As mentioned above, methylation can physically prevent transcrip- tion factors from binding to their recognition sequences (Watt & Molloy, 1988). For example, the glial ﬁbrillary acidic protein gene is activated during astrocyte differentiation by the demethylation of a CpG dinucleotide located in a signal transducer and activator of transcription 3 binding element (STAT3) (Takizawa et al., 2001). Although methylation normally leads to transcriptional silencing, methylation of repressor protein-binding elements, as in the imprinted insulin-like growth factor 2 (Igf2) gene, can increase its expression (Eden et al., 2001; Murrell et al., 2001). Methylation signals can be interpreted directly by methyl-CpG- binding proteins that can alter gene transcription (Klose & Bird, 2006). In vertebrates, there are two families of methyl-CpG-binding proteins: the methyl-binding domain (MBD) family and the Kaiso family (reviewed in Bogdanovic & Veenstra, 2009). Five members of the MBD family have been identiﬁed: MeCP2, MBD1, MBD2, MBD3 and MBD4, although MBD3 presents a mutation on the MBD domain and is unable to bind methylated CpGs (Hendrich & Bird, 1998). Unlike the MBD members, the Kaiso family lacks an MBD, but binds to methyl-CpGs through a zinc ﬁnger domain (Prokhortchouk et al., 2001). All members of the MBD family, except MBD4, have been shown to act as transcriptional repressors in vitro (Hendrich & Bird, 1998). In contrast, MBD4 has been associated with minimizing mutations at 5-methylcytosine and in DNA repair (Bellacosa et al., 1999; Hendrich et al., 1999). A number of studies have shown that members of the MBD family play different roles throughout development and in disease. This is attributed to the capability of the MBD proteins to interact with repressing complexes such as Nucleosome Remodeling and histone Deacetylation (NuRD), RE1-silencing transcription factor (REST) and REST corepressor 1 (CoREST), as well as with various chromatin remodelers such as histone deacetylases (HDACs), Dnmt1, Polycomb, Brahma and Alpha thalassemia/mental retardation syndrome X-linked (ATRX) (Jones et al., 1998; Nan et al., 1998, 2007; Zhang et al., 1999; Lunyak et al., 2002; Fuks et al., 2003; Kimura & Shiota, 2003; Harikrishnan et al., 2005). The interaction of MBDs with HDAC complexes (Prokhortchouk et al., 2001) contributes to condensing the chromatin into higher order structures that are transcriptionally silent (Ng et al., 1999; Wade et al., 1999; Zhang et al., 1999; Feng & Zhang, 2001). Therefore, the MBDs provide a link between DNA methylation-mediated transcriptional repression, histone deacetylation and chromatin remodeling. Among the different histone post-transla- tional modiﬁcations, acetylation on different residues of histone 3 and histone 4 as well as methylation on histone 3 lysine 4 (H3K4me) are normally associated with an open chromatin state, whereas histone 3 methylation on lysine 9 and lysine 27 (H3K9me and H3K27me) is associated with a closed chromatin state (Kouzarides, 2007). There are some cases in which DNA methylation has been associated with histone marks (Vaissiere et al., 2008). For example, reduction of cytosine methylation leads to an increase of histone 3 acetylation on lysine 9 and lysine 14 (H3K9Ac and H3K14Ac), whereas H3K4 methylation (H3K4me) causes a decrease in H3K9me in mammals (Nguyen et al., 2001; Bachman et al., 2003). However, it is still unknown whether a functional relationship exists between DNA methylation and histone methylation to control gene expression. Methyl-CpG binding protein 2 and disease Methyl-CpG binding protein 2 was one of the ﬁrst members of the MBD family to be discovered (Lewis et al., 1992). Apart from the MBD domain, MeCP2 has a transcriptional repressor domain that is able to interact with other co-repressors (like deacetylase complexes), MeCP2 target genes and Rett syndrome 1565 two nuclear localization signals and a tryptophan-tryptophan domain in the C-terminus that facilitates binding to nucleosomal DNA and is thought to mediate protein–protein interactions (reviewed in Bienvenu & Chelly, 2006; Fig. 3). Methyl-CpG binding protein 2 is required for correct brain function and development. Loss of MeCP2 has been shown to delay neuronal maturation and synaptogenesis, and cause Rett syndrome (RTT; reviewed in Matijevic et al., 2009). RTT is an X-linked, neurodevel- opmental disorder that is characterized by a normal period of development from 6 to 18 months, followed by a subsequent loss of acquired speech and motor skills. Patients with RTT can also develop mental retardation, stereotyped hand movements, ataxia, seizures, microcephaly, autism and respiratory dysfunctions (Hagberg et al., 1983). RTT is mostly caused by mutations on the MeCP2 gene (Fig. 2), but mutations for cyclin-dependent kinase-like 5 (CDKL5) have also been associated with RTT (reviewed in Matijevic et al., 2009; both genes are located on the X chromosome). Although CDKL5 has been shown to be able to phosphorylate MeCP2 and cause its release from the DNA (Mari et al., 2005; Bertani et al., 2006), a direct functional relationship between these two molecules in RTT is controversial (see below). Methyl-CpG binding protein 2 brain levels in newborn mice are very low but increase signiﬁcantly with development, reaching a maximum at 5 weeks, after which MeCP2 expression remains constant (Skene et al., 2010). RTT has mostly been attributed to gene deregulation caused by a lack of MeCP2 expression in adult neurons (Luikenhuis et al., 2004). This correlates with the fact that patients with RTT develop normally through the ﬁrst 6–18 months of age after which they start presenting the characteristics of the disease (Hagberg et al., 1983). This idea is supported by the phenotype of the MeCP2-null mice. MeCP2-null mice develop normally until 5– 6 weeks of age, when they begin to show neurological symptoms, hind limb clasping and irregular breathing. They also present a reduction in weight and neuronal cell size, and normally die between 6 and 12 weeks (Chen et al., 2001; Guy et al., 2001). Even though RTT has normally been attributed to neuronal MeCP2 deﬁciency (Luikenhuis et al., 2004), several studies have begun to implicate non-neural cells in the development of the disease (Maezawa & Jin, 2010; Ballas et al., 2009; Maezawa et al., 2009; Table 1). Recently, it has been shown that MeCP2 is not only expressed in neurons, as previously thought (Skene et al., 2010), but is also expressed in embryonic and adult stages in all glial cell types, including astrocytes, oligodendrocyte progenitor cells, oligodendro- cytes and microglia. Analysis of the RTT mouse brain (Ballas et al., 2009) shows loss of MeCP2 expression in astrocytes as well as low H3K9me3 levels, whereas H3Ac is enriched. Co-culture of neurons and astrocytes demonstrates that RTT astrocytes are unable to support normal dendritic morphology in wild-type (WT) hippocampal neu- rons, which show fewer and shorter dendrites. Culturing WT hippocampal neurons in astrocytic conditioned media from RTT astrocyte cultures results in aberrant dendritic morphology and a reduced neuronal density, whereas RTT neurons cultured in WT astrocytic conditioned media show normal dendritic morphology, suggesting that MeCP2 regulates the expression of soluble factors that are required for neuronal survival. Another study has demonstrated that astrocytes from MeCP2+ ⁄ ) mice show only 30% of the MeCP2 protein level with respect to WT, whereas MeCP2+ ⁄ ) neurons show 70–80% compared with WT (Maezawa et al., 2009). Astrocytic levels of MeCP2 do not correspond with the expected values for X chromosome inactivation, suggestive of a non-cell-autonomous mechanism of MeCP2 expres- sion in astrocytes. In agreement with this, MeCP2 expression was ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 33, 1563–1574