Cell Tissue Res (2014) 356:539–552 DOI 10.1007/s00441-014-1842-8

REVIEW

Histone methylation during neural development Deborah Roidl & Christine Hacker

Received: 26 November 2013 / Accepted: 3 February 2014 / Published online: 13 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Post-translational modification of histone proteins, such as the methylation of lysine and arginine residues, influences the higher order of chromatin and leads to gene activation or silencing. Histone methyltransferases or demethylases actively add or remove various methylation marks in a celltype-specific and context-dependent way. They are therefore important players in regulating the transcriptional program of a cell. Some control of the various cellular programs is necessary during the differentiation of stem cells along a specific lineage, when differentiation to alternative lineages needs to be suppressed. One example is the development of neurons from neural stem cells during neurogenesis. Neurogenesis is a highly organized process that requires the proper coordination of survival, proliferation, differentiation and migration signals. This holds true for both embryonic and neural stem cells that give rise to the various cell types of the central nervous system. The control of embryonic and neural stem cell selfrenewal and differentiation is achieved by both extrinsic and intrinsic signals that regulate gene expression precisely. Recent advances in neuroscience support the importance of epigenetic modifications, such as the methylation and acetylation of histones, as an important intrinsic mechanism for the regulation of central nervous system development. This review summarizes our current knowledge of histone

The authors are grateful to the grantholder, Prof. T. Vogel and the SFB992 Medical Epigenetics “MEDEP” (Deutsche Forschungsgemeinschaft) for funding our research. D. Roidl : C. Hacker (*) Department of Molecular Embryology, Institute of Anatomy and Cell Biology, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany e-mail: [email protected] D. Roidl Hermann Staudinger Graduate School (HSGS), Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany

methylation processes during neural development and provides insights into the function of histone methylation enzymes and their role during central nervous system development. Key words Epigenetics . Histone lysine methyltransferase (KMT) . Histone lysine demethylase (KDM) . Neural development . Chromatin state

List of abbreviations 5hmC CNS COMPASS DOT1L ESC EZH2 H3 H3K4 (same scheme for other modifications) H4R3me2a/s HDAC K KDM KMT KO JARID JHDM JmjC me me1/2/3

5-Hydroxymethylcytosine Central nervous system Complex of proteins associated with Set1 Disruptor of telomeric silencing 1-like Embryonic stem cells Enhancer of zeste homolog 2 Histone 3 Histone 3 lysine 4 Histone 4 arginine 3 asymmetric/ symmetric dimethylation Histone deacetylase Lysine Histone lysine demethylase Histone lysine methyltransferase Knockout Jumonji/ARID domain JmjC domain-containing histone demethylase Jumonji C Methylation Mono- /di- /trimethylation

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The mammalian central nervous system (CNS) comprises varied cell types, such as neurons, astrocytes and oligodendrocytes. These all develop from common neural stem cells (NSC) in a temporo-spatially regulated manner (Fig. 1) during embryonic development. NSC are derived from embryonic stem cells (ESC) that, similar to NSC, have a multilineage differentiation potential that is progressively limited during the development of specific cell types. Both ESC and NSC have the ability to self-renew and must balance their proliferation and differentiation potential in order to produce the correct amounts of differentiated somatic cell types (for a review, see Niwa 2007). In addition, the timing of NSC differentiation into neurons and glial cells is regulated in a manner such that astrogenesis starts only when neurogenesis is almost complete. Moreover, the proper specification of neuronal subtypes must be regulated. Different transcriptional programs need to be activated at different times in order to

promote proliferation at the expense of differentiation and to suppress alternative cell fates during terminal differentiation. These cellular programs depend on specific gene expression patterns, which are tightly connected with the chromatin state, in the cells. Chromatin structure is influenced by posttranslational histone modifications, of which a wide variety are associated with either active transcription or gene silencing (for a review, see Zhou et al. 2011). The modulation of chromatin structure by histone methylation is a fundamental mechanism in ESC and NSC maintenance, in the commitment of ESC to the neural lineage and NSC terminal differentiation, in the the neurogenic-astrocytic switch and in postnatal neurogenesis. Histone modifications are epigenetic mechanisms and poise the cell for the execution of a specific differentiation program upon stimulation by external signals. Epigenetic mechanisms enable both short- and long-term changes in gene expression and cellular function as a response to external conditions. They are therefore now acknowledged as prominent mechanisms for cells to adapt quickly and robustly to changes in the environment. This increases the plasticity of stem and progenitor cells but is also crucial for terminally differentiated cells, as it allows them to continue to interact with their environment. These mechanisms are especially important for postmitotic neurons that are exposed to a variety of environmental stimuli transmitted via the synapse. Mature neurons are responsible for memory formation, learning and experience throughout life. Neurons in particular are therefore highly dependent upon epigenetic mechanisms, which are a prerequisite for the variety of neuroplastic states that are necessary for neuronal morphology, elaborate circuit formation and experience-dependent behavior. Although this review focuses on the impact of histone methylation during neural development, epigenetic mechanisms are clearly of equal importance for the maintainance of postmitotic neurons. Decoding these mechanisms in neural development will help in understanding disease states not only of developmental neurological disorders but also of neurodegenerative diseases, learning disabilities and addiction.

