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MeCP2: the long trip from a chromatin protein to neurological disorders Juan Ausio´1,2, Alexia Martı´nez de Paz1, and Manel Esteller1,3,4 1

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de L’Hospitalet 199–203, L’Hospitalet del Llobregat, Barcelona, Catalonia, Spain 2 Department of Biochemistry and Microbiology, University of Victoria, British Columbia, V8W 3P6, Canada 3 Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain 4 Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Catalonia, Spain

Since the discovery of its fundamental involvement in Rett syndrome, methyl CpG binding protein 2 (MeCP2) has been the focus of an exhaustive biochemical and functional characterization. It is now becoming apparent that the intrinsic highly disordered nature of MeCP2, which is amenable to a plethora of post-translational modifications (PTMs), allows it to recognize a large number of protein interacting partners, including histones. MeCP2 is highly abundant in the brain and it is an important component of neuronal chromatin; nevertheless, the organization and implications of its involvement in terms of DNA methylation binding dependence and effects on transcription are still not well understood. Recent results have shown that MeCP2 plays an important role in brain development, aging, and in neurological disorders. MeCP2: an intrinsically disordered protein chromatin protein with manifold neurological implications MeCP2 (see Glossary) first appeared in vertebrates, a group of organisms in which CpG DNA methylation plays a very important epigenetic role. In these organisms, DNA methylation is involved in the fine-tuning of gene expression, the silencing of retrotransposable elements, and in genomic stability. MeCP2 belongs to the large family of intrinsically disordered proteins (IDPs) [1] and, as is typical of all the members of this family, it has many interacting partners. It is ubiquitously distributed throughout all tissues and hence plays an important role in many of the diseases that involve dysregulated DNA methylation, such as cancer [2]. Importantly though, MeCP2 is expressed at extremely high levels in the brain, where it is an abundant component of chromatin, particularly in neurons [3,4]. Mutations in MeCP2 cause neurodevelopmental alterations that account for more than 95% of Rett syndrome cases. The presence of one molecule of MeCP2 for every two nucleosomes in neuronal chromatin [3,4] suggests that alterations of this protein may be involved in many Corresponding authors: Ausio´, J. ([email protected]); Esteller, M. ([email protected]). Keywords: MeCP2; intrinsically disordered proteins; chromatin; DNA methylation; neurological disorders. 1471-4914/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/ j.molmed.2014.03.004

neuropathological disorders resulting from alterations in DNA methylation in this tissue. This review explores some of the poorly understood mechanisms of regulation of MeCP2 expression that can

Glossary Autistic spectrum: a group of neurodevelopmental disorders characterized by deficits in social and communicative interaction and stereotypic behaviors. Chromatin: a nucleoprotein complex formed by the interaction of histones and other chromosomal proteins with genomic DNA in eukaryotes. Chromatin repeat length: the average distance in nucleotides between two adjacent nucleosomes for the chromatin of a given tissue. Core histones (H2A/H2B/H3/H4): small (100–130 amino acid) histones consisting of an intrinsically disordered N-terminal domain (tail) and a structurally organized dimerizing C-terminal ‘histone fold’ domain. Two H2A–H2B dimers are associated with a H3–H4 tetramer forming the protein ‘core’ of the nucleosome. Histones: one of the most abundant chromosomal protein components of chromatin. These are basic proteins which are rich in arginine and lysine amino acids. Intrinsically disordered proteins (IDPs): a group of proteins that do not exhibit any secondary or tertiary structure in a solution but that can acquire such organization upon interaction with other protein partners or nucleic acids. Linker histones (histone H1): lysine-rich proteins (30% lysine) of approximately 200 amino acids. They consist of a central globular winged helix domain (WHD) flanked by N- and C- terminal disordered domains. They bind to the DNA at the entry and exit sites of the nucleosome and to the linker DNA regions of the chromatin fiber. Methyl CpG binding protein 2 (MeCP2): a member of the methyl-binding domain (MBD) family of proteins that binds to methylated symmetrical 50 CpG pairs. Neurodegenerative disease: disease resulting from a progressive partial or complete loss of neuronal function. Neurodevelopmental disease: disease arising from alterations of the brain and central nervous system that occur during development and result in an impairment of the proper growth and function of these organs. Nucleosome core particle: the fundamental repeating subunit of chromatin consisting of approximately 146 DNA bp wrapped about the histone core in 1.75 left-handed superhelical turns. Nucleosome core particles are connected within the chromatin fiber by a variable length DNA region (20–70 bp) known as ‘linker DNA’. Post-translational modifications (PTMs): modifications of proteins that take place after their synthesis in the ribosome. They usually involve the covalent attachment of biochemically functional groups (acetyl, methyl, phosphate, etc.) or other proteins (i.e., ubiquitin) to specific amino acids. The reactions are carried out by highly specialized enzymes such as acetyl/methyl transferases, kinases, or ubiquitin ligases. These modifications carry important functional and/or structural implications. Proline–glutamic acid, serine, threonine (PEST) rich sequences: protein regions, consisting of at least 12 amino acids in length, that are highly enriched in proline (P), glutamic (E), serine (S), and threonine (T) residues. It is hypothesized that they typically signal (to the proteins containing them) for rapid proteolytic degradation by the 26S ubiquitin proteasome system (UPS). Ubiquitin proteasome system (UPS): a multimeric adenosine triphosphate (ATP)-dependent protein complex (26S proteasome) that catalyzes the degradation of multiubiquitinated proteins.

