1570 S. D´ıaz de Leo´n-Guerrero et al. In addition to Dnmt–HDAC interaction, recent evidence shows the existence of speciﬁc interactions between Dnmts and histone methyl- transferases that incorporate repressive histone marks. Dnmt3 together with the H3K9 methyltranferase G9A cooperatively ensures proper neurogenesis in zebraﬁsh (Rai et al., 2010). Whether the Dnmt3 family in mammals displays a similar Dnmt–histone methyltransferase relationship will be a matter for future investigations. Signals controlling methyl-CpG binding protein 2 gene expression and function A precise regulation of MeCP2 levels is needed as both deﬁcient and overexpressing brains exhibit RTT-like phenotypes (Ramocki et al., 2009). Quantiﬁcation studies have estimated 16 · 106 molecules of MeCP2 per nucleus in neurons (Skene et al., 2010). The estimated number of methylated CpG sites is about 40 · 106 sites per nucleus, meaning that there are enough MeCP2 molecules to bind approxi- mately half of the CpG sites. Histone H1 is present at a ratio of one molecule per nucleosome, although in neurons this relationship is reduced to one H1 molecule for every two nucleosomes (Pearson et al., 1984). MeCP2 can compete with H1 for binding to methylated chromatin, functioning as a linker histone (Nan et al., 1997). In agreement with this, MeCP2-null mouse brain shows a twofold increase in H1, supporting the idea that, in the brain, MeCP2 binds half of the nucleosomes and H1 binds the other half (Skene et al., 2010). This suggests that MeCP2 deﬁciency could also lead to an overall chromatin decondensation that would result in gene deregu- lation as seen in previous screens. The MeCP2 gene is known to have multiple polyadenylation sites (Reichwald et al., 2000); the longer transcript (approximately 10 kb) is predominantly transcribed in the brain and its 3¢ untranslated region contains target motifs for different miRNAs. One of these sites is for miR-132, which plays a role in neurite outgrowth mediated by Bdnf (Vo et al., 2005). Overexpression of miR-132, as well as treatment with Forskolin and KCl (which induce miR-132 expression through CREB), in primary cortical neurons decreased MeCP2 protein levels (Klein et al., 2007). Bdnf levels are increased both by MeCP2 overexpression and by blocking miR-132 with an antisense oligo- nucleotide. MeCP2 overexpression induces Bdnf expression, which in turn increases miR-132 transcription that downregulates MeCP2 protein production. This suggests a homeostatic mechanism for maintaining correct MeCP2 levels (Klein et al., 2007); however, little is known about additional factors regulating the expression of MeCP2 gene in the brain. Few reports are available regarding extracellular signals regulating MeCP2 function. Recently, it has been shown that MeCP2 is phosphorylated by Calcium/calmodulin-dependent protein kinase II (CaMKII) on Ser421 in response to neuronal depolarization and Bdnf (Zhou et al., 2006). This phosphorylation releases MeCP2 from the promoter IV of Bdnf resulting in increased Bdnf expression (Chen et al., 2003). Consistent with this, expression of an MeCP2 S421A mutant prevents neurite arborization and reduces Bdnf expression (Tao et al., 2009), indicating that MeCP2 phosphorylation at this residue promotes MeCP2 release from its targets allowing their expression. Although MeCP2 is expressed in many adult tissues it is phosphory- lated at Ser421 almost exclusively in the adult brain (Zhou et al., 2006), suggesting that phosphorylation of MeCP2 at this residue is required for homeostatic brain functions. MeCP2 phosphorylation at the amino-terminus also modulates its DNA binding and silencing activity. In contrast to Ser421, phosphorylation at Ser80 is required for MeCP2 binding to DNA and correct regulation of target genes like gene trap locus 2 (Gtl2) and proopiomelanocortin (Pomc) among others (Tao et al., 2009). Transgenic knock-in mice carrying the S80A mutation show reduced