Fig. 1 Overview of cellular states during neurogenesis. Embryonic stem cells (ESC) have the ability to self-renew and the potential for pluripotent differentiation. During neurogenesis, ESC differentiate into neural stem

cells (NSC) that also have self-renewal ability. These tissue stem cells initially produce neuronal cell types during the neurogenic phase and later differentiate into glial cells during the gliogenic phase

MLL NSC NTD PRC1 PRC2 R RE REST RNAPII SAH SAM SET TSS Trx Trr

Mixed-lineage leukemia Neural stem cells Neural tube defects Polycomb repressive complex 1 Polycomb repressive complex 2 Arginine Neuron-restrictive silencer element RE1-silencing transcription factor RNA polymerase II S-adenosyl-homocysteine S-adenosyl-L-methionine Su(var)3-9, Enhancer of Zeste, Trithorax Transcription start site Trithorax Trithorax-related

Introduction

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General insights into histone methylation Epigenetics is defined as the “study of structural adaptations of chromosomal regions that register, signal or perpetuate altered transcriptional states” (Meaney and Ferguson-Smith 2010). This includes the transient and easily reversible posttranslational modifications of histones. Chromatin consists of nucleosomes, which are octamers of four core histones (H2A, H2B, H3, H4) around which 147 base pairs of DNA are wrapped (Fig. 2; Luger et al. 1997). Chromatin can alternate between transcriptionally repressive (heterochromatin) and transcriptionally active (euchromatin) states and thereby controls gene expression. Among other mechanisms, the post-translational modifications of histones mediate these changes. Histones can be post-translationally modified at over 60 sites by acetylation, methylation, phosphorylation, ubiquitination, sumoylation and ADP-ribosylation (Zhang et al. 2013).

Histone methylation marks and their regulation of transcription Histone lysine methylation regulates transcription and modulates other chromatin-related processes such as replication, recombination (for a review, see Zhang and Reinberg 2001) and the DNA-damage response (Sanders et al. 2004). Further, histone methylation has been implicated in heterochromatin formation, X inactivation, genomic imprinting and the silencing of homeotic genes, demonstrating the important role for histone methylation in many biological processes (for a review, see Martin and Zhang 2005).

Fig. 2 Model of a nucleosome. Chromatin consists of histone proteins (blue) around which DNA (gray) is wrapped. Both the N-terminal tails and cores of histones can undergo various modifications including the methylation of lysines (K) and arginines (R). K4, K9, K27 and K36 are found in the N-terminal tail of histone 3 (H3) whereas K79 is localized in the core domain of H3. Histone 4 (H4) harbors R3. Only the modifications described in this article are shown here

Fig. 3 Chemical structure of methylated lysine and arginine. Methyltransferases catalyze the addition of methyl groups to lysine and arginine whereas demethylases remove the methyl mark. Depending on the number of added methyl groups, modifications include mono-, di- and trimethylated lysine residues and mono- and dimethylated arginine residues (R residue)

The lysine (K) residues of histones H3 and H4 can be mono (me1)-, di (me2)- and tri (me3)-methylated, whereas arginine (R) can be mono- or dimethylated (Fig. 3). Modifications are named after the histone, the lysine, or the arginine residue and the amino acid position on which they occur, e.g., H3K4me3 signifies the lysine trimethylation at position four of histone H3. The effect of the various methylation states on transcription depends on the position of the modification within the histone. Known methylation sites include H3R2, H3K4, H3K9, H3R17, H3R26, H3K27, H3K36, H3K79, H4R3 and H4K20 (Fig. 4; for a review, see Kouzarides 2007). Specific histone methylation marks are associated with transcriptional activation and/or repression. Generally, methylation on H3K4 and H3K36 is linked to actively transcribed genes, whereas methylation on H3K9, H3K27 and H4K20 is involved in transcriptional repression. H3K79 methylation is associated not only with transcriptional activity but also with transcriptionally inert genomic regions. Histone marks are distributed in a specific manner over target genes. Within transcribed genes, the H3K4me1/2/3 marks are mostly found at the promoter region or the transcription start site (TSS) but H3K4me1/2 are also associated with enhancers. Whereas H3K79me1/2/3 enrichment is generally found downstream of the TSS within the gene body, H3K36me3 increases toward

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Fig. 4 Site-specific methylation of histone proteins. Specific lysine and arginine residues on H2A, H3 and H4 can be mono-, di-, or trimethylated (yellow). Enzymes that catalyze the methylation are called lysine (K) or

arginine (R) methyltransferases, whereas demethylases remove the methylation mark (term terminal)

the 3′end of active genes (for reviews, see Lee and Mahadevan 2009; Ernst et al. 2011). For repressed genes, such as the pluripotency genes Oct4, Nanog and Sox2 in differentiated cells, H3K27me3 is enriched at but not limited to, the promoter region, whereas H3K9me3 occupies the first exon independently of gene expression levels (Barrand et al. 2010). The presence of histone methylation marks is often interdependent upon other epigenetic marks, such as histone acetylation and DNA methylation. Therefore, accurate insights come not from the analysis of one single methylation mark but rather from the analysis of the whole network of histone modifications. For example, the gain of 5hmC (5hydroxymethylcytosine) DNA methylation is often accompanied by the loss of H3K27me3 during neuronal differentiation (Hahn et al. 2013). On the other hand, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation (Mohn et al. 2008). H2B ubiquitination drives monomethylation (McGinty et al. 2008) and the di- and trimethylation of H3K79 (Zhu et al. 2005; Kim et al. 2005; Mohan et al. 2010) and is required for resolution of the bivalent H3K4me3/H3K27me3 mark (see below; Karpiuk et al. 2012).

