DNA Methylation

Brain Plasticity xx (20xx) x–xx 1
DOI 10.3233/BPL-160034
IOS Press
1 Literature Review
2 DNA Methylation and Adult Neurogenesis
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3 Emily M. Jobea,b and Xinyu Zhaoa,b,c,∗
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a Cellular
and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, WI, USA
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b Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
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c Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA
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7 Abstract. The role of DNA methylation in brain development is an intense area of research because the brain has particularly
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8 high levels of CpG and mutations in many of the proteins involved in the establishment, maintenance, interpretation, and
9 removal of DNA methylation impact brain development and/or function. These include DNA methyltransferase (DNMT),
10 Ten-Eleven Translocation (TET), and Methyl-CpG binding proteins (MBPs). Recent advances in sequencing breadth and
11 depth as well the detection of different forms of methylation have greatly expanded our understanding of the diversity
12 of DNA methylation in the brain. The contributions of DNA methylation and associated proteins to embryonic and adult
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13 neurogenesis will be examined. Particular attention will be given to the impact on adult hippocampal neurogenesis (AHN),
14 which is a key mechanism contributing to brain plasticity, learning, memory and mood regulation. DNA methylation influences
15 multiple aspects of neurogenesis from stem cell maintenance and proliferation, fate specification, neuronal differentiation and
16 maturation, and synaptogenesis. In addition, DNA methylation during neurogenesis has been shown to be responsive to many
17 extrinsic signals, both under normal conditions and during disease and injury. Finally, crosstalk between DNA methylation,
18 Methyl-DNA binding domain (MBD) proteins such as MeCP2 and MBD1 and histone modifying complexes is used as an
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19 example to illustrate the extensive interconnection between these epigenetic regulatory systems.
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20 Keywords: DNA methylation, adult neurogenesis, neural stem cell, neuronal differentiation, DNA methyltransferase (DNMT),
21 Ten-Eleven Translocation (TET), and Methyl-CpG binding proteins (MBPs), MeCP2, MBD1
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22 INTRODUCTION cell functions depend on dynamic regulation of epige- 33
netic programs in addition to maintaining epigenetic 34
23 The term epigenetics was first used by Conrad H programs through cell division. DNA methylation, 35
24 Waddington in 1942, before the age of DNA, to define one of the core epigenetic modifications along with 36
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25 the “study of those processes by which genotype gives histone modification, can be transmitted to daughter 37
26 rise to phenotype” [1]. Later iterations of this defi- cells and was considered a largely permanent DNA 38
27 nition required that epigenetic modifications lead to modification until recent discovery of the Ten-Eleven 39
28 heritable changes in gene expression and function Translocation (TET) enzymes in 2009 [3]. This dis- 40
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29 that are maintained across cell divisions or genera- covery, coupled with the advent of more sophisticated 41
30 tions [2]. Thus, epigenetic regulation is particularly techniques to measure and sequence DNA methyla- 42
31 relevant in proliferative cells, such as those involved tion and other derivatives has greatly expanded our 43
32 in AHN. Recent advances have shown that certain knowledge of this epigenetic mark and the various 44
roles it plays in the cell. 45
∗ Correspondenceto: Xinyu Zhao, Waisman Center and Depart-

Likewise, the discovery and characterization of 46
ment of Neuroscience, University of Wisconsin-Madison School adult neurogenesis in humans and many other species 47
of Medicine and Public Health, Madison, WI 53705, USA. Tel.: over the past twenty years has caused a paradigm 48
+1 608 263 9906; E-mail: xinyu.zhao@wisc.edu. shift in the field of neuroscience: you are not born 49
ISSN 2213-6304/16/$35.00 © 2015/2016 – IOS Press and the authors. All rights reserved
This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC 4.0).
2 E.M. Jobe and X. Zhao / DNA Methylation and Adult Neurogenesis
50 with all of the neurons you will ever have. In small X syndrome (FXS) and Rett syndrome (RTT) [7, 8]. 99
51 regions of the brain stem cells continuously give Indeed, many anti-depressants and other medications 100
52 rise to new neurons which play important roles in are known to increase neural stem cell prolifera- 101
53 learning, memory and mood regulation. In addition in animal models and humans. Because there 102
54 tion, adult neurogenesis is also altered in many is a distinct cellular progression of adult neuroge- 103
55 disease states—but can also be manipulated by nesis, and defined behavioral outcomes associated 104
56 pharmacological treatment and other interventions, with altered neurogenesis, adult neurogenesis is an 105
57 making it a promising avenue for intervention. The excellent model to study regulatory mechanisms, 106
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58 role of DNA methylation in brain development is interventions, and functional outcomes. 107
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59 an intense area of research because the brain has Neurogenesis is defined as process that leads 108
60 particularly high levels of CpG and mutations in to the generation of new functional neurons. This 109
61 many of the proteins involved in the establishment, process includes the proliferation and fate specifi- 110
62 maintenance and interpretation of DNA methy- cation of neural stem cells and the differentiation 111
63 lation impact brain development and/or function. and integration of newly generated neurons into the 112
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64 Epigenetic pathways, including DNA methylation, existing neural circuitry. Mammalian neurogenesis is 113
65 influence multiple aspects of neurogenesis from stem divided into two phases: embryonic/developmental 114
66 cell maintenance and proliferation, fate specifica- neurogenesis and adult neurogenesis. Embryonic 115
67 tion, neuronal differentiation and maturation, and neurogenesis encompasses the generation of neurons 116
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68 synaptogenesis. In addition, DNA methylation dur- in the context of the formation of the central ner- 117
69 ing neurogenesis has been shown to be responsive to vous system (CNS). In the adult brain, multipotent 118
70 many extrinsic signals, both under normal conditions NSCs remain and continue to generate new neurons. 119
71 and during disease and injury. There are two niches of the adult mammalian CNS 120
confirmed to have ongoing neurogenesis: the sub- 121

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granular zone (SGZ) of the dentate gyrus (DG) in 122
72 NEUROGENESIS the hippocampus and the SVZ bordering the lateral 123
ventricles [9]. New neurons have also been observed 124
73 The epigenetic networks regulating neurogene- in other regions of the brain, but these reports are 125
74 sis are highly connected with each other and other more variable and may depend on the species and 126
signaling pathways and regulatory networks. To physiological or pathological state (reviewed in [10]).
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75 127
76 date, embryonic cortical neurogenesis and adult hip- aNSCs in both the DG and the SVZ derive from 128
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77 pocampal neurogenesis (AHN) are the best-studied embryonic NSCs. In mice, the DG begins to develop 129
78 systems. Although they have many similarities and at E13.5 from NSCs located adjacent to the cortical 130
79 are regulated by many of the same networks, there hem and continues to develop until early postna- 131
80 are also many differences that warrant investigation. tal stages [11]. Some NSCs retain their multi-potent 132
81 Embryonic brain development must integrate growth, properties and transform into radial glia like cells 133
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82 patterning and differentiation signals to generate neu- (RGLs), so named because their cell bodies reside 134
83 rons, astrocytes, and oligodendrocytes (and sub-types in the subgranular zone (SGZ) and their radial pro- 135
84 of cells) in a spatial and temporally-specific man- cesses extend through the DG molecular layer in 136
85 ner while still maintaining populations of stem cells. manner reminiscent of RGs. RGLs, also known as 137
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86 aNSCs also generate multiple cell types; moreover type 1 cells, give rise to intermediate or transit- 138
87 they are responsive to environmental inputs, phar- amplifying progenitors (type2a/b, type 3) which 139
88 macological interventions, and are a major source of subsequently differentiate into neurons. A small num- 140
89 plasticity in the adult brain [4]. Adult neurogenesis ber of astrocytes are generated from NSCs, but it 141
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90 contributes to the behavioral problems and learn- is unclear if they arise directly from RGLs or if 142
91 ing deficits observed in many neurodevelopmental, they pass through a non-committed transit amplify- 143
92 neurodegenerative, and injury-based disorders [5, 6]. ing intermediate [12, 13]. Cells in different stages of 144
93 Animal models have provided an invaluable resource neurogenesis can be defined by a combination of mor- 145
94 to evaluate adult neurogenesis and the mechanisms phology, cell-type specific markers, and proliferative 146
95 that regulate it. Several promising results indicate that capacity [14]. Throughout the process of neuroge- 147
96 it may be possible to improve function through treat- nesis, DNA methylation changes as cells proceed 148
97 ment at post-natal and adult stages, even for disorders through different cell stages and respond to different 149
98 with partial developmental etiology, such as fragile inputs.

