1568 S. D´ıaz de Leo´n-Guerrero et al. its target genes. In agreement with this idea, chromatin immunopre- cipitation assays show that MeCP2 is complexed with CREB1 in the promoter region of genes activated by MePC2, whereas a repressed gene did not show CREB1 enrichment. Interestingly, expressing both MeCP2 and CREB1, but not MeCP2 alone, enhances transcription of the MeCP2 target genes showing functional synergy. Thus, the effect of MeCP2 on gene expression depends on the proteins present at the promoter region of MePC2 target genes; when MeCP2 binds to co- repressors and HDACs it results in transcriptional repression. Nevertheless, a more thorough study of the methylation state in the genes activated by MeCP2 will help to shed more light on this issue, as well as looking for co-activators interacting directly with MeCP2 other than CREB1. A similar study performed in the cerebellum identiﬁed 1180 and 1102 altered genes in the MeCP2-tg and MeCP2-null mice, respec- tively (Ben-Shachar et al., 2009). The fold change in most cases was subtle, between 1.2- and 2-fold, with the exception of the gene Prl2c2 (prolactin-like protein), which was overexpressed 12-fold in the MeCP2-tg mice with respect to WT mice. This gene was not reported in the previous hypothalamus screening (Chahrour et al., 2008). As in the hypothalamus, MeCP2 activated the majority of the altered genes in the cerebellum. These results further support the idea that MeCP2 may act as a positive regulator of gene expression in the central nervous system and that this function is not restricted to the hypothalamus. Interestingly, whereas in the hypothalamus the screen found thousands of genes that were differentially expressed in the MeCP2-Tg and MeCP2-null mice (Chahrour et al., 2008), in the cerebellum only hundreds of misregulated genes were identiﬁed (Ben-Shachar et al., 2009). This difference indicates that, although some of the targets in different brain regions might be the same, there is still a considerable amount of genes that are differentially affected between different regions of the brain and suggests speciﬁc mecha- nisms of gene regulation that depend on the neuronal phenotype. This also supports the importance of epigenetic regulation and the greater impact it has on determining speciﬁcity for each tissue and cell type during development. It is also possible that MeCP2 might act preferentially as an activator on some cell types, whereas on others it might act as a repressor. Thus, looking at speciﬁc regions of the brain can provide more insights about MeCP2 targets and their regulation. However, it remains to be determined whether all of these genes are direct targets of MeCP2 or are regulated indirectly. Methyl-CpG binding protein 2 and microRNAs Recently, the role of miRNAs in a number of pathologies has started to be elucidated. To decipher the impact of miRNAs downstream of MeCP2, Urdinguio et al. (2010) used a speciﬁc miRNA microarray to determine the expression proﬁle in the MeCP2-null mice compared with WT mice. Sixty-ﬁve miRNAs showed differential expression between the two conditions. Among these, 45 were downregulated in MeCP2-null mice, whereas 18 were upregulated. Bisulﬁte sequencing and chromatin immunoprecipitation assays showed that, among four of the miRNAs identiﬁed, MeCP2 binds to methylated CpGs present on the 5¢ regulatory region of three of them. MeCP2 binds to the methylated 5¢ region of microRNA-29b (miR-29b), which is upreg- ulated in MeCP2-null mice, correlating with the role of MeCP2 as a repressor for miR-29b. However, both miR-146a and miR-146b were downregulated in the MeCP2-null mice and had MeCP2 enrichment at their methylated 5¢ ends, supporting a role for MeCP2 as a transcriptional activator. Both of these miRNAs target IRAK1 (interleukin-1 receptor-associated kinase; Taganov et al., 2006), which is upregulated in RTT mouse models. Therefore, downregulation of miR-146a and miR-146b could lead to higher levels of IRAK1. Thus, enhanced interleukin-1b signaling in glia cells resulting from elevated IRAK levels might contribute to the release of neurotoxic agents (Thornton et al., 2006) leading to neuronal death as previously described (Ballas et al., 2009; Maezawa et al., 2009; Maezawa & Jin, 2010). The miRNAs prove a very interesting method of regulation as they can act at both the post-transcriptional level and the translational level (Filipowicz et al., 2008). This suggests that the subtle changes in the mRNA levels of potential MeCP2 targets observed in different studies may not reﬂect the protein level. Thus, a proteomic approach in these models could certainly provide valuable information to fully under- stand RTT. Other studies have also focused on the regulation of miRNAs by MeCP2. One of these miRNAs is miR-184, which is found on an imprinted region of chromosome 9 (Nomura et al., 2008). Its expression is restricted to brain and testis and the paternal allele is the only one that is expressed in the brain. A CpG-rich region located downstream of the miR-184 transcript has been identiﬁed as hypermethylated only in brain (Nomura et al., 2008). KCl treatment increases miR-184 expression similarly to Bdnf in cortical neurons. Chromatin immunoprecipitation assays have shown that after KCl treatment the enrichment of MeCP2 in the miR-184 CpG-rich region was reduced in the paternal allele only, correlating with miR-184 speciﬁc paternal expression. The MeCP2-deﬁcient brain showed reduced miR-184 levels, which suggest a regulation similar to Bdnf. Interestingly, MBD1 also binds to this locus in the brain but, contrary to MeCP2, MBD1 deﬁciency causes miR-184 overexpression, as would be expected after the loss of a repressor (Liu et al., 2010). These observations point out the distinct mechanisms used by the different members of the MBD family to regulate gene expression in the brain. Furthermore, miR-184 results in an interesting MeCP2 target as it has been shown that this miRNA plays a role during neural development by inducing proliferation and inhibiting both neuronal and astrocytic differentiation (Liu et al., 2010). miR-184 targets Numblike, which has been shown to increase neuronal differentiation. These results suggest that MeCP2 might also regulate neuronal differentiation by indirectly targeting Numblike through miR-184. Interestingly, miR-184 is expressed in the two known brain regions (hippocampus and subventricular zone) of adult neurogenesis in vivo. However, it remains to be determined whether adult neurogenesis is altered in the MePC2-deﬁcient mice. Another miRNA that is regulated by MeCP2 is miR-137 (Szulwach et al., 2010). Overexpression of miR-137 enhances proliferation, whereas inhibition leads to a decreased proliferating capacity. Adult neural stem cell (aNSC) differentiation correlates with increased levels of miR-137. Consequently, MeCP2 deﬁciency promotes premature miR-137 expression before aNSC differentiation, consistent with MeCP2’s role as a repressor (Matijevic et al., 2009). Chromatin immunoprecipitation assays using MeCP2 antibodies show enrich- ment in the 5¢-region of miR-137 relative to the mice (MeCP2) ⁄ y) that correlates with increased methylated CpGs (Szulwach et al., 2010). Analysis of different chromatin marks in MeCP2) ⁄ y mice showed an increase in H3K4me3 and H3K9Ac in the region surrounding the miR-137 gene compared with WT mice (Szulwach et al., 2010); both histone marks are associated with an open chromatin state. During aNSC differentiation the levels of H3K4Me and H3K9Ac are increased, supporting MeCP2 deﬁciency leading to the premature establishment of an open chromatin conﬁguration. Additionally, an enrichment of Sox2 in the upstream region of miR- 137 was observed, which is lost in the MeCP2-null mice, suggesting 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