identified (Tables 1, 2). One major exception is the methylation of arginines; although it is dynamic, a demethylating enzyme has not yet been described in vivo. Deamination could be one possible antagonistic mechanism. The balance between active methyltransferases and demethylases regulates the degree of methylation at a given histone mark. Regulation of their expression therefore enables the fine-tuning of methylation states and gene transcription in the cell. Histone lysine methyltransferases (KMTs) transfer one, two, or three methyl groups from S-adenosyl-L-methionine (Fig. 5) to the 1-amino group of a lysine residue. With the exception of the H3K79 methyltransferase KMT4/DOT1L, all known KMTs contain a conserved SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain, which mediates the enzymatic activity. Histone lysine demethylases (KDMs) remove methyl groups from lysine residues. The majority of KDMs contain either the Jumonji C (JmjC) domain (JmjC domain-containing histone demethylases, JHDM) or the Jumonji/ARID domain (Jumonji/ARID domain-containing proteins, JARID; for a review, see Klose et al. 2006). Whereas the JmjC domain is necessary for the binding of the cofactors Fe2+ and αketoglutarate, the ARID domain enables binding to DNA. KDM1A/LSD1 and KDM1B/LSD2 do not contain a JmjC domain and represent a distinct family of histone demethylases. KMTs and KDMs can act in combination to coordinate histone modifications at distinct histone residues. Mixedlineage leukemia (MLL) family members that methylate

Enzymes regulating histone methylation Histone methylation is reversible and enzymes that specifically add and remove the methylation mark have been

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Table 1 Summary of histone lysine methyltransferases (KMTs) and demethylases (KDMs). These enzymes catalyze the addition or removal of one, two, or three methyl groups to the 1-amino group of histone lysine residues. The eight KMT and seven KDM subfamilies involved in histone methylation are shown, including their enzymatic activities and substrate specificities. Synonyms of the enzymes are shown in brackets Histone target

Writer KMTs

Eraser KDMs

H3K4me1/2/3

KMT2A (MLL1) KMT2B (MLL2) KMT2C (MLL3) KMT2D (MLL4) KMT2E (MLL5) KMT2F (SET1a) KMT2G (SET1b) KMT2H (ASH2l) KMT3C (SMXD2) KMT3D (SMYD1) KMT3E (SMYD3) KMT7 (SET7/9) KMT1A (SUV39H1) KMT1B (SUV39H2) KMT1C (G9a, EHMT2) KMT1D (GLP/EHMT1) KMT1E (ESET/SETDB1) KMT1F (SETDB2) KMT8A (PRM2/RIZ1)

KDM1A (LSD1) KDM1B (LSD2) KDM5A (JARID1A) KDM5B (JARID1B) KDM5C (JARID1C) KDM5D (JARD1D)

H3K9me1/2/3

H3K27me1/2/3 KMT6A (EZH2) KMT6B (EZH1)

H3K36me1/2/3 KMT3A (SET2) KMT3B (NSD1) KMT3C (SMYD2)

H4K20me1

H3K79me2/3

KMT3B (NSD1) KMT5A (SET8) KMT5B (SUV420h1) KMT5C (SUV420h2) KMT4 (DOT1L)

KDM1A (LSD1) KDM1B (LSD2) KDM3A (JHDM1A) KDM3B (JHDM1B) KDM4A (JMJD2A) KDM4B (JMJD2B) KDM4C (JMJD2C) KDM4D (JMJD2D) KDM7A (JHDM1D) KDM7B (PHF8/JHDM1F) KDM7C (PHF2) KDM6A (UTX) KDM6B (JMJD3) KDM7A (JHDM1D) KDM7C (PHF2) KDM2A (JHDM1A) KDM2B (JHDM1B) KDM4A (JMJD2A) KDM4B (JMJD2B) KDM4C (JMJD2C) KDM7C (PHF2)

H3K4 can recruit H3K27me-specific histone demethylases (M.G. Lee et al. 2007b; for a review, see S. Lee et al. 2009). While the H3K4me1 mark is implemented by KMT2C/D (MLL3/4), the H3K27me3 mark is simultaneously removed to activate gene transcription. Vice versa, the H3K4me3 demethylase KDM5A/JARID1A associates with the H3K27 methyltransferase KMT6/EZH2-containing polycomb repressive complex 2 (PRC2; Pasini et al. 2008). Another H3K4me3 demethylase, KDM5C/JARID1C forms a complex with the H3K9 methyltransferase KMT1C/G9A (Iwase et al. 2007; Tahiliani et al. 2007). Therefore, the synchronized actions of histone-modifying enzymes result in an open or condensed chromatin structure.

