4.4 Histone PTMs and writers/readers
In the developing brain and during the neurogenic phase when NPCs produce different neuronal
subtypes, the neurons of each cortical layer are produced sequentially, from an inner to outer layer
manner [57]. The time switch for the development of the different cortical layers is synchronized among
the NPCs and the transcription factor IKAROS has been suggested to regulate the deeper layer neuronal
formation. This is based on studies showing that IKAROS can condense chromatin by recruiting
chromatin remodeling complexes like NuRD. Other studies also suggest that IKAROS can activate
transcription by recruiting SWI/SNF [22].
During lineage commitment acetylation levels drop globally, proving histone de-acetylases essential for
ESC differentiation [2]. What’s more, during differentiation some gene transcripts get downregulated
and this upregulates the transcription levels of other genes [2]. In order to maintain NSC proliferation,
neurally expressed HDACs interact with the TLX protein, suppressing its target genes. Inhibition of these
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specific HDACs by specific drugs results in loss of the ESCs proliferating state and differentiation into
neurons, and the conditional loss of HDAC1 and 2 in neural precursor cells inhibits differentiation [6].
As would be expected, the Polycomb complexes also play an important role during neural cell line
commitment. PRC1 and PRC2 promote histone H2A ubiquitination and tri-methylate H3K27 respectively,
while both assist in chromatin condensation and maintain the undifferentiating state of the NPC while
also regulating the differentiation potential [2, 22].
Various knockout and overexpression studies have been conducted concerning various PRC1 and PRC2
components, underlining the importance of the complexes. The PRC1 component BMI-1 proved trivial
for the self-renewal of NSCs in the peripheral and central nervous systems after being studied in vitro.
When overexpressed however, no increased neurogenesis or proliferation could be detected in vivo [58,
59]. In human, absence of BMI1 leads to progressive postnatal growth retardation and neurological
defects [58]. In EZH2 (PRC2 component) knockout mice, early neurogenesis was promoted instead of
self-renewal in the cortex [60].
Another important factor in neural differentiation is the JMJD3 H3K27me3 demethylase which enables
the expression of neuronal differentiation genes like Nestin and the Hox genes, as well as the
H3K4me2/3 demethylase JARID1B which promotes neuronal lineage commitment [61, 62]. Other neural
genes such as NGN genes, PAX6, SOX1, SOX3, MASH1 and NKX2.2 also get rid of their repressive bivalent
marks when ESCs move towards NPCs [2].
What’s more, high levels of HMGA proteins have been linked to a globally open chromatin state in NPCs
and to their proliferation capacities, as well as promoting neurogenesis and inhibiting astrogenesis [22].
It is worth mentioning that the let-7b microRNA targets HMGA2 and the microRNA-296 targets HMGA1
[63, 64]. In addition, MYC and LIN28b regulate the neurogenic potential of NPCs and this fact, in
combination with the findings that MYC regulates HMGA1 and 2 and the possibility of an existing
positive feedback3 loop between HMGA and MYC, has led to hypothesis that the MYC-HMGA pathway is
active during neural development [22]. HMGAs are also negatively regulated by the Polycomb complex
and when BMI1 (regulator of NPC proliferation) of the PRC1 complex is depleted, HMGA2 gene
expression goes up which could mean that the pathway involving PcG and HMGA proteins can have a
bigger role than we know in differentiation [22]. The BAF complexes npBAFs and nBAFs are also
important in the NSC, contributing to self-renewal or differentiation, depending on the combination of
recruited proteins [6].
Other proteins that help in neural lineage commitment are the zinc finger nuclear protein ZFP521
promotes lineage committed differentiation of ESC by associating with its co-activator p300 and
activating early neural genes [65]. Self-renewal, growth and differentiation in NSC has been found to be
regulated by a number of factors including SOXB1 and GLI families, CBF-1, HMGA2, HES1/5, BMI-1,
HESR1/2, TLX, MASH1, PAX6 and REST [66].