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the nine N-terminal amino acids encoded by exon 2. In the brain, where MeCP2 expression is highest [3], E1 is present at ten times the amount of the shorter E2 isoform [9]. As seen in Figure 1, MeCP2 has very low levels of secondary structure organization, both as predicted by bioinformatics analysis and as determined experimentally [1,10]. In fact, this protein has been defined as a bona fide member of the IDP family [1]. Proteins of the IDP family [11] have gained important recognition in recent years, as they have a very low level of secondary structure, which allows them to interact with a large variety of macromolecular partners. Such interactions often result in the adoption of secondary structure upon the association of defined regions of the protein with defined regions of other proteins, or nucleic acids such as DNA and RNA. In addition to methylated DNA [6], MeCP2 can also interact with RNA [12] and with a plethora of other proteins [6]. MeCP2 has been shown to interact with HP1 (heterochromatin protein 1) transcriptional corepressors cSki (first isolated

be coupled to its intrinsic IDP nature. We next focus on what is currently known about the interaction of MeCP2 with DNA within the chromatin context and the relevance of DNA methylation. The specific chromatin organization resulting from the significant accumulation of the protein in neurons and its functional implications are also discussed. Finally, we describe how disruption in this chromatin interaction can participate in many neurodevelopmental and neurodegenerative pathologies with implications that largely transcend those solely resulting from the MeCP2 mutations involved in Rett syndrome [5]. The structure of MeCP2: the importance of being disordered In mammals, MeCP2 exists in two different isoforms produced by alternative splicing [6]: E1 [7] and E2 [8]. E1, which skips exon 2 entirely, contains 24 N-terminal amino acids encoded by exon 1; this is a different N terminus to E2, which does not utilize exon 1, but instead begins with

(A)

N C

(B)

50 24 25

Key: 73

Random coil

100

150

92 94

200

250

131 142 147 161

α-helix

AGKAETSESSGSAPAVPEASASPK

269

350

400

241 245 261 268 283 286 317-9 333

Extended strand 96

300

K-acetylaon

RKAEADPQAIPKKRGRK 285

450 413

S-phosphorylaon

435

K-ubiquinaon

TKAPMPLLPSPPPPEPESSEDPISPPEPQDLSSSICK

390

426

(C)

NTD 1

MBD

PEST1 92

176 196

TRD

ID

NLS

215

TGRGRGRPKGS

(D)

CTDα

205 276

326

IPKKRGRKPG

371

CTDβ PEST2

498

285

AT hook 78 92

176 215

MBD

276 285 308

DNA-binding domain

384 385

467

Binary chroman -binding sites

(E) 158

304 325

Dimerizing domain

498

WDR TRENDS in Molecular Medicine

Figure 1. Structural domains of human MeCP2 E1. (A) The tertiary structure of the methyl-binding domain (MBD) is shown, as obtained from the crystallographic information in [15]. (B) The predicted secondary structure of MeCP2 E1, determined using Hierarchical Neural Network (HNN) analysis is shown. Experimentally determined post-translational modifications (PTMs) are shown along the predicted structure [33,35,36]. (C) The different structural domains of MeCP2, as defined in [47], are depicted along with corresponding amino acid numbers. The sites and sequences corresponding to the PEST (enriched in proline–glutamic acid, serine, and threonine sequences) [3] and nuclear localization signal (NLS) [21] domains are also indicated. (D) The DNA- and chromatin-interacting domains of MeCP2 are shown, along with corresponding amino acid numbers. DNA-interacting domains include AT (adenine–thymine-rich DNA) hooks [16] (purple boxes, sequences are also shown) and the MBD [15] (dark brown), and chromatin-interacting domains include the DNA-binding domain [17] (orange) and binary chromatin-binding sites [18] (light brown). (E) The protein–proteininteracting domains of MeCP2 are shown, including the dimerizing domain [19] (blue) and the WW domain-binding region (WDR; pink) [20]. Abbreviations: NTD, N-terminal domain; ID, intervening domain; TRD, transcriptional repression domain; CTD, carboxy terminal domain; MeCP2, methyl CpG binding protein 2.