Histone methyltransferases and demethylases demonstrate relatively high specificity for the histone residue and the methylation state that they catalyze. Nevertheless, they are also able to target non-histone proteins, amongst them P53, P65, VEGFR and various transcription factors such as RARα, STAT3, E2F1 and NFκB (for a review, see Zhang et al. 2011).

Histone methylation during neurogenesis The bivalent histone mark in stem cells In ESC loci of important developmental regulators carry both the activating H3K4me3 and the repressive H3K27me3 mark at the same time, described as a “bivalent mark” (Fig. 6; Bernstein et al. 2006). The bivalent mark keeps key developmental genes repressed in ESC but poised for later activation upon differentiation into the neural lineage (Mikkelsen et al. 2007). During differentiation along this lineage, the promoters of many neural genes lose the H3K27me3 mark of the bivalent state but retain the H3K4me3 modification, resulting in transcriptional activation (Bernstein et al. 2006; Azuara et al. 2006; for a review, see Bernstein et al. 2007). In addition, however, some neuron-specific genes gain the H3K27me3 and H3K4me2 bivalent marks de novo when ESC become NSC (Mohn et al. 2008); the bivalent state is later resolved during terminal neuronal differentiation. To resolve the bivalent state, demethylases have to be upregulated, whereas methyltransferases are downregulated, demonstrating the synchronization of antagonistic enzymatic activities. H3K4 methylation Methylation at H3K4 is catalyzed by a group of KMTs that contains the catalytic SET domain: KMT2A/MLL1, KMT2B/ MLL2, KMT2C/MLL3, KMT2D/MLL4, KMT2E/MLL5, KMT2F/SET1A, KMT2G/SET1B and KMT7/SET7/9. The MLL/SET1-family proteins are catalytic subunits of multiprotein complexes. In contrast to yeast in which Set1 Table 2 Summary of arginine methyltransferases. Arginine methyltransferases target the arginine residues of histones Histone target

Arginine methyltransferases

H3R2 H3R8 H3R17 H3R26 H4R3

CARM1 PRMT5 CARM1 CARM1 PRMT1 PRMT4 PRMT5 PRMT6

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Fig. 5 Chemical structure of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). SAM is the cofactor for lysine and arginine methyltransferases and forms SAH during the methyl transfer reaction (R residue)

mediates all H3K4 methylation, Drosophila possess the three distinct H3K4 methyltransferases dSet1, Trx and Trr (Trithorax-related; Miller et al. 2001; Mohan et al. 2011). Mammals have two homologs of each of these three H3K4 methyltransferases found in Drosophila, thus totalling six different COMPASS (complex of proteins associated with Set1)-like complexes. These are, respectively, the dSet1 homologs KMT2F/SET1A and KMT2G/SET1B, the Trx homologs KMT2A/MLL1 and KMT2B/MLL2 and the Trr homologs KMT2C/MLL3 and KMT2D/MLL4. The various COMPASS-like complexes share several core subunits, including WDR5, ASH2, RBBP5 and DPY30,

which are necessary for the methylation activity of the complexes (Table 3) but also associate with class-specific subunits. When any single MLL family member is ablated in vivo, global levels of H3K4 methylation are only slightly reduced (Lubitz et al. 2007; Wang et al. 2009). By contrast, H3K4 methylation levels are significantly decreased when any of the core subunits of the complex is ablated (Dou et al. 2006). Depletion of DPY30 or RBBP5 in mouse ESC reduces H3K4 methylation and leads to defects in specification of the neural lineage, whereas self-renewal is not affected (Jiang et al. 2011).

Fig. 6 Distribution of H3K4 and H3K27 methylation on various gene classes during neurogenesis. H3K4me3 marks genes that are actively transcribed, whereas H3K27me3 is a repressive mark. Addition and removal of these marks at promoters change gene transcription. Pluripotency-associated genes (e.g., Oct4) carry the H3K4me3 mark in embryonic stem cells (ESC). They are silenced upon ESC differentiation into neural stem cells (NSC) by removal of the H3K4me3 mark and addition of the repressive H3K27me3 mark. In contrast, neural precursor genes, e.g., nestin (Nes) and paired box transcription factor 6 (Pax6) simultaneously carry both marks in ESC, in the so-called “bivalent” state and are thus kept poised for later activation. Their promoters lose the

repressive H3K27me3 mark of the bivalent state upon differentiation of ESC along the neural lineage but retain the H3K4me3 activating mark, leading to increased gene expression. They are silenced in terminal differentiated cells in which they exhibit the repressive H3K27me3 mark instead of the H3K4me3 mark. Neuron-specific genes including Syt1 and Grid1 are still kept poised when ESC are committed to become NSC but are activated later by the removal of the H3K27me3 mark during terminal differentiation (green histone residue H3K4, red H3K27, yellow methyl mark, blue cross promoter kept in the poised state, red cross gene is repressed). Modified after Hirabayashi and Gotoh (2010)

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Table 3 COMPASS-like complex members (complex of proteins associated with Set1; Herz et al. 2012) Core subunits