4.5 ncRNAs
NcRNAs are in general enriched in neuronal cells. In mouse neocortex, interference with the miRNA
pathway by conditional Dicer knockout results in smaller cortex, higher levels of neuronal apoptosis and
impaired cortical layering [6]. The microRNA miR-145 targets the 3’ UTR of the OCT4, SOX2 and KLF4
transcripts thus promoting ESC differentiation [67]. Other miRNAs that are selectively expressed at
different stages of NPC lineage progression are miR-9, miR-124, miR-92b and miR-23. MiR-124 especially
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is the most abundant miRNA in the central nervous system and regulates lineage progression from
transit-amplifying cells of the SVZ till they become neuroblasts [6]. What’s more, miR-9 can be found in
high levels in NPCs of mouse, human and others. It has both functions in embryonic and adult
neurogenesis by promoting differentiation. Both the miR-9/9* and miR-124 can promote the transition
of human fibroblasts to neurons. They seem to cooperate with the neural-specific transcription factors
ASCL1 and MYT1L during this process [6].
4.6 Extrinsic signals
Factors that contribute to the maintenance of multipotency of the NPCs include the Notch Signaling
family, Transforming Growth Factor-β (TGF-β) superfamily members, FGFs, Neuregulins (NRG) and
Platelet Derived Growth Factors (PDGF). What’s more EGF and FGF2 regulate neural programming, Betacellulin promotes proliferation into neural cells and IGF2 regulates adult hippocampal neurogenesis [2].
13
5. Epigenetics from NPCs to fully differentiated neurons
During the final differentiation of NPCs into mature neurons, neurogenesis precedes gliogenesis
(astrogenesis) and this associated with tight spatiotemporal changes in the epigenetic marks of the
genome.
5.1 DNA methylation and hydroxymethylation
DNA methylation serves as a dynamic regulatory mechanism in the process of gene expression and
silencing. DNMT3 silences genes by methylating the proximal promoter but allows expression of
neurogenic genes by antagonizing the repressive PRC complexes through non-proximal promoter
methylation [5]. As NSCs move from lineage-committed cell fate towards neurogenesis and gliogenesis,
loss of DNMT3a leads to inaccessible gene regions around the neurogenic genes DLX2, NEUROG2 and
SP8 therefore restricting their expression, while causing genes linked to astrogenesis and neurogenesis
to be expressed [6]. These mechanisms occur in post-natal NSCs, but are not limited to these since it’s
highly likely they also play a role in development as shown in mESCs [5].
During neurogenesis, methylation of the astrocytic genes like GFAP, keeps the NPCs from differentiating
into astrocytes and ensures the birth of neurons. Later on with the help of the gliogenic signals LIF and
CNTF, the neurogenesis process switches to gliogenesis and generates astrocytes. In NPCs that are
destined to become astrocytes, the STAT3 protein of the JAK-STAT pathway, binds to the GFAP promoter
and activates the gene. In NPCs destined to be neurons however, the STAT3 protein is unable to bind to
GFAP due to methylation marks. DNMT1 helps maintain those methylation marks in the neurogenic
phase and can be removed by the NF1A (nuclear factor 1 A-type) [22]. It’s probable that NF1A removes
DNMT1 from other gene promoters too for a simultaneous expression of astrocytic genes, since a
methylation decrease is observed in the transition from neurogenic to astrocytic phase [22]. A
conclusion can be drawn by saying that DNA methylation governs the process of neuronal cell-fate
determining.
On the other hand, hydroxymethylation has been proposed to be linked with neuronal development.
More specifically, the model states that hmC accumulation at different developmental stages could
mean that it regulates neural development in a stage-specific manner: from early stages where hmC
marks the intragenic regions of differentiation and migration-specific genes, later at genes responsible
for neuronal attributes, and finally subtype-specific genes during subtype-specification [22]. The
importance of hydroxymethylation in developing neurons is highlighted by the case of a RTT mutation of
MeCP2 which cast it impossible to bind to hmC, leading to Rett syndrome [68]. What’s more, the
conversion of mC to hmC can be prevented by the binding of the MBD domain of MeCP2, suggesting
MeCP2 has a role in regulating hmC levels as well. In adult mouse brain, hmC readers were identified
with some of the proteins involved being Ronin, MeCP2 and HMG1/2 [22, 68]. There is a difference in
hydroxyl readers between NPCs and neurons, suggesting a difference in hmC function depending on the
stage of differentiation [22].
5.2 Readers/writers
Chromatin remodeling complexes like SWI/SNF also play a role in the final differentiation of NPCs. An
example is the SWI/SNF component BRG1 that is expressed predominantly in neurons and in progenitor
cells of the VZ and the SVZ. It has also been suggested that BRG1 is essential for gliogenic cell
14
maintenance [6]. Not only is BRG1 involved in the SWI/SNF complex, but it can also -independently of its
ATPase activity- regulate Shh signaling in the forebrain [6].