E.M. Jobe and X. Zhao / DNA Methylation and Adult Neurogenesis 3
150 Function of adult hippocampal neurogenesis in non-neuronal cells, supporting earlier studies of 199
individual genes [32]. Multiple mechanisms likely 200
151 The hippocampus was recognized as being nec- contribute to gene repression by DNA methylation, 201
152 essary for certain types of learning and memory including recruitment of repressive complexes by 202
153 based on cognitive studies of memory impairment methyl-CpG binding proteins (MBPs) or through 203
154 in humans and hippocampal lesion studies in model blocking the binding of pro-neuronal transcription 204
155 animals [15]. Further studies determined that the factors (TFs). Though many studies have analyzed 205
156 hippocampus is required for spatial and contex- cell-type specific transcriptomes [33–37], the number 206
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157 tual learning and memory [16]. Evidence for the of studies that combine transcriptome and genome- 207
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158 importance of NSCs—and thus the formation of wide methylation is more limited. However, the 208
159 new neurons—in hippocampal learning and memory growing feasibility of this type of study will allow 209
160 came from studies that ablated adult neural progen- researchers to ask questions about the role of DNA 210
161 itors via genetic means, anti-proliferative drugs, or methylation in the cell. Such as, are DNA methyla- 211
162 focal irradiation [17–22]. Hippocampal neurogenesis tion patterns cell-type specific? Do they correspond 212
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163 contributes to hippocampal plasticity and both play a to histone modifications or the binding patterns of 213
164 role in hippocampal-dependent cognitive function. other repressive complexes? Is methylation always 214
165 AHN represents a unique source of plasticity in associated with repression? 215
166 the brain. Plasticity, or the ability to respond and

adapt to stimulus, is generated by AHN in the DG
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167 DNA methylation and DNMTs 216
168 by two ways: 1) how many new cells are produced
169 and 2) how these cell integrate into existing net- DNA methylation is well known for its role in 217
170 works. First, increased NPC proliferation represents long-term gene silencing; it serves as the basis 218
171 a major mechanism through which more neurons can of imprinting, X chromosome inactivation, and the 219
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172 be generated. The quiescence of RGLs, the cell cycle establishment of cell fate [38–40]. DNA methy- 220
173 progression and differentiation of IPCs, and the mat- lation involves the covalent addition of a methyl 221
174 uration of new neurons are all tightly controlled by group from the cofactor SAM (S-adenosyl-l- methio- 222
175 intricate molecular networks that consist of intrin- nine) to C5 of cytosine in CG dinucleotides (also 223
176 sic genetic and epigenetic programs modulated by referred to as CpG, 5mC, or mCG). This addition 224
extrinsic physiological and pathological conditions is catalyzed by a family of DNA methyltrans-

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177 225
178 [14, 23, 24]. The second mechanism driving adult ferases (DNMTs). DNMT3a and DNMT3b establish 226
neurogenesis-mediated plasticity is the integration of

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179 de novo methylation, whereas DNMT1 maintains 227
180 new cells to existing networks. These new neurons methylation patterns in newly synthesized DNA by 228
181 pass through a ‘critical period’ of enhanced synaptic recognizing hemi-methylated DNA and methylating 229
182 and dendritic plasticity 3–6 weeks post-mitosis that the unmodified strand [41]. 230
183 is dependent upon the inputs cells receive [25–27]. In addition to CG methylation, other dinucleotide 231
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184 Disruption of cells during this window of time dis- pairs containing cytosine can be methylated, referred 232
185 rupts hippocampal-dependent learning and memory to as CH or CpH methylation, where H = A/C/T. 233
186 [19, 21, 28, 29], revealing that cells in this stage are Recent studies have shown that CH methylation 234
187 vital to the functional output of adult neurogenesis. (mCH) is high in the brains of humans and mice [42, 235
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43]. And within the brain, non-CG methylation is 236
188 DNA METHYLATION much more prevalent in neurons than non-neuronal 237
cells and is estimated to account for 25–38% of 238
189 Epigenetic mechanisms, including DNA methy- total methylation [44–46]. CH methylation has been 239
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190 lation, are critical for establishing the diverse cell shown to accumulate dramatically in neurons but not 240
191 fates present in the central nervous system. Different astrocytes during postnatal development, a critical 241
192 regions of the human brain (cerebral cortex, cere- period of neuronal maturation and synaptogenesis 242
193 bellum, and pons) each have a characteristic DNA [46]. There is evidence that DNMT3A is responsible 243
194 methylation signature [30]. And even within brain for the deposition of mCH, and that non-CG methy- 244
195 regions like the hippocampus, global methylation lation is also associated with gene repression [46]. 245
196 varies between neuronal subtypes [31]. There is good The growing awareness of non-CG methylation has 246
197 support from genome-wide methylation studies that the potential toyield novel insights into the role of 247
198 DNA methylation globally represses neuronal genes DNA methylation in regulating brain development 248
249 and plasticity prominent 5hmC peak at the 5’splice site boundary 299
250 Though many studies have analyzed cell-type [55, 56]. One of the key remaining questions is how 300
251 specific transcriptomes [33–37], the number of stud- 5hmC patterns are ‘read’ and interpreted by the cell. 301
252 ies that combine transcriptome and genome-wide One possibility is through recruitment or exclusion 302
253 methylation is more limited. However, the growing of DNA-methyl binding proteins. 303
254 feasibility of this type of study will allow researchers

255 to ask questions about the role of DNA methylation DNA methylation readers: MBPs 304
256 in the cell. Such as, are DNA methylation patterns
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257 cell-type specific? Do they correspond to histone Three families of proteins are known to bind 305
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258 modifications or the binding patterns of other repres- to methylated DNA, including the methyl binding 306
259 sive complexes? Is methylation always associated domain (MBD) family, the zinc finger/Kaiso fam- 307
260 with repression? ily, and SET and RING associated (SRA) domain 308
family. In addition, recent work using quantita- 309
261 TET proteins and demethylation tive proteomics has also allowed for the unbiased 310
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detection of proteins that interact with specific 311
262 Until recently, methylation was thought to be a DNA sequences including methylated and hydrox- 312
263 static DNA modification, with demethylation occur- ymethylated sequences [57–59]. These methods have 313
264 ring only passively upon the reduction of DNMTs. confirmed the binding properties of many MBPs 314
and identified many novel methylated DNA binding
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265 However, the discovery of 5-hydroxymethyl cytosine 315
266 (5hmC) and the subsequent elucidation of the cyto- proteins, such as RBP-J [58], a transcription factor 316
267 sine demethylation pathway substantially changed the within the Notch pathway. These findings highlight 317
268 view of DNA methylation [47]. The regulation of a growing appreciation of the contribution of DNA 318
269 DNA methylation and methylation derivatives is now methylation to transcription factor binding, in addi- 319
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270 known to be a dynamic and active process, thought tion to mediating the binding of the established MBP 320
271 the biological functions of this process are not yet families. So far, only members of the MBD family 321
272 entirely clear [48]. Active DNA demethylation is have been implicated in human disorders affecting 322
273 a multi-step process in which the methyl group is the brain [60], but many other MBPs are involved in 323
274 modified before the entire base is replaced via base cancer [61]. 324
excision repair (BER) pathways (reviewed in [3, 49]).