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Review at Sloan Kettering Institute) and N-CoR (nuclear receptor corepressor 1), Sin3A (Switch-independent 3A), and transcriptional coactivators CREB (cAMP response elementbinding protein), YB1 (Y box-binding protein), TDP-43 (TAR DNA-binding protein 43), and Dnmt1 [DNA (cytosine-5)-methyltransferase 1] [6]. In this way, IDPs are involved in important regulatory functions and in signaling [13]. Thus, it is not surprising that single amino acid mutations distributed throughout the whole protein, in addition to its methyl-binding domain (MBD), may have structural and functional consequences that result in Rett syndrome phenotypes with different extents of severity [6,14]. Despite its disordered nature, MeCP2 consists of many well-defined structural domains [6] such as DNA-interacting domains, including the MBD, one of the few regions with organized tertiary structure [15], and several adenine–thymine (AT) hooks [16]. Also present are several methylation-independent chromatin-binding DNA domains that include the DNA-binding domain [17] and the binary chromatin-binding sites [18]. Moreover, there are protein–protein-interacting regions present within these domains, including a dimerizing domain [19] and a WW domain-binding region (WDR) [20], which are responsible for some of the protein–protein interactions of MeCP2. MeCP2 also has a nuclear localization signal (NLS) [21] and two PEST (enriched in proline–glutamic acid, serine, and threonine) sequences [22]. PEST sequences are sequentially phosphorylated at a serine within the motif; this is followed by ubiquitination of a nearby lysine [23]. Such ubiquitination targets the protein for rapid degradation by the 26S ubiquitin proteasome system (UPS). Hence, PEST sequences and their phosphorylation may be involved in protein turnover, and thereby responsible for maintaining an adequate level of MeCP2 within the nucleus. IDPs have a fivefold higher propensity to contain PEST sequences, and their protein half-life is on average shorter than that of structured proteins [24]. It is interesting to note that the predicted half-life based on the N-end rule [25] for E1 MeCP2, the more abundant mammalian form, is approximately 4 h, whereas that of the shorter E2 form is approximately 100 h. Although this needs to be confirmed experimentally, such drastically different turnover rates could also contribute to the relative abundance of the two isoforms in the cell at any given time. This would explain how sequence variation in the brief N-terminal amino acid sequences could have physiological significance, instead of the two isoforms being functionally redundant, as has previously been assumed. In addition, the temporal steady state regulation of their expression by differential methylation of regulatory elements within the promoter and intron 1 regions [26] could also affect differential expression of the isoforms. In this regard, it is interesting to note that it is the E1 form which has been held mainly responsible for Rett syndrome [27,28]. However, the relevance of this observation to the putatively shorter lifetime of E1 is not clear, and a better understanding of the differential functionality between the two isoforms is needed. Interestingly, the IDP nature of MeCP2 could also help explain some of the still unexplainable functional attributes

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of this protein. For instance, the observation that both the presence of too much or of too little MeCP2 might have deleterious effects for proper brain function [29] remains unexplained. Although males are most likely to suffer from MeCP2 disorders, their mothers often carry the same mutation (which is subject to skewed X chromosome inactivation). These females may express evidence of obsessive– compulsive disorder or depression [30]. Such tight regulation of MeCP2 expression has important implications for therapies such as those designed to rescue the Rett type phenotype by activation of MeCP2 in a mouse model [31]. Overexpression and underexpression of MeCP2 have deleterious effects on dendrite formation and trigger opposite effects in synaptic transmission, and hence tightly regulated expression of MeCP2 is critical for neuronal homeostasis [32]. The mechanistic nature of these phenomena remains obscure. However, because of their ability to interact with a plethora of multifaceted cell partners, IDPs, and therefore MeCP2, have a ‘concentration restriction’, and altered availability of these proteins has been associated with several pathological conditions [13]. It has been proposed that the disordered domains are prone to create promiscuous molecular interactions, and hence when their expression is altered, these proteins are likely to have toxicity [24]. Thus, maintenance of a tightly defined dosage appears to be imperative for their proper function, as seems to be the case with MeCP2. Finally, PTMs play a critical role in the interaction of IDPs with their macromolecular partners. As such, MeCP2 has been shown to undergo many PTMs including acetylation [33,34], phosphorylation [33,35–37], ubiquitination [33], and sumoylation [38] (Table 1). The functional roles of MeCP2 PTMs remain largely unclear in many instances. It is likely that the interacting partner specificity imparted by these marks can explain the interaction of MeCP2 with activating complexes such as CREB or with repressor complexes associated with Sin3A and histone deacetylases (HDACs). Indeed, the initial concept of this protein being a transcriptional repressor that binds to transcriptionally repressive methylated CpG dense regions [39] has been recently changed to that of a transcriptional regulator [6]. The ability of the protein to associate with many different partners in such a PTMdependent manner could potentially account for apparent functional disparity. MeCP2 histone H1-binding competition and DNA methylation-binding dependence A very elegant experiment in the initial studies of the interaction of MeCP2 with chromatin used nucleosomes reconstituted onto methylated and nonmethylated plasmid DNA templates of different sizes [40]. The chromatin constructs were assembled using Xenopus extracts and radiolabelled histone H1. It was shown that MeCP2 could displace histone H1 in a DNA methylation-dependent way. These results raised several important questions regarding the interaction of MeCP2 with nucleosome-organized chromatin, which 15 years later have not yet been satisfactorily resolved. Firstly, is the interaction of MeCP2 with DNA within the chromatin template in the in vivo setting exclusively DNA methylation-dependent? Secondly, how 3

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Table 1. MeCP2 PTM and functiona PTM type

Site (amino acid number): human E1/E2 determined (*)/predicted(**) m = mouse

Function known (*)/predicted (**)

Refs

S25/S13 (*) S80/S68 (**) S92-mS95/S80 (*) S161/S149 (*) S241/S229 (*) S286/S274 (*) S413/S401 (*) mS416/mS399 (*) S421/S409 (**) mS438/mS421 (*) mS441/mS424 (*)

Unknown Regulation of PEST sequence activity (**) Interaction with Sin3A and YB-1. Transcriptional repression (*) Unknown Interaction with Sin3A, HP1, and SMC3 (*) Unknown Unknown Unknown Regulation of PEST sequence activity (**) Generic chromatin response to neuronal activity (*) Unknown

[33] [22] [33,35] [40] [36,40] [33] [33] [35] [22] [36,37] [35]

K317/K305 (*) K319/K307 (*) K333/K321 (*) mK464/mK447 (*)