What’s more, MLL1 is essential to promote expression of neural-specific genes during neurogenesis by
resolving bivalent loci, and MLL1 deficient SVZ cells fail to differentiate properly [69]. In NPCs of the
adult mouse hippocampus, the GADD45B nuclear factor demethylates the promoters of neurogenesis
genes like FGF and BDNF, thus promoting differentiation [70]. What’s more, NFκB signaling is essential
for early neurogenesis in NSCs and its inhibition causes Nestin, SOX2 and glial fibrillary acidic protein to
accumulate in vivo [71]. In addition, the histone acetyltransferases Querkorpf (QKF) is used as a marker
for self-renewing NSC and is concurrently required for regular adult neurogenesis [2].
During the specification of the neuronal subtype, higher chromatin structure in postmitotic neurons has
been shown to be regulated by SATB (special AT-rich sequence binding) proteins SATB1 and SATB2
which cause chromatin looping helping the recruitment of chromatin remodeling complexes like
SWI/SNF and NuRD and HDACs respectively, hereby changing gene expression [22]. SATB2 in particular,
represses the transcription of the CTIP2 gene, vital for subcerebral projection neuron (SCPN) fate,
specifying thus the upper layer callosal projection neuron (CPN) over the SCPN [22]. Another factor
involved in the above processes is SKI, which recruits HDAC1 to the CTIP2 genes hereby contributing to
the CPN identity. This is confirmed by results showing loss of axon projection across the corpus callosum
in SKI knockout mice, an effect similar to the SATB2 knockout mice [22]. The SATB1 protein on the other
hand, specifies the somatostatin (SST)-expressing neuron subtype in the post-mitotic MGE-derived
interneurons by acting downstream of the LHX6 gene [22]. MGE is a transitory brain structure that
guides cell and axon migration.
Another protein playing an important role in differentiated neurons is the methyl-CpG binding protein
MeCP2 which is much more abundant in the brain in relation to other tissues and more specifically in
neurons and not glia or other cell types. It regulates neural development genes like BDNF (brain-derived
neurotrophic factor), while also playing a role in determining dendrite morphology and synapse
formation [22]. The importance of MeCP2 in formation and maintenance of neuron connection during
development is highlighted by the mutated version of MeCP2 that leads to Rett syndrome, a
neurological disease almost exclusively affecting females. It’s a serious disease causing severe
intellectual disability, continuous stereotypic hand movements with decline of purposeful hand use, loss
of language skills, autistic features, gait abnormalities, breathing defects, seizures, hypotonia, scoliosis
and autonomic dysfunctions.
MeCP2 levels increase later in development and in 5-6 week old neurons binds to methylated DNA on a
global scale, providing more proof on its role in post-mitotic development [22]. MeCP2 has been shown
to compete with histone H1 in mC binding, leading to a hypothesis of H1 being replaced in nucleosomes
by MeCP2. This would lead to gene repression because of the HDAC recruitment but also because of the
lack of repressive PTMs thanks to MeCP2. It has also been shown that the NCoR nuclear receptor corepressor interacts with MeCP2 most probably leading to the abovementioned reduction of histone
acetyl marks, making the interaction rather important for the function of MeCP2 [22]. Another possible
mechanism by which MeCP2 influences chromatin structure is based on its AT-hook domains. Their
absence causes neurodevelopmental symptoms, underlining once more the importance of MeCP2 [72].
What’s more, MeCP2 can get phosphorylated at numerous sites as a result of environmental cues like
membrane depolarization or other pathways, leading to a possible MeCP2 working model: the
15
phosphorylation caused by neuronal activity could result in either location changes of the MeCP2
interaction partners or alteration of chromatin structure [22]. This is however still contradicted by
various findings, suggesting that further investigation upon the subject is needed [22]. Finally, the LIN1
retrotransposon element has been suggested to play an important role in the regulation of neural
development by MeCP2. This is because MeCP2 knockout NPCs showed an increase of LINE1 and this
retrotransposon has been observed to occur often in neurons, influencing their mosaicism [22].