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275
276 First, members of the TET family of proteins, includ- MBD Family 325
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277 ing TET1, TET2, and TET3, catalyze the conversion DNA methylation-induced gene repression is 326
278 of methylated cytosine to 5hmC and subsequently primarily mediated by proteins containing a methyl- 327
279 to other derivatives such as 5-formylcytosine (5fC) CpG binding domain (MBD) [62]. Among MBPs, 328
280 and 5-carboxylcytosine (5caC) which are removed by MBDs were discovered first and remain the best- 329
281 BER glycosylases [50]. A second pathway, which is studied to date. This family includes MBD1-6 and 330
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282 still controversial, involves deamination of 5hmC by MeCP2 (Methyl CpG Binding Protein 2) (Fig. 1) 331
283 AID/APOBEC to 5hmU, followed by base excision [62]. In addition to the MBD domain, members of 332
284 repair [51, 52]. Although this second pathway may the MBD family possess diverse functional domains 333
285 be important in certain situations, such as neuronal that enable them to bind to chromatin modifying pro- 334
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286 activity induced demethylation (described below), it is teins and other DNA-methylation specific proteins 335
287 considered unlikely that AID and APOBEC are gener- (Fig. 1). For example, MBD1 possesses a transcrip- 336
288 ally involved in 5hmC-dependent demethylation [3]. tional repression domain (TRD) that enables binding 337
289 Mounting evidence indicates that 5hmC methyla- to multiple histone-related proteins (Table 1). Both 338
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290 tion may have biological function beyond acting as a MBD1 and MeCP2 are highly expressed in the brain 339
291 chemical demethylation intermediate. For example, and play important roles in neurodevelopment and 340
292 5hmC has a unique distribution pattern across the plasticity [60]. MBD1 Members of the MBD fam- 341
293 genome, leading to the question of how it is deposited ily play significant roles in the regulation of adult 342
294 and maintained. Compared to 5mC, 5hmC is rel- neurogenesis, which will be discussed below. 343
295 atively abundant at CG islands (CGIs), promoters, A critical step in understanding the function 344
296 and within gene bodies (exons), but low in intergenic of MBDs is to identify their binding specificity 345
297 regions [53, 54]. In addition, 5hmC is relatively abun- and preferred sequences. The initial description of 346
298 dant in constitutively expressed exons and displays MeCP2 noted that it is localized to methylated DNA 347
A comparison of genome-wide promoter methyla- 357
tion and RNA Pol II enrichment showed that active 358
transcription is opposite to that of MeCP2, MBD1, 359
MBD2, and MBD4, confirming the general role of 360
these MBDs in gene repression [65]. MBD1 has 361
multiple splice isoforms with the longest form con- 362
taining both MBD domain that can bind to methylated 363
CpG and a CXXC zinc finger domain that can bind 364
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to unmethylated CpG. A shorter version of MBD1, 365
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called MBD1v3, contains only MBD domain and 2 366
CXXC domains that do not bind to DNA [66]. Thus 367
far, MBD5 and MBD6 have not been found to bind to 368
methylated DNA, although they are localized to het- 369
Fig. 1. A schematic overview of the MBD family of methyl erochromatin [67]. This localization may be mediate 370
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binding proteins (MBPs) including known protein domains in by co-factors such as the PR-DUB Polycomb com- 371
(MBD, methyl-CpG binding domain; TRD, transcriptional repres-
plex [68]. 372
sion domain; AT hook; CXXC, zinc finger Cys-x-x-Cys domain;
PWWP, Pro-Trp-Trp-Pro). The discovery of 5hmC in the brain led multiple 373
groups to question whether or not known methylated 374
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348 and pericentromic heterochromatin [63]. Since then, DNA binding proteins also bound to 5hmC. Multiple 375
349 site-specific analysis has shown that these initial labs have shown that the MBD domain is specific for 376
350 observations hold true: MeCP2 binds throughout the 5mC and that the presence of 5hmC reduces binding 377
351 genome and tracks with mCG density [64]. Genomic of MeCP2 and MBD1 [59, 69–72]. There is some 378
352 mapping of the MBD proteins MeCP2 and MBD1- conflict over the ability of MeCP2 to bind to 5hmC 379
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353 4 revealed that MBD binding correlates with DNA [71]; however, some binding may be mediated by 380
354 methylation, peaking at regions of dense methy- hemi-hydroxymethyled DNA as MeCP2 is already 381
355 lation, such as methylated CG islands, except for known to bind to hemimethylated DNA [73, 74]. It 382
356 MBD3 which does not bind to methylated DNA [65]. is likely that the in vivo binding affinity of MeCP2 383
Table 1
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MBD1 interacting proteins

Reference Interactor Function Methods Required domains
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Watanabe et al. [227] MPG DNA damage repair Y2H (TRD) CoIP (HeLa) TRD
Reese et al. [228] CHAF1A Histone octamer assembly on Y2H CoIP (HeLa) MBD
DNA during replication
Fujita et al. [229] MCAF1 Transcriptional co-factor Y2H TRD
Sarraf et al. [230] SETDB1 H3K9 methyltransferase, PCR2 Y2H TRD
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component
CHAF1A Y2H MBD
Ichimura et al. [225] MCAF2 Transcriptional co-factor GST pull-down Co-IP TRD
SETDB1 H3K9 methyltransferase, PCR2 Co-IP
component
SP1 Zinc-finger TF, gene activation Co-IP
co
HP1 Pericentromic heterochromatin Colocalization

Lyst et al. [222] PIAS1/PIAS3 E3 SUMO (small ubiquitin-like Y2H See [230]
modifier)-ligases
Uchimura et al. [231] MCAF1 Transcriptional co-factor Y2H
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Villa et al. [221] HDAC3 Histone deacetylase CoIP TRD

Sakamoto et al. [224] HPC2 PRC1 component Y2H (Cxxc domain)
Co-IP (HeLa)
RING1B PRC1 component
Xu et al. [232] ␥H2AX DNA-damage associated histone Co-IP (PANC1)
MDC1 Cell cycle checkpoint Co-IP (PANC1)
CHAF1A, Chromatin Assembly Factor 1, Subunit A (P150), also known as CAF-1; ␥H2AX, phosphorylated Histone H2A.X; HDAC3,
histone deacetylase 3; HPC2 also known as CBX2; MCAF1, MBD1-containing chromatin-associated factor 1, also known as AFT7IP1 or
AM; HP1, heterochromatin protein 1; MDC1, Mediator of DNA damage checkpoint 1; MPG, N-methylpurine-DNA glycosylase; MCAF2,
MBD1-containing chromatin-associated factor 2, also known as AFT7IP2; PIAS1/3, protein inhibitor of activated STAT-1/3; SETDB1, SET
Domain, Bifurcated 1; SP1, specificity protein 1; RING1B, Ring Finger Protein 1B.
384 and other MBDs may depend on the local chromatin others have found that it binds to both 5mC and 5hmC 434
385 environment or DNA sequence. [59]. Kaiso acts as a transcriptional repressor [80] and 435
386 Based on the observation that mCH increases has been identified as a co-factor of N-CoR [83]. 436
387 specifically in the brain during postnatal develop- Other members in the Zinc finger/ Kaiso 437
388 ment, Gabel et al. investigated how MeCP2, which family include ZBTB38 (ZENON) and ZBTB4 438
389 also exhibits high postnatal expression, binds to dif- (KIAA1538). The expression of ZBTB38 is restricted 439
390 ferent methylated dinucleotide pairs [75]. They found to the brain and specifically to differentiating 440
391 that MeCP2 binds to mCG, mCA and 5hmCA with neurons—it is highly expressed during development 441
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392 relatively high affinity and to mCC and mCT with and continues in adult [86, 87]. ZBTB4 is expressed 442
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393 low affinity. Moreover, they found that mCA was in multiple adult tissues, but not in embryonic stages 443
394 enriched in longer genes, longer genes are more [87]. Binding of both ZBTB38 and ZBTB4 is methy- 444
395 enriched in the nervous system, and that changes lation dependent [87] and ZBTB4 also binds to the 445
396 in gene expression in the absence of MeCP2 corre- Kaiso non-methylated consensus sequence [88]. 446
397 sponds with both of these factors [75]. The correlation
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398 between MeCP2 loss and upregulation of long genes SRA domain family 447
399 has also been confirmed in expression profiles from

400 various neuronal subtypes [76]. Another study found UHRF1 and UHRF2 are ‘hub’ proteins that contain 448
401 that genes that acquire more mCH methylation after multiple epigenetic interaction domains including a 449
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402 birth are more likely to be differentially expressed in ubiquitin-like (UBL) domain, tandem Tudor domain 450
403 MeCP2 models, adding further support to the idea (TTD), plant homeodomain (PHD) finger domain, 451
404 that binding of MeCP2 to non-CG methylation is SET and RING associated (SRA) domain, and really 452
405 critical for gene regulation [77]. In addition, both interesting new gene (RING) finger domain [89]. The 453
406 mCG and mCH appear to be associated with nucleo- TTD and PHD domains are responsible for interac- 454
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407 some positioning [44, 46, 78], indicating that non-CG tions with other epigenetic proteins such as DNMT1 455
408 methylation may be linked to other epigenetic mech- and histones [90]. The SRA domain is responsible for 456
409 anisms. These studies indicate that it is important recruitment to hemi-methylated DNA [91]. Crystal- 457
410 to consider more than just CG methylation when ization of the SRA domain of UHRF1 revealed that 458
411 evaluating MBP binding or changes in DNA methy- binding of UHRF1 to DNA is not sequence depen- 459
lation. DNA methylation is increasingly viewed as a