Unknown Unknown Unknown Repression of BDNF. Deacetylation by SIRT1 increases transcription of this gene (*)

[33] [33] [33] [34]

K24/K12 (*) K73/K61 (**) K94/K82 (*)(**) K131/118 (*) K142/K130 (*) K147/K135 (*) K245/4233 (*) K261/K249 (*) K283/K271 (*) K333/K321 (*) K389/K377 (**) K426/K414 (**)

Unknown Regulation Regulation Unknown Unknown Unknown Unknown Unknown Unknown Unknown Regulation Regulation

[33] [22] [22,33] [33] [33] [33] [33] [33] [33] [33] [22] [22]

K73/K61 (**) K389/K377 (**)

Unknown Unknown

Phosphorylation

Acetylation

Ubiquitination of PEST sequence activity of PEST sequence activity (**)

of PEST sequence activity (**) of PEST sequence activity (**)

Sumoylation [22] [22]

a

Abbreviations: MeCP2, methyl CpG binding protein 2; PTM, post-translational modification; PEST, proline–glutamic acid, serine, and threonine rich sequences; Sin3A, Switch-independent 3A; YB-1, Y box-binding protein 1; HP1, heterochromatin protein 1; SMC3, structural maintenance of chromosomes 3; BDNF, brain-derived neurotrophic factor; SIRT1, sirtuin 1.

does the methylation-dependent histone H1 displacement take place? Many attempts have been made to answer the first question both in vitro [18,41,42] and in situ [3,42–45]. In vitro studies showed that MeCP2 has a higher preference for binding methylated DNA, especially in the presence of competitor DNA [8], a situation similar to that found in the nucleus. This result has been recently corroborated, and the binding affinity of MeCP2 for methylated DNA was found to be approximately threefold higher than for nonmethylated DNA [42]. This was also confirmed by sedimentation equilibrium [44]. The binding affinity of MeCP2 for specific methylated DNA compared with generic nonmethylated DNA was found to decrease by approximately tenfold [46], suggesting that the presence of histones decreases the preference for methylated DNA. Thus, in vitro, MeCP2 is able to bind both specifically to methylated DNA and nonspecifically to unmethylated DNA [18,41,47]. For this in vitro work, it is important to keep under consideration that MeCP2 is a highly basic chromosomal protein (pI 10.0) that will indiscriminately bind to DNA under sufficiently low ionic strength conditions. 4

Recent studies have shed additional insight into this problem within the in situ context [43,45]. In the first approach, Baubec et al. looked at the genome-wide distribution of MBD proteins. In their analysis, they used triple knockout (TKO) embryonic stem (ES) cells to analyze the methylation-dependent and -independent targeting of MBD proteins in the genome. TKO cells lack the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b, and despite the absence of genomic CpG methylation can grow normally [48]. Baubec et al. showed that MBD proteins exhibit a linear correlation with the extent of local methylation density, and that DNA methylation is the primary determinant of their chromatin binding [43]. However, in TKO cells MeCP2 still retains its characteristic chromocentric association, which suggests that mechanisms other than DNA methylation may be responsible for such localization. Of interest, despite the complete lack of DNA methylation, TKO cells retain their pericentromeric heterochromatin domains that are marked by methylation of histone H3 lysine 9 (H3K9) [48]. The interaction of MeCP2 to these domains is not surprising, as MeCP2 has been

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shown to bind to nucleosomes containing H3K9/H3K27methylated histone marks, which label heterochromatin [3]. In the second in situ approach [45], the experimental strategy used a mouse model of Rett syndrome that expresses MeCP2 with a functionally impaired truncated MBD. Although the MBD-impaired MeCP2 can bind to chromatin in vivo, this binding is very weak and exhibits a highly disperse, ill-defined nuclear localization. This study further suggested that whatever MeCP2 domain is responsible for the localized binding to chromocenters in TKO cells, it must be close, if not coincidental, with the MBD of native MeCP2. These two studies clearly indicate that in the in vivo setting, most MeCP2 binds to methylated DNA. Any methylation-independent binding, although possible, is of a secondary nature and under normal conditions it is probably used to strengthen or synergize with the MBD driven binding. The DNA methylation-independent interactions arise from structural features of the MeCP2 protein itself which, in addition to MBD, contains several other DNA-binding domains (Figure 1) [16]. Such is the case of the three AT hook DNA-binding domains. Mutations in one such domain

(AT hook at amino acids 276–285) can determine the clinical course of Rett syndrome-related pathologies [16]. Also, the in vitro characterization of the interaction of MeCP2 with nucleosomal templates has provided evidence for the existence of additional chromatin-binding sites at the C-terminal end of the protein. In particular, the presence of binary chromatin-binding sites within the same terminal region of MeCP2 [18] or the DNA-binding domain of the intervening domain (ID) [17] may play a role in internucleosomal interactions leading to the formation of functionally active chromosomal loops [49]. The issues regarding the DNA–methylation chromatinbinding dependence of MeCP2 are mirrored by the unresolved questions about binding competition between MeCP2 and histone H1, and are equally puzzling. Despite their completely different amino acid sequence, and secondary and tertiary structure organization (Figure 2), these two proteins exhibit a striking amount of functional convergence. For instance, similar to histone H1, MeCP2 exhibits a preference for DNA cruciform structures which are reminiscent of the DNA organization at the entry and exit region of the nucleosome. Also, similar to histone H1,