Enzymatic active unit

Class-specific subunits

WDR5 ASH2 RBBP5 DPY30

Trr/LPT(KMT2C/MLL3, KMT2D/MLL4)

NCOA1 PTIP (PAXIP1) PA1 (PAGR1A) dUTX (KDM6B)

Trx (KMT2A/MLL1, KMT2B/MLL2)

MNN1

dSet1 (KMT2F/SET1A, KMT2G/SET1B)

WDR82 CXXC1

Notably, histone methylation is usually gene- and cell-typespecific and the removal of specific histone methyltransferase or demethylase activities will more likely change methylation at the level of specific loci instead of at the global level. In the case of key transcription factors, this specific effect can still have a broad impact: KMT2A/MLL1-deficient postnatal NSC show impaired postnatal differentiation and yet the production of glial cells is not affected. KMT2A/MLL1 is essential for bivalent state resolution of the Dlx2 gene, a key transcription factor for the generation of interneurons (Lim et al. 2009). In KMT2A/MLL1-deficient cells, the repressive H3K27me3 mark is still present at the Dlx2 locus. This implies that, in addition to its methyltransferase activity at H3K4, the ability of KMT2A/MLL1 to recruit an H3K27me3 demethylase is also needed during neurogenesis. The activity of H3K4me demethylases is also directed to specific gene loci. The H3K4me and H3K9me demethylase KDM1A/LSD1 is expressed in NSC and needed for their proliferation. Together with the histone deacetylase HDAC5, this demethylase is recruited to the promoters of TLX target genes to repress their expression. TLX is an essential stem cell regulator and its target genes are regulators of proliferation (Sun et al. 2010). KDM5B/JARID1B, another H3K4me3 demethylase, functions as a transcriptional repressor in ESC and plays a crucial role in regulating the balance between proliferation and differentiation while being dispensable for ESC self-renewal. Forced overexpression of KDM5B/ JARID1B results in reduced numbers of differentiated cells and increased numbers of proliferating progenitors (Dey et al. 2008). Specifically, KDM5B/JARID1B is required for the efficient generation of NSC from ESC. It binds at the transcription start sites of developmental regulators, maintaining a low level of H3K4me3 marks (Schmitz et al. 2011). Impaired differentiation in KDM5B/JARID1B-deficient mutants is caused by a failure to silence pluripotency genes, because of elevated H3K4me3 levels (Albert et al. 2013). The X-linked mental retardation protein KDM5C/JARID1C demethylates H3K4me3, even as far as the monomethylated state and forms a complex with other regulators (RING2, NCOR1, HDAC1,

HDAC2, G9A). In ESC, KDM5C/JARID1C localizes to both promoter and enhancer regions but binding to enhancer-like regions is lost upon differentiation into NSC (Outchkourov et al. 2013). KDM5C/JARID1C suppresses the expression of neuronal genes in non-neuronal cells by associating with the transcriptional repressor REST. REST targets neuronal genes that carry the RE1 element. KDM5C/JARID1C and REST cooccupy the RE1 elements in the promoters of a subset of REST target genes. When KDM5C/JARID1C is repressed in vitro, H3K4me3 levels at the promoters of REST target genes are increased, resulting in increased gene expression (Tahiliani et al. 2007). Therefore, the main function of H3K4me3 demethylases in stem cells seems to be the transcriptional repression of target genes such as transcription factors and, thereby, the repression of differentiation along alternative lineages. However, in addition to removal of the H3K4me3 mark to suppress previously activated or poised genes, H3K4me3 must also be added at other loci, e.g., Dlx2 for neurogenesis to occur efficiently. H3K27 methylation H3K27 methylation is performed by either KMT6B/EZH1 or KMT6A/EZH2, the methyltransferases of PRC2 and is associated with gene repression (for a review, see Cao and Zhang 2004). The H3K27me3 mark is recognized by PRC1, which then prevents RNA polymerase II (RNAPII)-dependent transcriptional elongation and thus represses transcription. PRC1 complex members are actively transcribed during the proliferation and differentiation phases of neurogenesis. The variable composition of the complex suggests a cell-specific finetuning of PRC1 complexes during neural development (Vogel et al. 2006). The PRC2 complex contains the subunits EED, SUZ12, RBAP46/48 and either KMT6B/EZH1 or KMT6A/EZH2. In the mouse brain, expression of Kmt6b/Ezh1 and Kmt6a/ Ezh2 mRNAs are inversely regulated during development: whereas Kmt6a/Ezh2 is expressed during early development and is reduced during neuronal maturation, the expression of Kmt6b/Ezh1 is low during embryogenesis and high in the adult brain (Laible et al. 1997). In the hippocampus, KMT6B/EZH1 and KMT6A/EZH2 proteins are both abundantly expressed in immature cells but KMT6B/EZH1 is the predominant form in mature cells (Henriquez et al. 2013). Thus, KMT6B/EZH1 and KMT6A/EZH2 are correlated with distinct developmental states. Whereas KMT6B/EZH1 is an important regulator in mature neuronal cells, KMT6A/EZH2 is the only known H3K27 methyltransferase expressed in neural progenitor cells and is therefore a key regulator of stem cell renewal, maintenance and differentiation (E.R. Lee et al. 2007a; Sher et al. 2008). ESC, NSC and differentiated neurons show distinct H3K27me3 profiles. In ESC, lineage-specific genes are