Another methyl-CpG-binding protein playing a role in neurogenesis is the protein MBD1 which is
expressed in higher levels in neural cells and NPCs in the adult brain, and has been found to play an
important role in adult neurogenesis in the SVZ and the dentate gyrus of the hippocampus. In absence
of MBD1 no developmental defects are detected, though MBD1 knockout mice show reduced genomic
stability and risen expression of the IAP repeated element. The knockout mice also show deficits in
spatial learning and long-term potentiation in the dentate gyrus of the hippocampus and adult
neurogenesis [73]. MBD1-deficit NSCs show reduced methylation at the promoter of the FGF2 mitogen,
leading to increased FGF2 expression and reduced neural differentiation [74]. Loss of MBD1 also causes
adult neural stem cells to differentiate into neural cells less efficiently. This is due to the role of MBD1 in
promoting differentiation of NPCs by being part of a regulatory loop involving microRNA-195 [75].
5.3 miRNAs
miRNAs have a modulatory role in neural regulation and one important miRNA is the mir-124a which is
essential for neural differentiation, because it targets the neural stem cell proliferation-promoting factor
Sox9 [2]. Mir-124a and mir-125b can both increase differentiation of neuroblastoma cells of the SH-SY5Y
line, with neurite outgrowth [2]. Another neural differentiation-promoting mi-RNA is mir-9 which
negatively regulates the nuclear receptor TLX and prevents it from assisting in the maintenance of the
ESCs self-renewal status, thus creating a balance between proliferation and differentiation [76]. Mir-9
also promotes neurogenesis by forming a negative feedback loop with REST, and is downregulated in
Huntington’s disease [77]. What’s more, mir-132 targets the translation of methyl-CpG-binding protein
MeCP2 which might interact with REST for repression. This is of importance because when MeCP2 levels
are up or downregulated, neurodevelopmental defects can occur [78]. The miR-184 has been shown to
be repressed by MBD1 in the NPCs of the adult brain and MBD1 also regulates Numbl which is a
regulator of the brain development [6, 79].
5.4 General neural cell types
5.4.1 Astrocytes (glial cells)
Astrocytes are characteristic star-shaped cells mostly found in the brain and spinal cord. They are known
to have several functions in the brain, including the secretion or absorption of neural transmitters and
maintenance of the blood–brain barrier.
During differentiation of neural progenitor cells into astrocytes both inner and outer signals play a role:
the Janus kinase (JAK) pathway is initiated by cytokines of the interleukin (IL)-6 family such as the
leukemia inhibitory factor (LIF), the ciliary neurotrophic factor (CNTF) and cardiotrophin 1 (CT-1) and is
followed by the activation of the STAT transcription factors, forming the so-called JAK-STAT pathway [80,
81]. The transcription factor STAT3 stimulates the expression of the astrocyte-specific genes like GFAP
and S100β in NPCs and when its expression is prevented astrogenesis doesn’t occur [81, 82]. The
16
promoters of the GFAP and S100β genes lose their methylation marks through the course of
differentiation, making them accessible for transcription [2].
This differentiation pathway is in constant competition with the neurogenesis pathway in which
neurogenic factors like Neurogenin1 are in charge and prevent astrogenesis by binding to STAT3 coactivators [81]. What’s more, during the neurogenic period, the promoters of astrocytic genes are
methylated thus blocking differentiation and making the retinal ganglion cells (RGCs) unresponsive to IL6 cytokines. Once removal of the methylation mark occurs, astrogenesis can take place.
Another astrogenic-promoting pathway is Notch signaling which modulates removal or addition of the
methylation mark on the promoters of astrocytic genes with nuclear factor 1 which binds to astrocytic
genes promoters, but only if at the same time the JAK-STAT3 pathway is active [83]. Once the first
astrocytes have formed, they start secreting gliogenic cytokines which promote faster differentiation of
the remaining NPCs [80].
Histone modifications also promote the astrocyte fate, as it was reported that FGF2 induces H3K4me
and suppresses H3K9me of the GFAP promoter resulting in more open chromatin structure that allows
transcription factors activated by CNTF, to bind [6]. Also, PcG proteins play a role in regulating the
transition from neurogenic to astrocytic state and do so by repressing neurogenic genes like NEUROG1
and NEUROG2 in astrogenic NPCs of the neocortex [22]. This repression mechanism can be blocked by
the ncRNA UTNGN1 which originates from an enhancer element of the NEUROG1 gene, resulting in
NEUROG1 expression. From this study we can postulate that it’s possible for this ncRNA to inhibit PcG
proteins [22].