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412 dent, as the SRA domain only makes contact with the 460
413 dynamic modification that can be interpreted by the hemimethylated CpG [92–94]. UHRF1 (also known 461
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414 cell in a variety of ways to mediate gene regulation. as Np95) is essential in maintaining DNA methyla- 462
415 Binding by MBPs to DNA is a major mechanism that tion through cell division as it recruits DNMT1 to 463
416 cells use to interpret methylation status. hemi-methylated sites [95, 96]. UHRF2, which is also 464
known as Np95/ICBP90-like RING finger protein 465
417 Zinc ﬁnger family (NIRF), in contrast, does not maintain DNA methyla- 466
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tion during DNA synthesis [97] but is involved in cell 467
418 Kaiso (also known as ZBTB33) is a member of cycle progression and DNA damage repair. UHRF1 468
419 the BTB/POZ family of zinc-fingers (ZF) and con- was shown to bind to 5mC and 5hmC with similar 469
420 tains three copies of a Krüppel-like C2 H2 zinc finger. affinity [70]. This finding was confirmed by [59] who 470
co
421 Kaiso was originally identified as a binding partner also showed that UHRF2 does not have any affinity 471
422 of p120 catenin [79]. It was later found to be part for methylated DNA. Although further research is 472
423 of the MeCP1 complex—a methylated DNA bind- needed, this result is consistent with UHFR’s role in 473
424 ing complex that also contains MBD2 and NuRD the DNA damage response which is connected to the 474
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425 [80]. Kaiso is unique in that it binds to mCpG sites demethylation and BER pathways. 475
426 in a methylation-dependent manner but also binds

427 to a specific sequence (TCCTGCNA) similar to a Methylation-speciﬁc transcription factors 476
428 transcription factor [80–83]. Recent crystallization of
429 the zinc-finger added insight into how two different DNA methylation may also be interpreted/ 477
430 types of binding are mediated by the same domain influenced by transcription factors that are sensitive 478
431 [84]. There are conflicting reports on Kaiso’s abilty to DNA methylation, or that require DNA methy- 479
432 to bind to hydroxymethylated DNA as some groups lation to bind. Because many TF display cell and 480
433 have found that it does not bind to 5hmC [85] while developmental-stage specificity, they may represent 481
482 a mechanism for fine-tuning the DNA methylation the adult. Technological advancements, such as the 531
483 profile of different cell types. The relationship ability to detect DNA modifications beyond classical 532
484 between TF and DNA methylation may function CpG methylation, have greatly expanded our knowl- 533
485 according to several different models (reviewed in edge of the function of DNA methylation in the brain. 534
486 [Reference 98]). Briefly, TFs can project specific Mounting evidence indicates that altered neuro- 535
487 sequences from DNA methylation. For example, genesis contributes to many pathological conditions 536
488 deletion of the TF SP1, which binds to the consensus [5, 6]. Adult neural stem/progenitor cells play a 537
489 motif CCGCCC, has been shown to increase DNA major role in normal brain functions and the brain’s 538
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490 methylation at a promoter with SP1 binding sites response to injury and disease. Many neurodevelop- 539
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491 and to decrease gene expression [99]. Second, some mental, psychiatric, and neurodegenerative disorders 540
492 TF complexes may promote DNA methylation. are characterized by symptoms that have been linked 541
493 Although not a transcription factor itself, SETDB1, to reduced adult neurogenesis, such as depressive 542
494 a H3K9 methyltransferase, interacts with DNMT3A behaviors and memory and learning deficits [107]. 543
495 and DNMT3B and is recruited to shared sites via Knockout and conditional deletion models have been 544
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496 TRIM28 and associated zinc-finger TFs [100, 101]. used to elucidate how the loss of DNA methylation- 545
497 Third, there is some evidence that TF binding related proteins effects neurogenesis. Although the 546
498 can reverse methylation. In REST knock out ES picture is not entirely complete, it is known that 547
499 cells, sites surrounding REST binding motifs were MBDs, DNMTs and TET proteins play a role in 548
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500 methylated while they were not in WT cells, and some stages of AHN ranging from stem cell main- 549
501 reintroduction of REST into these cells removed the tenance and proliferation to neuronal differentiation 550
502 inappropriate methylation [102]. Finally, binding and maturation (Fig. 2, Table 2). 551
503 of TFs can reinforce repression of methylated

504 regions. CpG methylation influences how E2F TFs DNA methylation 552
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505 bind to their recognition sites, which contain two
506 possible methylation sites (T/CTTC/GG/CCGC/G); Neurogenesis occurs before astrogenesis during 553
507 bi-methylation inhibits binding of all E2F TFs, but embryonic brain development; therefore, suppressing 554
508 methylation of one site inhibits binding of E2F1 but astrocytic genes in NSCs is a vital step in the tempo- 555
509 not of E2F2-E2F5 [103]. E2F TFs are well known as ral fate specification of these cells. DNA methylation 556
regulators of the cell cycle, but during neurogenesis is a major mechanism through which this occurs. For
d
510 557
511 E2F3 and E2f4 bind to the promoters of many path- example, during neurogenic phases the STAT3 bind- 558
cte
512 ways involved in cell fate and differentiation such as ing sites in the promoters of Gfap (Glial Fibrillary 559
513 members of the Notch, Wnt, and Fgf pathways [104]. Acidic Protein) and s100␤ are highly methylated, 560
but at later stages that corresponding to astrogenesis 561
they become demethylated, allowing STAT3 to bind 562
514 DNA METHYLATION AND [108, 109]. A similar phenomenon occurs in ESCs: 563
rre
515 NEUROGENESIS the Gfap promoter is highly methylated but becomes 564
demethylated in ESC-derived astrocytes [110, 111]. 565
516 The epigenetic landscape of the brain is unique: it Binding of NFIA (Nuclear Factor I/A), a down- 566
517 has a specific pattern compared to other tissues [105] stream mediator of Notch and JAK/STAT activation, 567
co
518 characterized by high levels of non-CpG methyla- at the Gfap promoter has been shown to lead to DNA 568
519 tion and hydroxymethylation, which are exclusive to demethylation and gene activation via dissociation of 569
520 the brain [106]. In addition, many epigenetic factors, DNMT1 [112]. 570
521 such as MBPs, are highly and/or uniquely expressed The early embryo undergoes large-scale DNA 571
Un
522 in the brain during development and beyond. These methylation remodeling, but the changes that occur 572
523 observations indicate that establishing, maintaining in DNA methylation during brain development are 573
524 and interpreting DNA methylation serves an impor- less clear. A large study that used in vitro neural dif- 574
525 tant function in the brain. These roles can be divided ferentiation showed that the DNA methylation state 575
526 into two general processes: 1) to help establish the of a cell undergoes large changes as mouse embry- 576
527 diverse cell fates found in the central nervous sys- onic stem cells (mESCs) transition to restricted neural 577
528 tem and 2) to alter gene expression programs based progenitor cells and differentiated neurons [113]. 578
529 on inputs received by the cell. These two processes Newly-methylated genes are associated with pluripo- 579
530 occur in neurogenesis during development and in tency, early embryonic development, or germ line 580
f
roo
rP
tho
d Au
Fig. 2. Schematic drawing illustrates the two neurogenic regions in the adult rodent brains and the stages of neurogenesis regulated by DNA
methylation-related epigenetic proteins. Top, Adult neurogenic niches in the brain include the dentate gyrus (DG, blue) and the subventricular
cte
zone (SVZ, purple). Bottom, the stages of DG neurogenesis and regulation by epigenetic proteins.
581 development while the majority of the demethylated is high at embryonic day 10.5–13.5, correspond- 599
582 genes are brain-related, supporting the hypothesis ing with peak neurogenesis, but is not detectable 600
583 that DNA methylation in general acts to repress after E13.5 [118]. In contrast, in human ESCs 601
rre
584 genes. Another important finding of this study is that RNAi-mediated knockdown of DNMT3B resulted in 602
585 patterns of DNA methylation correlate more strongly accelerated maturation of the neuroepithelium and 603
586 with histone modifications than with the underly- precocious expression of neuronal markers such as 604
587 ing genetic code, highlighting the interplay between NEUROD1 coupled with loss of Polycomb repressive 605
co
588 these two systems [114]. complex (PRC) component EZH2 at the promoters 606
of early neuronal genes [119]. Does DNMT3B pro- 607
589 DNMTs mote or suppress neuronal programs? Are the effects 608
specific for developmental windows? DNMT3A-null 609