(A)

Key:

mC

hm/mCpG.[A/T]≥4

H1 MeCP2 200 bp

MeCP2 MBD AAATT

(B) (1)

N

HistoneH1 WHD

(C)

A 165 bp

(3)

H1 Repeat length bp

(2)

MeCP2

165 bp 200

L

A

180

N 160 0

7

Days

14

TRENDS in Molecular Medicine

Figure 2. Chromatin organization in astrocytes and neurons during brain development. (A) The tertiary structures of the methyl-binding domain (MBD) of MeCP2 [15,117] and of the winged helix domain (WHD) of linker histones (histone H1) [118], and their preferred binding sites [42,119] and consensus sequences of interaction in the nucleosome [120] are shown. DNA (blue) is depicted as a wound around a nucleosome, with MeCP2 shown in red, binding to hm/mCpG.[A/T] 4 in orange, whereas the histone H1 WHD is shown in green, binding to AAATT in red. (B) A proposed model for chromatin organization in (1) astrocytes (A) and in (2), (3) neurons (N). A chromatin repeat length of approximately 200 bp is observed in astrocytes where the levels of MeCP2 are lower than in neurons (1). In mature neurons where MeCP2 is very abundant and present at approximately one molecule for every two nucleosomes [3,4], the chromatin length decreases to approximately 165 bp [55]. Two putative models are put forward. In the first model, the MeCP2 molecules are evenly distributed throughout the chromatin, binding to methylated DNA sites (2). The replacement of histone H1 by MeCP2 may impart a decrease in the indicated repeat length. An alternative possibility may arise from MeCP2 binding to more densely methylated long non-nucleosomal DNA regions [3], resulting in a ‘squeezing’ of the neighboring nucleosome domains (3). (C) Variations of MeCP2, histone H1, and nucleosome repeat length during mouse development. The blue line (L, liver), the pink line (A, astrocytes), and the green line (N, neurons) represent the change of the chromatin repeat length during mouse development (in days) before and after birth (day 0). The higher proportion of MeCP2 to histone H1 results in a shorter repeat length as indicated in (B) [55]. Abbreviations: hm/mCpG, hydroxymethylated/methylated CpG; MeCP2, methyl CpG binding protein 2.

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Review MeCp2 is able to form homodimers and heterodimers with MBD2 [17,19]. Such interactions may play a role in the interfiber chromatin associations that have been observed [41]. Heterodimerization with MBD2 may help explain the functional synergy observed for these two proteins at certain promoters [50] and their potential functional redundancy [51]. Neuronal chromatin organization and transcriptional regulation The high abundance of MeCP2 in the brain, particularly in neurons where the protein coexists with stoichiometric amounts of histone H1 [4], imparts chromatin with a highly specific, yet undefined chromatin organization in this tissue (Figure 2) [3]. Micrococcal nuclease digestion analysis of nuclei obtained from unfractionated mammalian brain cortex cells showed that approximately 50–60% of MeCP2 eluted with the chromatin fraction most readily accessible to micrococcal nuclease [3], which is probably associated with open chromatin domains. Such a result was very unexpected considering that MeCP2 had been initially thought to be a global transcriptional repressor [40]. With such a role, the protein would have been expected to be part of a very tightly folded chromatin organization, probably such as that observed in the in vitro reconstituted oligonucleosome arrays [41]. However, the nuclease sensitivity result is more in agreement with the notion of MeCP2 operating as a modulator of transcription that can bind to both coactivator and corepressor factors [52], and hence associate with both transcriptionally active and repressed genes [52,53]. A potential insight into the chromatin conformation of such MeCP2-rich chromatin may be gained from the accumulation of this protein during neuronal development. There is a gradual increase in the levels of MeCP2 during neuronal and brain development in the first 7 days after birth (in mice) both in vitro and in vivo (Figure 2) [9,54]. Such an increase is important for synaptogenesis during brain maturation [9]. Concomitant with this change, there is a decrease in the apparent repeat length (distance from the center of a nucleosome to the center of its neighbor) of chromatin from approximately 200 bp before birth to 165 bp in adult rat brain neurons [55]. This phenomenon was extensively studied at a time when the existence of MeCP2 was not yet known and hence the significance of these changes was not fully understood. It was noticed that chromatin in the mature mammalian brain neurons contained approximately half of the histone H1 complement of that of typical somatic cells, and that granulocytes and astrocytes, which appeared to undergo an opposite transition in their chromatin repeat length [55], contained normal amounts of histone H1 [56]. Although the nature of these chromatin transitions is not clear, it is tempting to speculate that it is the result of the presence of MeCP2, with several possible alternative nucleosome arrangements as depicted in Figure 2. In one of these arrangements, the substantial presence of MeCP2 preferentially binding to CpG-rich regions may result in the ‘squeezing’ of the distance between nucleosomes and alterations in nucleosome positioning, resulting in an average decrease of the repeat length [3]. 6