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trimethylated at H3K27, concomitant with the high expression of KMT6A/EZH2. Many neuron-specific genes are KMT6A/ EZH2 targets only in NSC and not in ESC (Mohn et al. 2008). KMT6A/EZH2 is still expressed at high levels in NSC and also in oligodendrocytes (Sher et al. 2008) but is downregulated during the differentiation of NSC to neurons and astrocytes. Forced overexpression in astrocytes results in the formation of proliferating cells that are able to form small neurosphere-like clusters and in the upregulation of NSCspecific genes (Sher et al. 2011). The suppression of KMT6A/EZH2 activity in premyelinating oligodendrocytes also leads to the re-expression of neuronal and astrocytic genes, whereas suppression in NSC impairs their proliferation. Genes involved in stem cell maintenance, cell cycle control and the suppression of neuronal differentiation are KMT6A/EZH2 target genes in mouse NSC, e.g., Cdkn2a and Neurod2 (Sher et al. 2012). Mice deficient for KMT6A/ EZH2 in the cortex show the loss of the repressive H3K27me3 mark in cortical progenitor cells. As a consequence, the balance between self-renewal and differentiation is shifted toward differentiation and neurons are overrepresented, whereas the stem cell pool is depleted (Pereira et al. 2010). During the switch from the neurogenic to the astrogenic phase in NSC, H3K27me3 levels usually increase at the Neurogenin promoter but in KMT6A/EZH2-deficient mice, Neurogenin-1-positive cells are not reduced in number in the late neurogenic phase (Hirabayashi et al. 2009). Conditional mutants for the loss of KMT6A/EZH2 in the dorsal hindbrain show the de-repression of Netrin1, abnormal migration and supernumerary nuclei integrating into the brain circuitry (Di Meglio et al. 2013). Thus, KMT6A/EZH2 plays a role in maintaining neural stemness, triggers the neurogenic to astrogenic fate switch and controls neuronal guidance and network integration. The Jumonji domain-containing proteins KDM6A/UTX, KDM6B/JMJD3, KDM7A/JHDM1D and KDM7C/PHF2 demethylate H3K27me3. KDM6B/JMJD3 is upregulated when ESC differentiate along the neural lineage (Burgold et al. 2008) and further during terminal differentiation into neurons (Jepsen et al. 2007). It controls the expression of key regulators of neurogenesis such as Nestin (Burgold et al. 2008). KDM6B/JMJD3 upregulation during neuronal development is accompanied by KMT6A/EZH2 downregulation. This effectively reduces H3K27me3 at specific loci and thus resolves the bivalent state of neuronal genes during neurogenesis. KDM7A is a dual demethylase for H3K9me1/2 and H3K27me1/2. In zebrafish, its inhibition leads to a decrease in the size of the tectum and a reduced number of neurons in this region. KDM7A functions as an eraser of repressive marks on chromatin, thus removing gene silencing during brain development (Tsukada et al. 2010).

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H3K9 methylation In addition to H3K27me3, the methylation at H3K9 is also a repressive mark. Although it is not involved in the bivalent state, it still has important functions during neurogenesis. H3K9me2 is associated with reversible gene repression, whereas H3K9me3 is located at pericentric heterochromatin and transposons, suggesting that it promotes long-lasting gene repression (Peters et al. 2002). When ESC differentiate into NSC, pluripotency-related genes and non-neural lineagespecific genes become di- and later tri-methylated at H3K9 (Golebiewska et al. 2009). During neurogenesis, H3K9me2/3 is required to suppress non-neuronal genes and to maintain the correct timing of differentiation, as demonstrated by the deletion of H3K9 methyltransferases in mouse and zebrafish. The H3K9me3 methyltransferase KMT1E/ESET (SETDB1) is highly expressed at early stages of mouse brain development and is essential for proper neurogenesis. Forebrain-specific knockout (KO) of Kmt1e/Eset leads to decreased levels of H3K9me3 and the upregulation of non-neuronal genes, resulting in severe brain defects and early lethality. Furthermore, the distribution of neurons in the layers of the cerebral cortex is disturbed and astrocyte formation is accelerated (Tan et al. 2012). As with the H3K4me3 demethylase KDM5C/ JARID1C, KMT1C/G9A is also recruited by REST to silence neuronal genes in non-neuronal cells (Roopra et al. 2004). In zebrafish, KMT1C/G9A cooperates with the DNA methyltransferase DNMT3 to regulate tissue-specific development and its knockdown leads to incorrect neurogenesis and a reduction in brain size (Rai et al. 2009). During differentiation of NSC to astrocytes, H3K9me2 levels decrease and H3K4me3 levels increase at the promoters of astrocyte-specific genes, including Gfap. This results in the activation of astrocyte-specific genes that have been repressed during neurogenesis (Song and Ghosh 2004). The main function of H3K9 methylation during neurogenesis is therefore the silencing of non-neural and pluripotency-associated genes. However, it is also crucial for the regulation of the neurogenic to astrocytic switch during later development. H3K36 methylation H3K36 methylation is not only associated with active transcription but also plays a role in dosage compensation, transcriptional initiation and repression, alternative splicing and DNA repair (for a review, see Wagner and Carpenter 2012). In ESC, the H3K36 demethylase KDM2B/JHDM1B is highly expressed and directly regulated by the transcription factors OCT4 and SOX2. Depletion of KDM2B/JHDM1B leads to premature differentiation via the de-repression of lineage-specific genes (He et al. 2013). Gliomas show the loss of function of the H3K36 methyltransferase KMT3A/SETD2 and reduced