Un
590 The establishment of DNA methylation during mice appear normal at birth, but die at around 4 weeks 610
591 development is essential as all DNMT null mutations of age [117]. The specific roles of DNA methyltrans- 611
592 (except DNMT2 and DNMT3L) are lethal in embry- ferases in development has been investigated using 612
593 onic stages or shortly after birth [115]. DNMT1-KO cell-type specific deletion models. 613
594 mice develop neural tube defects (NTDs) and die Deletion of DNMT1 in the entire CNS by E9-10 614
595 by E9 [116]. Likewise, homozygous mutations in via crosses with Nestin-cre mice results in exten- 615
596 DNMT3B, which is highly expressed in the embry- sive hypomethylation in cells across the CNS [120]. 616
597 onic neural ectoderm, leads to NTDs and death by These mice display precocious astrocytic differentia- 617
598 E9.5 in mice [117]. DNMT3B expression in the brain tion linked to hypomethylation of astrocytic genes 618
Table 2
DNA methylation related proteins in AHN
Mouse model AHN Stage Direction Details Ref
DNMT1 Nestin-CreERT2 cKO NSC maintenance No change Type 1 cells, 36 dpi Tamoxifen [124]
Proliferation No change Ki67+, BrdU+(24 hrs), 36 dpi
Tamoxifen
Survival ↓ BrdU+, 28 dpi BrdU
Synapsin1-Cre Maturation No change DCX+
DNMT1/DNMT3A CamK-Cre dKO Maturation ↓ Reduced hippocampal volume [126]
f
and impaired spatial memory
roo
DNMT3A KO, 4 week-old mice Proliferation No change Ki67+ [123]
Neuronal differentiation ↓ DCX+
Neuronal survival No change TUNEL+
TET1 KO NSC maintenance No change Type 1 cells [141]
KO and Nestin-CreERT2 cKO Proliferation ↓ Nestin-GFP+, BrdU+Ki67+
Nestin-CreERT2 cKO Neuronal differentiation ↓ 6 weeks pi Tamoxifen:
DCX+BrdU+ (7 dpi),
rP
NeuN+BrdU+ (21 dpi)
KO Behavior ↓ Learning and memory MWM
TET1 KO Neuronal diff/survival No change NeuN hippocampal density [142]
Behavior ↓ Memory extinction in MWM and
contextual fear conditioning
↓
tho
MeCP2 Nestin-Cre cKO Maturation Reduced neuronal cell body [166]
MeCP2 Retrovirus sh-MeCP2 Maturation ↓ Reduced dendritic branching and [170]
spine development
MeCP2 MeCP2S421A Proliferation ↓ Ki67, BrdU (24 hrs) [174]
Neuronal Differentiation ↑ NeuN+BrdU+ (4 weeks pi)
MBD1 KO Fate specification ↓ neurons BrdU+NeuN+, BrdU+GFAP+ [163]
↑ astrocytes
Au (4 weeks pi)
MWM, Morris Water Maze.
619 and activation of the JAK-STAT pathway [121]. also leads to defective neuronal differentiation and 644
620 This result mirrors the findings of the role of DNA eventual wide-spread neuronal death [125]. These 645
methylation in astrocyte promoter activation and results indicate that DNA methylation in postnatal
d
621 646
622 fate specification. Likewise, mice with conditional neurogenesis is required for neuronal differentiation 647
cte
623 deletion of DNMT3A in nestin-expressing cells are and survival, as opposed to embryonic neurogene- 648
624 apparently normal but lose DNA methylation at the sis where DNA methylation contributes to cell fate 649
625 Gfap promoter [122]. These mice die during young specification. However, differences between in vivo 650
626 adult hood, most likely due to defects in the neu- and in vitro models may also contribute to some of 651
627 romuscular junctions of the diaphragm that cause these differences. Further exploration of how DNA 652
rre
628 respiratory failure [122]. methylation is altered in these mutants is needed to 653
understand the mechanisms regulating neurogenesis. 654
629 DNMTs and adult neurogenesis DNA methylation also likely contributes to neu- 655
630 The observation that DNMT3A-null mice appear ronal maturation. In addition DNMT1 and DNMT3A 656
co
631 normal at birth but die by 4 weeks of age, suggests a may have overlapping functions in post-mitotic 657
632 role for DNMT3A in postnatal de novo methylation. neurons. Only Camk2a-cre double KO, which 658
633 A group that evaluated postnatal neurogenesis in the affects neurons in the hippocampus, resulted in an 659
634 DG and SVZ found that the number of DCX+ imma- observed phenotype [126]. These mice had smaller 660
Un
635 ture neurons is greatly reduced, though there are no hippocampi, impaired spatial memory, increased 661
636 changes in proliferation [123]. This group found that expression of genes associated with synaptogenesis, 662
637 DNMT3A promotes the transcription of a number of and reduced DNA methylation [126]. DNMT- 663
638 neurogenic genes by antagonizing PRC2-mediated mediated methylation in post-mitotic neurons is also 664
639 repression. DNMT1 is also involved in adult neu- important in other regions of the brain. For example, 665
640 rogenesis: deletion of DNMT1 in DG aNSC does Emx1-cre Dnmt1 conditional knockout mice fail to 666
641 not change cell proliferation, but does reduce new generate the correct structures in the somatosensory 667
642 neuron survival [124]. Retinal-specific Dnmt1 dele- barrel cortex and do not establish long term poten- 668
643 tion from the onset of neurogenesis by Chx10-Cre tiation (LTP) [127]. Deletion of Dnmt3a driven by 669
670 Sim1-Cre (single-minded1) causes obesity and over- defects in head development, indicating that TET1 720
671 growth phenotypes in mice [128]; SIM1 is expressed and TET2 are likely involved in neural development 721
672 in a subpopulation of in neurons of the paraven- [139]. Deletion of TET3 in mice causes perinatal 722
673 tricular hypothalamus (PVH) and amygdala that are lethality early in development, but it is not known 723
674 known to contribute to energy homeostasis [129]. whether or not neurological defects contribute to this 724
675 Because some phenotypes of DNMT3A overgrowth outcome [140]. Overexpression of TET2 or TET3 by 725
676 syndrome (discussed below) are recapitulated by loss in-utero electroporation enhanced cortical neuronal 726
677 of DNMT3A in SIM1 neurons, it suggests that de differentiation whereas knock-down via sh-RNA 727
f
678 novo methylation may be more critical to the func- reduced the progression of neuronal differentiation 728
roo
679 tion of specific subpopulations of neurons which may and resulted in the accumulation of progenitor cells 729
680 contribute to the disease phenotype in different ways. [136]. As described previously, other studies have 730
681 Interestingly, deletion of MeCP2 in SIM1 expressing found that DNA methylation is lost on neurogenic 731
682 cells also causes obesity, highlighting the importance genes during neuronal differentiation, which is con- 732
683 of both the establishment and interpretation of DNA sistent with a role for TET proteins in this process. 733
rP
684 methylation in this specific population of neurons
685 [130]. In Purkinje neurons, DNMT3B is responsi- TET and adult neurogenesis 734
686 ble for de-novo methylation of protocadherin genes Even though TET1 deletion is compatible with life, 735
687 that results in stochastic and monoallelic expression multiple groups have shown that its loss impacts adult 736
tho
688 of different cadherin isoforms in individual neurons, neurogenesis. In TET1-KO mice there are no changes 737
689 a mechanism hypothesized to contribute to dendritic in the proliferation of RGLs but proliferation of nIPCs 738
690 arborization by self-avoidance, synaptogenesis and is reduced, leading to the generation of fewer new 739
691 circuit formation [131]. These studies indicate that neurons [141]. TET1-KO animals also have learning 740
692 de novo DNA methylation is required in post-mitotic and memory deficits [142]. Multiple studies indicate 741
Au
693 neurons, thought the extent to which this is required that TET1 controls the expression of neuronal- 742
694 for maturation or integration of input signals partic- activity related genes such as Bdnf (Brain-derived 743
695 ularly in the hippocampus remains to be determined. neurotrophic factor) and c-Fos and that 5hmC is a key 744
intermediate in this regulation [51, 142–144]. How- 745
696 5hmC and TET ever, other labs have found that demethylation is not 746
necessarily associated with increased transcription of