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There is now a wealth of evidence that in the brain, the majority of methylated CpG islands occur in intragenic and intergenic regions, whereas only a very small amount of methylation (less than 3% of CpG islands) is found at 50 promoters [57]. Moreover, gene bodies and transcriptionally active genes in neurons are enriched in hydroxymethylated CpG (5hmC) [58], which bind MeCP2 and exhibit nuclease hypersensitivity [58]. Interestingly, the levels of 5hmC in mouse brains (at 0.5%) [59] are 17-fold higher than those in mouse ES cells (0.032%) [60]. Furthermore, the transition from 5mC to 5hmC increases during neuronal differentiation and brain development and during aging [59,61]. All of this evidence provides a new perspective to the MeCP2-mediated chromatin organization in the brain, and provides support to the chromatin organization shown in Figure 2 and to the early nuclease digestion studies carried out on brain chromatin [3]. Based on this evidence, in Figure 3 a model is proposed for the role of MeCP2 in the regulation of gene expression in neurons. This potentially accounts for the repressor and activator roles of MeCP2 regulation of gene expression, particularly as it pertains to genes expressed in neurons. The model takes into consideration the roles of DNA methylation and the effects of hydroxymethylation, as well as the antagonistic correlation of these DNA marks with a histone H2A.Z [62,63]. H2A.Z is a replacement histone H2A variant that is expressed throughout the whole cell cycle and replaces canonical histone H2A during different metabolic states of chromatin [64]. Similar to MeCP2, H2A.Z is an important modulator of gene expression and has been associated with repressed and activated chromatin domains [64]. MeCP2 in neurodevelopmental and neurodegenerative diseases In recent years, there has been an exponential increase of interest in the role of epigenetics in neurological diseases [65]. In particular, special attention has been focused on DNA methylation and histone methylation. These two chemical modifications are structurally and functionally linked [66], and both of them can play a role in the specific recognition of neuronal chromatin by MeCP2 [3,67]. Therefore, because of the high abundance of this protein in the brain, there is also an increasing recognition of its relevance in normal and altered functional states of this organ. In a normal brain, MeCP2 is directly involved in the regulation of GABAergic [68], DOPAMINergic [69], and SEROTONergic [69] neuron secretion, and controls glutamatergic synapse number [70]. Conversely, dopamine and serotonin can also differentially regulate MeCP2 phosphorylation [71]. Most of this is mediated by the interaction of MeCP2 with brain derived neurotrophic factor (BDNF), which exerts modulatory effects on glutamatergic and GABAergic synapses. Hence, it is not difficult to understand how dysregulation of MeCP2 is an important participant in many neurological disorders ranging from Alzheimer’s [72] and Huntington’s [73] diseases to Rett syndrome [74], schizophrenia [75], epilepsy [76], obsessive–compulsive disorders [77], depression and suicide [78], and drug addiction [79] (Table 2). MeCP2 can also adversely affect functional states of the brain resulting

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Promoter

Sin3A

HDAC

(1) TSS

H2A.Z

MeCP2 Gene body

H1 MeCP2 H2A.Z

Pol II

(2) TSS

Key:

mC

hmC

H2A.Z TRENDS in Molecular Medicine

Figure 3. Model for the regulation of MeCP2-mediated neuronal gene expression. (1) Binding of MeCP2 at methylated promoter regions in conjunction with other corepressors (Sin3A–HDACs) has a repressor effect on transcription [121]. DNA demethylation in promoter regions results in chromatin accessibility by nucleosome loss, a process that requires H2A.Z insertion [122] and also results in loss of MeCP2 binding. It has been shown that the proximity of a H2A.Z-containing nucleosome to the transcription start site (TSS) enhances gene expression in the brain [123] and sets the stage for the assembly of RNA polymerase initiation complex. (2) Unlike DNA methylation at promoter regions, DNA methylation in the gene body has the opposite effect on expression [124], with DNA methylation present in the gene bodies of moderately transcribed constitutive genes [125]. Gene bodies are extensively methylated in neurons [57] and are often hydroxymethylated. MeCP2 binds to these regions, providing nuclease accessible regions that are amenable to transcription and prevent histone H2A.Z from binding. A genome-wide anticorrelation between DNA methylation and histone H2A.Z occupancy has been well documented [62,63]. Abbreviations: Sin3A, Switch-independent 3A; HDAC, histone deacetylase; TSS, transcription start site; PolII, RNA polymerase II; mC, methylated C; hmC, hydroxymethylated C; MeCP2, methyl CpG binding protein 2.

from aging and/or altered states such as anxiety [80] and depression [81]. Aging Changes in chromatin organization and epigenetics play a crucial role during embryogenesis and aging [82]. Levels of DNA methylation decrease with aging, particularly in the brain [83] where this change is mainly restricted to euchromatin domains [84]. This process has been shown to be mediated by a decline of Dnmta3.2, a member of the Dnmta.3 family which is activated by NMDA (N-methylD-aspartate) receptor and calcium signaling neuronal activity [85], and is associated with transcriptionally active euchromatin regions [86]. It is thus possible that this results in an impairment of MeCP2 binding to these regions and a deleterious alteration of the neuronal chromatin organization. Neurodevelopmental disorders Since the discovery in 1999 by Huda Zoghbi’s group [87] that Rett syndrome is caused by mutations in MeCP2, this neurodevelopmental disorder has become the prototype for the study of the functional role of MeCP2 in the brain [88]. Although autism and Rett syndrome may show some overlap, a fundamental difference exists in that not all children with Rett demonstrate features of autism and, in most, this is a transient phenomenon. By age 5, girls with Rett syndrome are very socially interactive, a feature very distinct from autism, and they rarely have the motor dexterity of children with autism. It should be noted that Rett syndrome is an X-linked ‘dominant’ disorder that