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H3K36me3 levels (Fontebasso et al. 2013). These recent findings indicate the essential function of H3K36 methylation in the regulation of proliferation versus differentiation. Its involvement in neurogenesis will hopefully be further clarified in the near future.

H3K79 methylation H3K79 methylation is found in the body of transcribed genes (Schübeler et al. 2004; Steger et al. 2008) and is considered to be an activating mark. KMT4/DOT1L is probably the only H3K79 methyltransferase, as the KO of Kmt4/Dot1l in yeast, flies and mice results in the complete loss of H3K79 methylation (van Leeuwen et al. 2002; Shanower et al. 2005; Jones et al. 2008). KO of Kmt4/Dot1l in mice is lethal at embryonic day 10 (Jones et al. 2008). A brain-specific deletion of the enzyme has not yet been described. KMT4/DOT1L is required for correct mitosis during the early stages of ESC differentiation, as knockdown of KMT4/DOT1L leads to defects in differentiation and increased levels of aneuploidy (Barry et al. 2009). The impact of this finding on neurogenesis needs to be further explored. During the development of the cerebral cortex, KMT4/DOT1L is important for neuronal subtype specification. It interacts with AF9 to perform H3K79me2 modification of the transcription factor Tbr1 gene. This leads to the downregulation of TBR1 in upper layer neurons, since AF9 also attenuates RNAPII binding at the Tbr1 transcription start site (Büttner et al. 2010). KMT4/DOT1L also interacts with actively transcribing RNAPII. The binding of RNAPII and H3K79 methylation are both required for the expression of target genes including Nanog and Oct4 (Kim et al. 2012). In addition, yeast DOT1 and H3K79 methylation are required for global genomic DNA repair, whereby methylated H3K79 might serve as a docking site for the repair machinery on chromatin (Tatum and Li 2011). Moreover, KMT4/DOT1L associates with long non-coding RNAs to enhance gene activation (Yang et al. 2013). KMT4/DOT1L has diverse functions in transcriptional elongation, genomic DNA repair and gene activation and repression, depending on its binding partners. The way that these functions contribute to correct neurogenesis needs to be further elucidated. To date, no H3K79 demethylating enzyme has been described. Nevertheless, H3K79me2 levels vary throughout the cell cycle and development (Ooga et al. 2008; Schulze et al. 2009). One explanation for this might be an as yet undiscovered H3K79 demethylase but since H3K79 methylation accumulates on ageing histones, it could also be a cell timekeeping mechanism that couples cell-cycle length to changes in chromatin modification at the nucleosome core (De Vos et al. 2011).

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H4R3 methylation Dimethylation at H4R3 can be symmetric, giving H4R3me2s, with each of the two terminal nitrogen atoms carrying one methyl group, or, more frequently, asymmetric, giving H4R3me2a, one of the terminal nitrogen atoms carrying two methyl groups (Fig. 3). Asymmetric and symmetric methylation are catalyzed by class I and class II arginine methyltransferases, respectively. H4R3 methylation can be mediated by the class I enzymes PRMT1 and PRMT6 or the type II enzyme PRMT5 (Huang 2005; Zhao et al. 2009; Li et al. 2010). PRMT5-mediated symmetric H4R3 methylation is associated with gene repression (Xu et al. 2010) and is required for subsequent DNA methylation. H4R3me2s is a direct binding target of the DNA methyltransferase DNMT3A (Zhao et al. 2009). In contrast, asymmetric H4R3 methylation is essential for successive active histone acetylation patterns (Huang 2005). The two different H4R3me modifications also have distinct roles during neurogenesis: the H4R3me2s modification is associated with undifferentiated cortical NSC, whereas both H4R3me2a and H4R3me2s are observed in mature neurons and in oligodendrocyte precursors. The H4R3me2s mark probably maintains “stemness”, whereas the appearance of the H4R3me2a modification specifies the onset of terminal differentiation of the NSC (Chittka 2010; Chittka et al. 2012). In vitro, H4R3 methylation can be removed by JMJD6 (Chang et al. 2007) but no arginine demethylase has yet been described in vivo. A summary of the role of histone-modifying enzymes during neural development is presented in Fig. 7.