d
747
697 Multiple studies have shown that 5hmC is much neuronal genes during neuronal differentiation [136]. 748
cte
698 more abundant in brain tissues relative to other tis- There is growing evidence that 5hmC, irrespective 749
699 sues of the body [132–134]. In adult cells, 5hmC is of its role in DNA demethylation, may be involved 750
700 abundant in the neurons relative to other cell types in transcriptional regulation, maintenance of bivalent 751
701 or cancer cells, constituting 0.6% of all nucleotides chromatin states and neuronal differentiation. TET1 752
702 in Purkinje neurons and 0.2% in cerebellar granular most likely does not bind to methylated DNA on its 753
rre
703 neurons [135]. The abundance of 5hmC increases as own: it likely recruits or is recruited by other fac- 754
704 neurons differentiate and mature in both embryonic tors. So far, three mechanisms have emerged that 755
705 and adult models [136, 137]. Multiple studies indi- might couple 5hmC distribution, TET1 binding pat- 756
706 cate that genes that gain or are enriched for 5hmC terns, and gene expression. First, multiple reports 757
co
707 are important for neuronal differentiation [56, 136]. have shown that TET1 and Polycomb (PcG) binding 758
708 5fC and 5caC, derivatives of 5hmC, also accumu- overlaps significantly genome-wide and that TET1 759
709 late in differentiating human ESCs (hESCs), in mouse binds to PcG proteins targets [145–148]. PcG pro- 760
710 embryos during peak neurogenesis (E11.5-13.5), and teins play a critical role in regulating pluripotency 761
Un
711 during initial differentiation of aNPCs [138]. genes and TET1 and TET2 knock-down leads to 762
712 TET proteins have garnered significant interest decreased expression of pluripotency genes [53], 763
713 in recent years for their role in activity-mediate indicating that DNA demethylation may also be 764
714 demethylation, but they appear to be involved in early involved in resolving bivalent chromatin. Second, 765
715 neuronal development as well. All TET proteins are TET1 may be involved in recruiting the NURD 766
716 expressed in the brain; TET3 is expressed at the (Nucleosome Remodeling and Deacetylase) repres- 767
717 highest level, followed by TET2 and TET1 [132]. sive complex. There is evidence indicating that the 768
718 About half of TET1-KO; TET2-KO mice survive to NuRD complex is targeted to specific genomic loci 769
719 adulthood and are fertile; but those that die exhibit by TET1 and 5hmC. MBD3, which is part of NuRD 770
771 complex, co-localizes with TET1 across the genome, MBDs and neurogenesis 820
772 MBD3 knock down affects expression of genes with MeCP2 performs dual complementary functions 821
773 5hmC, and localization of MBD3 is TET1 dependent during embryonic fate specification of NPCs to 822
774 [149]. PRC2 and the MBD1-NURD complex also neuronal and astrocytic fates. MeCP2 represses astro- 823
775 co-regulate a set of common targets that are char- cyte genes during neurogenesis, and this repression 824
776 acterized by bivalent chromatin state (H3K27me3 must be released during astrogenesis. For example, 825
777 and H3K4me3), highlighting the possible interplay of during neurogenic phases, the s100␤ promoter is 826
778 multiple epigenetic pathways [150]. Third, a recent highly methylated, but at later stages that correspond 827
f
779 report indicates that TET1 may be recruited to a to astrogenesis, it becomes demethylated, releasing 828
roo
780 portion of its genomic loci in by Lin28Aa protein MeCP2 during astrogenesis [109]. Other evidence 829
781 which was previously thought of as an RNA bind- suggests that the presence of MeCP2 supersedes 830
782 ing protein [151]. Nor are the interacting partners of other fate specification cues because the expression 831
783 TET1 limited to the mechanisms described above. of MeCP2 in transplanted neuroepithelial progenitors 832
784 TET1 Co-IP identified methyl-CpG binding proteins promotes neuronal differentiation even when cells 833
rP
785 MeCP2 and UHRF1, histone modifying proteins are transplanted into non-neurogenic brain regions 834
786 including several HDACs, SIN3A and LSD1 (lysine [164]. In neurons, astrocyte induction cues were not 835
787 specific demethylase), and PCNA (proliferating cell sufficient to increase GFAP expression because the 836
788 nuclear antigen) [148]. Binding competition at 5mC promoters of astrocytic genes Gfap and s100␤ were 837
tho
789 may be one mechanism of TET1 regulation; one study found to be highly methylated and repressed by 838
790 found that MeCP2 inhibited TET1 activity [152]. MeCP2 [165]. It is still unclear if MeCP2 and other 839
791 In addition 5hmC levels in the mouse cerebellum MBPs inhibit astrocyte genes directly, or if they also 840
792 have been correlated with MeCP2 gene dosage [153]. regulate the major pathways that promote gliogene- 841
793 In summary, the precise role of TET proteins and sis. It is also possible that many of these experiments 842
Au
794 DNA demethylation at different stages of develop- may be of limited biological relevance because the 843
795 ment is still unclear. In addition, the crosstalk between expression of MeCP2 is relatively low during embry- 844
796 TET proteins and MBPs in the context of neural onic development relative to its postnatal expression. 845
797 development, whether it is facilitative (MBD3) or However, the majority of MeCP2 research involves 846
798 antagonistic (MeCP2), warrants further exploration. the loss of MeCP2, which is more relevant for neu- 847
ronal maturation rather than fate specification. NSCs

d
848
derived from the cortex of E13.5 MeCP2-KO mice 849
MBDs
cte
799 do not display altered fate specification for neurons, 850
astrocytes or oligodendrocytes relative to controls 851

800 MeCP2, MBD1 and MBD5 are highly expressed [159]. Nor do MeCP2-null brains display obvious 852
801 in the brain. MeCP2 is highly expressed in neurons structural deficits associated with altered cell fate 853
802 throughout the brain of adult rodents and primates and specification [166]. Rather, MeCP2-KO mice, sim- 854
rre
803 expression correlates with age [154–157]. MeCP2 ilar to humans with RTT [167–169], have neurons 855
804 is expressed in some nestin-positive precursor cells with smaller nuclei, reduced dendritic branching and 856
805 from the neuroepithelium [158] and its expres- immature spines [170]. 857
806 sion increases with neuronal differentiation [159]. Both MBD1 and MeCP2 play a role in aNSC 858
Although initial reports focused on MeCP2 was
co
807 fate specification. In NPCs, oligodendrocytes, and 859

808 expression in neurons [158, 159], subsequent reports astrocytes in vitro, both MeCP2 and MBD1 are 860
809 have identified it in astrocytes and oligodendrocytes localized to the RE1/NRSE region of the mGluR2 861
810 [160] and confirmed MeCP2 is not expressed in promoter, which is suppressed in non-neuronal cell- 862
Un
811 microglia [161]. MBD1 is expressed in the brain types [171]. This suggests that MBD1 and MeCP2 are 863
812 by P7 (postnatal day 7), but it is not known if it involved in suppressing neuronal genes in astrocytes, 864
813 is expressed earlier [162]. In adult mice, MBD1 is which is distinct from MeCP2’s function in embry- 865
814 expressed in neurons throughout the brain, though its onic NSCs. A role for MBD1 in fate specification is 866
815 expression is particularly high in the DG and CA1 of supported by differentiation analysis of MBD1-KO 867
816 the hippocampus [163]. MBD5 is highly expressed animals injected with DNA analog BrdU (5-Bromo- 868
817 in the brain relative to other tissues [67]. This expres- 2’deoxyuridine): 4 weeks after BrdU injection, the 869
818 sion pattern is strongly indicative of a role of MBDs number of BrdU-labeled neurons decreases greatly, 870
819 in brain development and function. while the number of astrocytes increases slightly
871 [163]. These mice also show signs of depression, and responsible for the regulation of DNA methylation and 920
872 exhibit impaired spatial learning, and reduced long- human disease are of great interest. Indeed, mutations 921
873 term potentiation believed to be a result of decreased in a proportion of DNA methylation associated-genes 922
874 adult neurogenesis [172]. However, in the function have devastating impacts on brain development and 923
875 of MBD1 in NSCs is unexplored and it is not clear function. Deficits in AHN may contribute to the eti- 924
876 what stage of NSC development leads to reduced ology of some of these disorders [179]. However, 925
877 neurogenesis. In summary, many questions remain mutations in others may be related to other brain 926
878 about how MeCP2 and other MBDs regulate stem regions and functions. But even in situations where 927
f
879 cell maintenance, proliferation, or fate commitment. there is no direct link between an epigenetic factor and 928
roo
880 Although MeCP2 seems to play a more promi- AHN in humans, studies of AHN can be used to eluci- 929
881 nent role in neuronal maturation, it may also function date the function of a protein in different cells ranging 930
882 in NSCs and other cell types. MeCP2 expression in from stem cells through new neurons. Moreover, AHN 931
883 astrocytes has recently been shown to have signifi- is a useful model system because aNSCs generate a rel- 932
884 cant impact on neuronal function and RTT pathology atively homogenous population of neurons that pass 933
rP
885 [160, 173]. Since astrocyte-“specific” manipulations through well-defined developmental stages with con- 934
886 utilize the promoter of Gfap, a gene that is also sistent timing. Information gained from such studies 935
887 expressed in NSCs, it is possible that some of these may provide important mechanistic insights into dis- 936
888 effects might be, at least in part, due to changes in orders that effect the brain, but which are not primarily 937
tho
889 NSCs. In addition, the phosphorylation-incompetent caused by defects in AHN, such as neurodegeneration 938
890 MeCP2 mutant S421A reduced proliferation of and caused by mutations in DNMT1 (discussed below). 939
891 enhanced neuronal differentiation in the adult dentate The scope of disorders linked to DNA methylation 940
892 gyrus, indicating that MeCP2 function is important in offers insight into the specific role of DNA methy- 941
893 NSC/NPCs [174]. lation in the brain compared to other cell types and 942
Au
894 MeCP2 has been shown to play an important tissues. 943
895 role in translating neuronal activity and signaling