occurs generally by de novo mutations in paternal germline cells and is rarely transmitted by mutations residing in germline cells. When Rett syndrome is inherited from the mother, the mother may carry the mutation, suppressed by unbalanced or skewed X inactivation; therefore, the syndrome predominantly affects females (as many as 1 in 15 000 to 1 in 10 000 females [87]), although in some rare cases, males can be affected [5]. Individuals with Rett syndrome are heterozygous for the mutation in the MeCP2 allele and are mosaic, due to the effects of X chromosome inactivation. Affected females appear to have a seemingly normal early postnatal development as per healthy children. Between ages 6 and 18 months, however, the development of Rett syndrome in females begins to regress and they lose the ability to retain communication function and motor skills [89]. Overall, physical growth also becomes retarded and is notably characterized by microcephaly. The disease phenotypes were initially thought to arise from functional neuronal alterations resulting from MeCP2 mutant forms. However, it has recently been shown that despite the much lower abundance of MeCP2 in astrocytes and in microglia, the resulting functional alterations in these cells also have important implications for the disease [90]. Additionally, as understanding of the biochemical aspects of MeCP2 increases, new knowledge on different aspects of the disease is gained. An example is the identification of new interacting partners, such as miRNA-132, which involve MeCP2 in the regulation of the circadian cycle [91] and may have important yet unrecognized implications for the sleeping disorders observed in Rett syndrome patients. 7

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Table 2. MeCP2 involvement in different neuropathological disordersa Neurological disorder/ syndrome/substance abuse Neurodevelopmental disorders Autism Rett syndrome Down syndrome (DS) Attention deficit hyperactivity disorder (ADHD)/ADD Mental retardation Schizophrenia Neurodegenerative disorders Alzheimer’s Huntington’s Parkinson’s Amyotrophic lateral sclerosis (ALS) (Lou Gehrig’s disease) Spinocerebellar ataxias

Other neuronal disorders Epilepsy

MeCP2 involvement: known (*)/predicted (**)

Refs

Overexpression/increased dosage of MeCP2 is related to core features of many ASDs (*) MeCP2 mutations (*) MeCP2 duplication (*) MeCP2 under-expressed in DS human and mouse brains (*) Alterations in the MEF2C expression levels result in diminished MeCP2 expression and hyperkinesis. MEF2C is a transcription factor that binds to E boxes at the MECP2 promoter and regulates its expression Mutations in MEF2C diminish MECP2 and CDKL5 expression (*) Increased MeCP2 binding to reelin and GAD67 promoters is responsible for their overexpression in GABAergic neurons, which is characteristic of psychotic disorders (*)

[92] [101]

Decrease of MeCP2 in the cortical region of primates (*) Alterations of the MeCP2 levels affect axonal transport of BDNF affecting the expression of Htt and huntingtin-associated protein 1 (Hap1) (*) Loss of MeCP2 in substantia nigra dopamine neurons results in alteration of the nigrostriatal pathway (*) MECP2 is a target of translocated in liposarcoma (TLS), which links transcription and pre-mRNA splicing. TLS targets the LISTERIN gene, which has been involved in neurodegeneration and presumably in ALS (**) DNA methylation in the core ataxin-2 gene (ATXN2) promoter, which is responsible for spinocerebellar ataxia type 2, is controlled by DNA methylation alterations which could probably result in anomalous MeCP2 targeting of the gene (**)

[100] [103]

Upregulated expression of MeCP2 in intractable temporal lobe epilepsy (*) Mild overexpression of MeCP2 deters neuron maturation and increases epileptic seizures(*) Alterations in the MeCP2 levels resulting from MEF2C haploinsuffiency (*)

[93] [94]

[95] [96]

[104] [97]

[105]

[76] [99]

[94] Bipolar mood disorder

Depression Substance abuse Fetal alcohol syndrome (FAS) /alcohol abuse Cocaine

MeCP2 repression of glial cell derived neurotrophic factor (GDNF) promoter (*) MeCP2-mediated silencing of ERa, pS2, and cyclin D1 promoters as a result of the use of valproate treatment to ameliorate the disorder (*) MeCP2 repression of GDNF promoter (*). S421 phosphorylation has an antidepressant action Decrease in MeCP2 expression/reduced spine density MeCP2 regulates genes involved in ethanol sensitivity and intake (*) Repeated cocaine intake leads to an increase of the promoter methylation of the PP1Cb catalytic subunit gene leading to its MeCP2-mediated repression. The activity of protein phosphatase type 1 (PP1) plays an important role in neuron plasticity (*) MeCP2 enhances compulsive drug use by attenuation of the increase in miRNA-212 in response to cocaine consumption (*) Phosphorylation of S421 in rat striatum nucleus accumbens (*)

[104] [81] [36] [111] [108]

[109] [36]

a

Abbreviations: ASD, autism spectrum disorder; MeCP2, methyl CpG binding protein 2; ADD, attention deficit disorder; MEF2c, myocyte enhancer factor 2c; CDKL5, cyclindependent kinase-like 5; GAD67, glutamic acid decarboxylase 67; ERa, estrogen receptor a; pS2, estrogen-inducible pS2 gene; PP1Cb, protein phosphatase 1 Cb, BDNF, brain-derived neurotrophic factor; Htt, huntingtin; GDNF, glial-derived neurotrophic factor.