MeCP2 binding to target genes and consequently aberrant gene expression (Tao et al., 2009). The homeodomain-interacting kinase 2 has been shown to phosphorylate MeCP2 at Ser80 (Bracaglia et al., 2009). Accordingly, both the homeodomain-interacting kinase 2-deﬁcient mice and S80A knock-in mice show locomotion defects, suggesting that phosphorylation at this residue by HIP2K regulates MeCP2 activity in vivo (Zhang et al., 2007). Interestingly, although signals that promote neuronal survival like neuronal activity and calcium ﬂuxes lead to Ser80 dephosphor- ylation and Ser421 phosphorylation, MeCP2 phosphorylation by HIP2K at Ser80 has been associated with cell death (Bracaglia et al., 2009). Thus, it remains to be determined whether phosphorylation at this site promotes or represses the expression of pro-apoptotic or antiapoptotic genes, respectively; and also whether the phenotypes observed in the S80A knock-in mice and homeodomain-interacting kinase 2-deﬁcient mice result from altered neural cell death. Never- theless, it seems that both Ser80 dephosphorylation and Ser421 phosphorylation are required to dissociate MeCP2 from DNA in response to neuronal activity and calcium inﬂux. As mentioned above, mutations in CDKL5 have been identiﬁed in patients with RTT and other encephalopathies (Matijevic et al., 2009). The fact that MeCP2–CDKL5 protein–protein interactions have been shown by two independent groups, and that mutations in CDKL5 found in patients with RTT alter its kinase activity and cellular distribution (Mari et al., 2005), suggest that these naturally occurring mutations alter the MeCP2–CDKL5 interaction and point out a functional role for these molecules in RTT development. Although it is not yet clear whether MeCP2 is a direct target of CDKL5, Chen et al. (2010) have shown that CDKL5 is required for normal neurite outgrowth, a process that is altered in RTT neurons (Jellinger et al., 1988). Interestingly, Bdnf regulates neurite outgrowth by promoting CDKL5 kinase activity leading to ras-related C3 botulinum toxin substrate 1 (rac-1) activation and neurite arborization (Chen et al., 2010). Thus, we propose that, during normal development, neuronal activity and other extracellular signals promote MeCP2 dephosphor- ylation at Ser80 and phosphorylation at Ser421 alleviating MeCP2 repression of the Bdnf gene. Bdnf is then produced, secreted and through its receptor initiates an autocrine loop that further inhibits MeCP2 function at two levels: (i) preventing MeCP2 DNA binding through phosphorylation mediated by CDKL5 and ⁄ or CAMKII and (ii) reducing MeCP2 levels by inducing miR-132 via CREB, thus ensuring the expression of Bdnf and other genes involved in neurite outhgrowth (Fig. 4). Brain-derived neurotrophic factor, through its speciﬁc receptor Neurotrophic tyrosine kinase receptor type 2 (TrkB), activates distinct signal transduction pathways [protein kinase C (PKC), v-akt murine thymoma viral oncogene homolog 1 (AKT) and mitogen-activated protein kinase (MAPK)] that regulate neural functions (Yoshii & Constantine-Paton, 2010). MeCP2 protein contains different domains that may be targeted by these pathways as demonstrated by a bioinformatic analysis (Motif Scan) performed in our laboratory. This analysis revealed potential phosphorylation sites for cyclin-dependent kinase 5 (CDK5) in the central region of the molecule (Ser164, Ser178, Ser341, Ser350 and Ser360), as well as putative sites for different members of the PKC family – at the amino-terminus for PKCa ⁄ b (Ser113) and in the central region for PKCe and PKCd (Ser292; Fig. 3). Interestingly, a potential AKT phosphorylation site (Ser357) is located within a putative MeCP2–14.3.3 interaction domain (SPKGRSSS357ASSPPKK). This domain also contains Ser350 and Ser360, which could be phosphorylated by CDKL5 (Fig. 2). 14.3.3 proteins regulate protein function by steric competition 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