896 cascades into epigenetic gene regulation. Because DNMTs 944
897 phosphorylation is a dynamic modification, MeCP2

898 phosphorylation can integrate activity-induced sig- Mutations in DNMT3A have recently been associ- 945
naling and gene expression [175]. For example, Chen ated with human disease. Researchers searching for
d
899 946
900 et al. 2003 found that depolarization of neurons led genes responsible for overgrowth disorders identi- 947
cte
901 to phosphorylation of MeCP2 and decreased occu- fied heterozygous de novo mutations DNMT3A in 948
902 pancy at the Bdnf promoter [176]. A complementary approximately 10% of overgrowth patients, all of 949
903 study by Martinowich et al. linked activity-dependent whom also displayed moderate intellectual disabil- 950
904 changes in DNA methylation with reduced MeCP2 ity [180]. Mutations in DNMT3A are also frequently 951
905 binding at the Bdnf promoter which corresponded found in acute myeloid leukemia (AML) [181] 952
rre
906 to increased BDNF expression [177]. BDNF signal- and other hematological cancers like myelodysplas- 953
907 ing via its receptor, TRKB (Tropomyosin receptor tic syndromes (MDS) [182]. Mutations that cause 954
908 kinase B), is also essential for AHN [178]. MeCP2 DNMT3A overgrowth syndrome have been found 955
909 phosphorylation also integrates Notch signaling with in all functional domains of the protein and mod- 956
co
910 neurogenesis: phosphorylation-incompetent MeCP2 eling suggests that many mutations interfere with 957
911 mutants S421A exhibited deficits associated with histone binding [180]. Interestingly, other epigenetic 958
912 reduced Notch signaling and could be rescued by proteins seem to play a dual role in growth syn- 959
913 overexpression of the Notch intra cellular domain dromes and myeloid neoplasms. Mutations in EZH2, 960
Un
914 (NICD) [174]. NSD1, and MLL (mixed lymphomic leukemia) cause 961
Weaver, Sotos, and Wiedemann-Steiner syndromes, 962
respectively, and are also associated with hematolog- 963
915 DNA METHYLATION IN DEVELOPMENT ical cancers [180, 183]. This suggests that loss of 964
916 AND DISEASE de novo methylation or the inability to interpret the 965
methylation state contributes to a cell’s oncogenic 966
917 Given the unique methylation landscape in the brain transformation. 967
918 and the contributions of epigenetics to brain develop- In addition, DNMT3A was identified as an autism 968
919 ment and function, connections between the factors spectrum disorder candidate gene by genome-wide 969
970 association from whole-genome sequencing [184]. disorders, including cases of Angelman syndrome, 1020
971 One study suggests that DNMT3A overgrowth Prader-Willi syndrome, autism, and non-syndromic 1021
972 syndrome and possibly the intellectual disability mental retardation [195]. Mouse models have repli- 1022
973 associated with this disorder may be comorbid with cated key phenotypes of RTT and revealed that MeCP2 1023
974 autistic symptoms: a study that integrated two large plays a critical role in neuronal maturation and synap- 1024
975 copy number variation (CNV) data sets identified togenesis, in part through the regulation of dendritic 1025
976 small de novo deletions of DNMT3A in 3 patients morphology, synaptic transmission, and long-term 1026
977 with autism who also had heights and weight charac- plasticity [196, 197]. Both clinically and in mouse 1027
f
978 teristic of over-growth syndromes [185]. models of RTT, deletion or loss of function MeCP2 1028
roo
979 Mutations in DNMT3B results in ICF1 syndrome and duplication both result in similar pathology, high- 1029
980 which is about 50–60% of the cases of ICF syndrome lighting the necessity for its precise control [195]. 1030
981 (immunodeficiency, centromere instability and facial As detailed above, changes in adult neurogene- 1031
982 abnormalities syndrome), a rare autosomal recessive sis have been described for MeCP2 mouse models 1032
983 disorder that leads to CG hypomethylation of peri- [174, 198]. A potential exciting avenue of research 1033
rP
984 centromic satellite regions of chromosomes 1, 9 and is the modulation of adult neurogenesis by various 1034
985 16 [186-188]. About of half of ICF1 patients also pharmacological and non-pharmacological interven- 1035
986 display a high incidence of intellectual disability tions. Because adult neurogenesis is responsive to a 1036
987 [189], indicating that de-novo methylation mediated variety of inputs, it may be possible to counteract 1037
tho
988 by DNMT3B is important for proper brain function some behavioral deficits by increasing or normaliz- 1038
989 and development. ing adult neurogenesis. For example, treatment of 1039
990 Mutations in DNMT1 have been linked to two FXS mice with a GSK3␤ inhibitor was capable of 1040
991 rare neurodegenerative syndromes: autosomal dom- restoring hippocampal-dependent learning and res- 1041
992 inant cerebellar ataxia-deafness and narcolepsy cuing neurogenesis in adult mice [8]. Reactivation 1042
Au
993 (ADCA-DN) and hereditary sensory neuropathy of MeCP2 in post-natal neurons also partially res- 1043
994 with dementia and hearing loss (HSN1E) [190, cues multiple pathologies observed in MeCP2-null 1044
995 191]. Mutations that cause HSN1E prevent bind- mice [7]. These studies suggest that some aspects 1045
996 ing of DNMT1 to heterochromatin during the G2 of neurodevelopmental-associated pathology may be 1046
997 phase of the cell cycle, leading to global DNA reversible and may offer useful information in the 1047
hypomethylation and selective hypermethylation development of therapies.

d
998 1048
999 [190]. Whole genome bisulfite sequencing of HSN1E MBD5 was identified as the critical region con- 1049
cte
1000 patients reveled that differentially methylated regions tributing to 2q23.1 microdeletion syndrome [199, 1050
1001 (DMR) are associated with neurological disorders 200]. Microdeletions of that disrupt MBD5 cause 1051
1002 and NAD+ /NADH metabolism [192]. Defects in a spectrum of neurodevelopmental phenotypes that 1052
1003 DNA methylation have been identified in many share some behavioral, developmental, and neu- 1053
1004 neurodegenerative diseases including Alzheimer’s rologic features with autism, Rett syndrome, and 1054
rre
1005 disease, Parkinson’s disease and Huntington’s dis- other disorders [200]. In addition to being a hotspot 1055
1006 ease, reviewed in [193]. for microdeletions, mutations in Mbd5 have been 1056
identified in ASD patients [185, 201–203]. Also, 1057
1007 MBPs duplications of MBD5 have been identified in patients 1058