As in a few instances of Rett syndrome [30], MeCP2 dosage is the underlying factor in many other autism spectra disorders [92] and has been implicated in other neurodevelopmental disorders such as Down syndrome [93], attention deficit hyperactivity disorder (ADHD) [94], mental retardation [95], and schizophrenia [96]. Neurodegenerative disorders A common repetitive theme in neurodegenerative illnesses is a decrease in the levels of MeCP2. However, these observations need to be taken carefully and need to be examined more closely, as many of the analyses are performed with brain tissue homogenates and may simply reflect neurodegeneration. Loss or atrophy of neurons may result in a 8

significant decrease in the amount of MeCP2 present in the brain. Translocated in liposarcoma (TLS) and TDP-43, two RNA–DNA-binding proteins involved in RNA processing bind to the promoter region of MECP2 and MeCP2, respectively, and are involved in neurodegenerative diseases, particularly in amyotrophic lateral sclerosis (ALS) [97]. It is important to recognize that alterations in DNA methylation, whether at promoters or other genomic regions, may play an important role in many neurological disorders [98]. Aberrant DNA methylation has been observed in epilepsy [99], Alzheimer’s disease [100], schizophrenia [101], and some autism disorders [102]. In such instances, in addition to its direct involvement, MeCP2 effects may be of an indirect nature, as in Huntington’s

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Review [103] and Parkinson’s [104] diseases and in spinocerebellar ataxias [105]. Substance abuse Chromatin organization may also mediate the effect of cocaine, as the drug increases the magnitude of histone acetylation levels [106] at the promoters of MeCP2-targeted genes such as Bdnf2, Cdkl5 (cyclin-dependent kinaselike 5), and Fosb (FBJ murine osteosarcoma viral oncogene homolog B) in the striatum [107]. Such an increase in acetylation, together with the S421 phosphorylation of MeCP2 may help release the inhibitory presence of MeCP2 at such promoters. An increase in DNA methylation resulting from cocaine intake can also result in MeCP2-mediated repression of the promoters of genes involved in brain plasticity, such as the gene of the catalytic subunit PP1Cb of protein phosphatase type 1 (PP1) [108]. However, the situation is not simple because cocaine overconsumption intake increases miRNA-212 expression and decreases Bdnf expression [109], in yet another example of MeCP2 functional duality. Ingestion of alcohol during pregnancy can increase the levels of Dnmt1, DNA methylation, and MeCP2, which affects gene expression in hypothalamic proopiomelanocortin (POMC) neurons of the embryo [110]. MeCP2 has been shown to have an important contribution to the regulation of ethanol sensitivity and intake [111]. Concluding remarks and future perspectives In this review, we explored the potential for the intrinsically disordered nature of MeCP2 and its two E1 and E2 isoforms to account for the multifaceted structural and functional roles of this protein. The structural and functional implications of the intrinsically disordered organization of MeCP2 deserve closer attention. It is very likely that, as in many other IDPs, the expression and availability of MeCP2 is very tightly regulated. In this regard, many outstanding questions still remain unanswered (Box 1). Despite the relatively extensive amount of PTMs described for this protein, their functional roles (beyond those of a few phosphorylation sites) remain completely unknown. At the chromatin level, the role of MeCP2 has been substantially examined [6]. Yet, when it comes to the overall organiza-

Box 1. Outstanding questions  What are the functional differences between the MeCP2 E1 and E2 isoforms?  What is the significance of the different amounts of the two MeCP2 isoforms in different tissues?  Are the average half-life times of the E1 and E2 isoforms as different as the bioinformatics analysis suggests?  What are the roles of PTMs already described, such as ubiquitination and acetylation in particular, and the yet to be uncovered MeCP2 PTMs?  Do the PEST sequences play a role in MeCP2 metabolism?  What are the precise roles of MeCP2 in neurodegenerative disorders? The answer to all these questions may provide the key to deciphering the implications for the plethora of mutations observed in Rett syndrome and for the emerging role of MeCP2 itself in other important neurological disorders.

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tion, resulting from its high abundance in neurons, and the high nuclease accessibility found in the tissue of some of the MeCP2-containing domains [3,58], the picture is still unclear. MeCP2 is expressed in all tissues, including the germinal cell line [3,9]. Indeed, MeCP2 plays an important role in cancer, in connection with its binding to methylated promoter CpG islands of tumor suppressor genes [51,112]. So far, most of the research carried out on this protein has mainly focused on its important role in Rett syndrome [6,88,113–116]. In the grand scheme of things, however, the substantial presence of MeCP2 in the brain suggests that any disruption of its activity in this tissue may have important, multifaceted pathological consequences beyond this syndrome. This research, however, represents only the tip of the iceberg when it comes to MeCP2 involvement in brain function, development, aging, and associated pathological states. There is a pressing need to further explore the role of MeCP2 in connection with all these diseases, especially as it pertains to the different aspects of its involvement at the molecular level. Acknowledgments We are very thankful to Jose Vicente Sanchez Mut for comments and for sharing his knowledge about brain organization and function. This work was supported by the European Research Council under grant agreement no. 268626 EPINORC Advanced grant (M.E.) and a Canadian Institutes of Health Research (CIHR) grant [MOP-130417] (J.A.). M.E. (Research Professor) is supported by the Catalan Institution for Research and Advanced Studies. We apologize to those authors whose work could not be cited here owing to space constraints.

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