Histone methylation and neurological disease The misregulation of neurogenesis can result in neurological disorders. One crucial step in early embryonic neural development is the closure of the neural tube. Failures in this step result in severe pathological birth defects called neural tube defects (NTD). Low maternal folate and vitamin B12 are known risk factors for NTD (Smithells et al. 1981; Mulinare et al. 1988; Bower et al. 2009). Since folate functions as a methyl donor in the cell, histone methylation could be affected by low folate levels and thus involved in the pathology of NTD. Indeed, several aberrant histone methylation patterns are associated with NTD. In embryos with a homozygous deletion of the transcription factor Pax3, cells from open caudal neural tubes show increased H3K27 methylation and decreased expression of the H3K27 histone demethylase KDM6B/JMJD3; this can be reversed by folate treatment (Ichi et al. 2010). Mice homozygous for mutations in the

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Fig. 7 Representation of the role of histone-modifying enzymes during neural development. Knockout and overexpression of histone-modifying enzymes in mouse and zebrafish have revealed their crucial role during neural development. Both histone methyltransferases and demethylases regulate differentiation from embryonic stem cells (ESC) into neural stem cells (NSC) and the generation of neurons, astrocytes and oligodendrocytes. Maintaining the balance between proliferation and differentiation is

crucial for these transitions (green boxes enzymes mediating the methylation or demethylation of H3K4, yellow boxes H3K9-modifying enzymes, red boxes H3K27-modifying enzymes, hatched boxes indicate enzymes with activities for two different modifications as follows KMT1C: G9A; KMT1E: ESET, SETDB1; KMT2A: MLL1; KMT6A: EZH2; KDM1A: LSD1; KDM5B: JARID1B; KDM5C: JARID1C; KDM6B: JMJD3; KDM7A: JHDM1D)

H3K4me demethylase KDM2B fail to undergo neural tube closure (Fukuda et al. 2011). In humans, H3K79me2 and H2BK5me1 levels are reduced in those with NTD and ESC deprived of folate display low H3K79me2 levels affecting target gene activation (Zhang et al. 2013). Whereas these results are hints that various activating and repressing histone modifications are involved in the proper development of the neural tube, no formal demonstration has been reported as to whether aberrant histone methylation is a cause or a consequence of NTD. Mutations in histone methyltransferases and demethylases are also associated with mental disorders such as autism (KMT2C/MLL3; Neale et al. 2012; O’Roak et al. 2012), X-linked mental retardation (KDM7B/PHF8; Laumonnier 2005; KDM5C/JARID1C; Adegbola et al. 2008), Weaver’s syndrome (KMT6A/EZH2; Tatton-Brown et al. 2011; Gibson et al. 2012), Kleefstra’s syndrome (KMT1D/GLP/EHMT1; Kleefstra et al. 2006; KMT2C/ MLL3; Kleefstra et al. 2012), Wiedemann-Steiner syndrome (MLL; Jones et al. 2012) and Kabuki’s syndrome (KMT2B/MLL2; Ng et al. 2010). The nonsense or missense mutations mostly occur in the catalytic SET or JmjC domains, thus severely affecting protein function. This confirms that the proper regulation of histone methylation is a prerequisite for correct neurogenesis.

Given their importance in regulating the balance between proliferation and differentiation, histone methyltransferases and demethylases unsurprisingly also play a role in the development of brain and other tumors (reviewed in DeCarlo and Hadden 2012; Spyropoulou et al. 2012).

Outlook Histone methylation is only one part of an extended interdependent epigenetic network that includes DNA methylation and histone acetylation and that regulates overall histone modification and higher order chromatin structure (Mohn et al. 2008; Huang and Dixit 2011; Herz et al. 2012; Hahn et al. 2013). Some crosstalk lines between these modifications have previously been elucidated but many other interactions will probably be uncovered in the future, as new modifications of the histone proteins are still being discovered. Additionally, our current knowledge is mainly based on work in mouse models. Whereas the general mechanisms in the regulation of neurogenesis by histone methylation are expected to be the same in humans, differences in specific details might occur. In this regard, interestingly, the first studies on histone methylation in human post-mortem prefrontal cortex have recently been published. They compare the H3K4me3 profiles of

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individuals in the age range of late prenatal age to 80 years and consistently report distinct profiles that change with age. The greatest changes occur during and in the first year after birth. Whereas modulation still occurs in the H3K4me3 profile during childhood and early adolescence, the profile is more stable in later life (Cheung et al. 2010; Shulha et al. 2013). In addition, histone methylation contributes to memory formation in adult hippocampus (Gupta et al. 2010). Mice deficient for the H3K4 methyltransferase KMT2B/MLL2 in excitatory adult forebrain neurons show impaired hippocampusdependent memory formation and reduced H3K4me2/3 levels (Kerimoglu et al. 2013). These results demonstrate that histone methylation is required not only for embryonic neurogenesis but also in adult brain functions. Despite only a few of the known histone methyltransferases and demethylases having been associated with the regulation of NSC differentiation, the proper regulation of histone methylation is clearly crucial for the maintenance of NSC, for coordinated differentiation, for cortical layering and for postnatal neurogenesis. In the future, large-scale genome-wide studies of histone modifications in mouse and human will hopefully further elucidate the role of histone modification in the complex process of neurogenesis. Acknowledgement The authors thank P.P. Bovio and S.C. Weise for graphical assistance, Dr. C.J. Hindley for language editing and the reviewers and editors for critical comments and fruitful discussions that have helped to improve the manuscript.

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