co
with similar features as those with the gene dis- 1059
1008 Mutations in MBDs have been linked to mul- ruption, indicating that gene dosage of MBD5, like 1060
1009 tiple neurodevelopmental disorders that manifest MeCP2, is critical for neural development [204, 1061
1010 with varying degrees of severity. A common thread 205]. Mice with MBD5 deletions display craniofa- 1062
Un
1011 between MBPs is autism-like symptoms, with dis- cial abnormalities and decreased motor function and 1063
1012 ruptions to MeCP2 representing the most severe and altered social interaction [206]. The contribution of 1064
1013 MBD1 the most mild [194]. The similar yet distinct developmental or adult neurogenesis to these pheno- 1065
1014 phenotypes associated with MBDs suggest that over- types has not been examined but may be affected. 1066
1015 lapping but distinct mechanisms contribute to disease In humans, mutations or polymorphisms in MBD1 1067
1016 etiology. have been identified in sporadic cases of autism 1068
1017 Mutations in MeCP2 are the cause of the neurode- spectrum disorder (ASD) [202, 207]. MBD1 is also 1069
1018 velopmental disorder Rett syndrome (RTT), but they contained within the region of del (18) (q12.2q21.1) 1070
1019 are also associated with a number of other neurological syndrome, which has also been identified in some 1071
1072 cases of atypical Rett syndrome [208, 209]. Mice network influences other pathways is vital in under- 1121
1073 with MBD1 deletion (MBD1-KO) exhibit behav- standing the system as whole. 1122
1074 ioral deficits associated with ASD, including learning MBD1 is important in linking DNA methylation 1123
1075 impairment, increased anxiety, reduced social inter- to the regulation of gene expression in combina- 1124
1076 est, and impaired sensorimotor gating [163, 172]. tion with other epigenetic and regulatory pathways. 1125
1077 These phenotypes are consistent with a role for MBD1 MBD1 binds directly to the promoter and reg- 1126
1078 in the fate specification and differentiation of aNSCs. ulates the expression of Fgf-2, a molecule that 1127
1079 So far, Kaiso has only been associated with can- promotes proliferation and is commonly used to 1128
f
1080 cer [210] and little is known about its function in the expand NSC populations. Furthermore, a DNA 1129
roo
1081 brain. Kaiso KO mice show no overt developmental methylation inhibitor blocked the effects of MBD1 in 1130
1082 phenotype, nor does its loss effect neural stem cell forebrain-derived aNSCs and increased Fgf-2 expres- 1131
1083 fate decision or stem cell maintenance [211]. How- sion, underscoring the important role the methylation 1132
1084 ever, another group that analyzed the behavior of the state of the promoter plays [217]. In part, MBD1 1133
1085 same mice found that the KO mice had increased regulates neurogenesis by silencing miR-184; inhibit- 1134
rP
1086 locomotion, exploratory behavior and sensorimotor ing miR-184 hinders proliferation and promotes 1135
1087 gating as well as shrunken ventricles [212]. A dou- the differentiation of hippocampal DG adult NSCs 1136
1088 ble KO of Kaiso and MeCP2 does not worsen the in vitro. In turn, miR-184 binds the 3’ UTR of 1137
1089 MeCP2-KO phenotype [213], indicating that Kaiso is Numblike (Numbl), a signaling protein required 1138
tho
1090 not functionally redundant to MeCP2. It is unknown if for differentiation in adult neurogenesis, and tar- 1139
1091 ZBTB38 and ZBTB4 have a brain-specific function. gets it for degradation [218]. Numbl is known to 1140
1092 UHRF2 is expressed in the adult brain at compara- inhibit the Notch pathway [219], which has a signifi- 1141
1093 ble levels to other tissues in the body, while UHRF1 cant impact on neurogenesis. Inhibition of miR-195, 1142
1094 is expressed primarily in pluripotent and proliferative a microRNA repressed by MBD1, increases neu- 1143
Au
1095 cells [214, 215]. Consistent with its function in DNA ronal differentiation in vivo, similar to miR-184 1144
1096 damage and repair, UHRF2 is frequently upregulated [220]. 1145
1097 in cancer [89]. During embryogenesis, UHRF1 is Similar to MeCP2, MBD1 has been found to inter- 1146
1098 highly expressed in proliferating NPCs around E11. act with multiple epigenetic co-factors and it is highly 1147
1099 In the adult hippocampus it is also expressed in likely that MBD1- mediated regulation of adult neu- 1148
proliferating NPCs, suggesting that it is involved in rogenesis requires cooperation from other epigenetic
d
1100 1149
1101 maintaining DNA methylation in this proliferating machinery. MBD1-interactors have been identified 1150
cte
1102 stem cell populations in the brain at multiple stages primarily in cancer cell lines and it is not known if 1151
1103 [216]. these interactions are relevant to MBD1 regulation in 1152
the context of adult neurogenesis (Table 1). HDAC3 1153
can facilitate the regulation of gene expression by 1154
1104 CROSSTALK BETWEEN EPIGENETIC MBD1 in cancer cells [221] but it is not clear gene 1155
rre
1105 PATHWAYS repression is mediated through the recruitment of 1156
HDACs by MBD1 as the HDAC inhibitor trichostatin 1157
1106 DNA methylation is heavily interconnected with A (TSA) does not always activate MBD1-repressed 1158
1107 other epigenetic and regulatory systems such as his- genes [222, 223]. However, MeCP2, MBD1, and 1159
co
1108 tone modifying complexes, and transcription factor HDACs have also been shown to target the RE1 REST 1160
1109 binding. The proteins that mediate the deposition, binding site in a subset of neuronal genes, reflecting 1161
1110 maintenance, interpretation and removal of DNA the possibility that both DNA methylation and histone 1162
1111 methylation are also highly connected with each remodeling are required to mediate gene repression 1163
Un
1112 other. For example, MeCP2 has been found to inter- [171]. In addition, MBD1 has been shown to interact 1164
1113 act with diverse proteins including, but not limited to with PRC1 components HPC2 and RING1B [224], 1165
1114 NCOR-SMRT (nuclear receptor co-repressor), HP1 and PRC2 component SETDB1 [225], indicating a 1166
1115 (heterochromatin protein 1), SOX2, TET1, p300, and possible role in the resolution of bivalent chromatin 1167
1116 CREB1 (cAMP responsive element binding protein states. In addition, in the absence of MBD1, the lev- 1168
1117 1) [197]. In addition, it is becoming increasingly clear els of activating and repressive histone marks are 1169
1118 that signaling pathways and transcriptional regulation altered at the promoter of miR-184 in aNSCs, sug- 1170
1119 pathways are highly connected to epigenetic regula- gesting that MBD1 can influence the recruitment of 1171
1120 tion. Understanding how disrupting one aspect of the histone-modifying proteins [218]. However, exactly 1172
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DNA Methylation

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Brain Plasticity xx (20xx) x–xx 1

2 DNA Methylation and Adult Neurogenesis

22 INTRODUCTION cell functions depend on dynamic regulation of epige- 33

netic programs in addition to maintaining epigenetic 34

roles it plays in the cell. 45

∗ Correspondenceto: Xinyu Zhao, Waisman Center and Depart-

confirmed to have ongoing neurogenesis: the sub- 121

ventricles [9]. New neurons have also been observed 124

98 with partial developmental etiology, such as fragile inputs.

individual genes [32]. Multiple mechanisms likely 200

166 the brain. Plasticity, or the ability to respond and

extrinsic physiological and pathological conditions is catalyzed by a family of DNA methyltrans-

neurogenesis-mediated plasticity is the integration of

179 de novo methylation, whereas DNMT1 maintains 227

43]. And within the brain, non-CG methylation is 236

cells and is estimated to account for 25–38% of 238

254 feasibility of this type of study will allow researchers

256 in the cell. Such as, are DNA methylation patterns

family. In addition, recent work using quantita- 309

and identified many novel methylated DNA binding

excision repair (BER) pathways (reviewed in [3, 49]).

A comparison of genome-wide promoter methyla- 357

tion and RNA Pol II enrichment showed that active 358

transcription is opposite to that of MeCP2, MBD1, 359

MBD2, and MBD4, confirming the general role of 360

these MBDs in gene repression [65]. MBD1 has 361

multiple splice isoforms with the longest form con- 362

taining both MBD domain that can bind to methylated 363

CXXC domains that do not bind to DNA [66]. Thus 367

methylated DNA, although they are localized to het- 369

groups to question whether or not known methylated 374

MBD1 interacting proteins

HP1 Pericentromic heterochromatin Colocalization

Villa et al. [221] HDAC3 Histone deacetylase CoIP TRD

397 sponds with both of these factors [75]. The correlation

399 has also been confirmed in expression profiles from

lation. DNA methylation is increasingly viewed as a

known as Np95/ICBP90-like RING finger protein 465

tion during DNA synthesis [97] but is involved in cell 467

426 in a methylation-dependent manner but also binds

503 of TFs can reinforce repression of methylated

but at later stages that corresponding to astrogenesis 561

they become demethylated, allowing STAT3 to bind 562

demethylated in ESC-derived astrocytes [110, 111]. 565

of early neuronal genes [119]. Does DNMT3B pro- 607

specific for developmental windows? DNMT3A-null 609

understand the mechanisms regulating neurogenesis. 654

intermediate in this regulation [51, 142–144]. How- 745

necessarily associated with increased transcription of

ronal maturation rather than fate specification. NSCs

derived from the cortex of E13.5 MeCP2-KO mice 849

799 do not display altered fate specification for neurons, 850

astrocytes or oligodendrocytes relative to controls 851

807 fate specification. In NPCs, oligodendrocytes, and 859

895 role in translating neuronal activity and signaling

897 phosphorylation is a dynamic modification, MeCP2

Weaver, Sotos, and Wiedemann-Steiner syndromes, 962

respectively, and are also associated with hematolog- 963

methylation state contributes to a cell’s oncogenic 966

hypomethylation and selective hypermethylation development of therapies.

identified in ASD patients [185, 201–203]. Also, 1057

1007 MBPs duplications of MBD5 have been identified in patients 1058

with similar features as those with the gene dis- 1059

1016 etiology. have been identified in sporadic cases of autism 1068