5.4.2 Oligodendrocytes
The key role of oligodendrocytes is granting support and protection to axons of the central nervous
system and they do this by creating a myelin sheath, reducing ion leakage and increasing signal
transmission along the axons.
Differentiation into oligodendrocytes takes place after astrocytes have been formed. Through Shh
signaling oligodendrocytes develop in the ventral part of the developing spinal cord [84]. Another
reported contributing factor to oligodendrogenesis is Olig1/2 (oligodendrocytes transcription factors 1
and 2) in whose absence oligodendrogenesis is stalled [85, 86]. In different parts of the developing brain
oligodendrocyte progenitors express specific transcription factors such as NKX2.1, GSX2 and EMX1 [87].
The majority of mature oligodendrocytes however derives from EMX1-expressing progenitors [81]. The
transition from progenitors to differentiated oligodendrocytes requires the intervention of HDAC1 and 2
which inhibit Wnt signaling thus activating Olig2 and differentiation [88]. Stimulation of the HDACs to
deacetylate, repress anti-differentiation genes and consequentially lead to differentiation has been
reported to be extrinsic and Shh signaling is such an example [89]. Oligodendrocyte precursor cells
(OPCs) are NG2+ cells and the specification during differentiation is in a large deal determined by Olig2
as loss of Olig2 leads to astrocyte production instead [90].
5.4.3 Ependymal cells
These cells form the cellular blockade separating the CSF (cerebrospinal fluid) and the adjacent
parenchyma in the human brain and even after differentiation during late embryonic and perinatal
period they maintain their ability to proliferate [81]. Although there is not much known about their
development, in new ependymal cells the FOXJ1 transcription factor is expressed, as well as the
17
homeobox transcription factor Six3 [81]. Interesting is also the fact that Numb/Numblike proteins that
play a negative role in Notch signaling during neurogenesis are important for the development of the
ependymal cells [91].
6. Potential in medicine
Adult neurogenesis is trivial in synaptic plasticity, playing a big role in spatiotemporal learning, memory
and mood. Several neurological conditions like depression, anxiety, schizophrenia have been linked to
impaired hippocampal function. This is thought to be due to the lack of proliferation and renewal of the
adult NPCs located in the subgranular zone of the hippocampal dentate gyrus. Understanding the role
epigenetics have in the development of these conditions can prove useful for therapeutic strategies
What’s more, directing ESCs towards differentiation in neurons can prove useful for transplantations
(where pure populations of a differentiated cell type are needed) or regenerative medicine. Thus,
understanding the impact epigenetics have on self-renewal and differentiation processes in stem cells
and neural progenitors is a promising field for cell-based therapies. These could improve treatment
and/or prevention of human neurodegenerative diseases such as Parkinson’s and Alzheimer’s that can
be caused by altered epigenetic machinery that in turn cause abnormal methylation and histone
acetylation resulting in abnormal transcriptional expression [2, 6]. How exactly the alterations in
epigenetic control play a role in the etiology and progression of these illnesses is yet to be found out.
Since the role of ncRNAs in the epigenetic landscape is still quite unclear, studies on the microRNA
fluctuations could help understand the balances between normal and abnormal neurological expression.
What’s more, different model systems for studying could be used, including single cells and organoids.
Single cells can be useful to study in the case of progenitor and stem cells, in which cases one single cell
can give rise to a whole new population during differentiation. Any abnormal behavior in one single cell
could lead to problematic differentiation possibly resulting in an illness or syndrome. Understanding the
mechanisms by which single cells control the intake of extrinsic and intrinsic signals is therefore crucial.
Organoids on the other hand are in vitro ideal model of the human brain development since they can
represent the brain in much smaller scale and complexity. An example of such a organoid model is the
one developed in the Knoblich lab called “cerebral organoid” which forms different regions, including a
progenitor zone–just like the human brain does, making it ideal for studying brain disorders like primary
microcephaly [92].
18
7. Figures
Figure 1: Major epigenetic regulators involved in the differentiating process from embryonic stem cells into neural
stem cells and the timing of cell fate determination of neural stem cells (neurogenesis and gliogenesis) [6].
19
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