It had been thought that the causative gene for RTT should encode a synapse-associated molecule based on its pathogenesis. However, the gene in which most RTT patients have mutations does not encode a synapse molecule, but encodes an epigenetic regulation protein. This raises the question about which synaptic molecules are regulated by MeCP2, and directly contribute to its neuropathogenesis. To address this question, several attempts to identify MeCP2 target genes have been performed. The initial study using a expression microarray demonstrated that subtle expression changes occur in the brain of Mecp2-null mice , indicating that the accumulation of subtle changes affect brain function and that brains are less tolerant of background transcriptional noise than other organs . To date, several neuronal molecules regulated by MeCP2, such as BDNF, IGFBP3 and CRMP1 have been identified by candidate gene approaches or MeCP2 target screenings using expression microarrays [[12–16]]. To our knowledge, this is the first attempt to identify MeCP2 target genes using ChIP-on-BAC array approach using a genome microarray. We identified two genes PCDHB1 and PCDH7 that encode molecules associated with neuronal function.
However, we did not detect the previously reported MeCP2 target genes probably because our in-house array only covers one third of the human genome and the genomic loci of previously identified genes might not be located within the overlapping regions with MeCP2 binding, DNA methylation and repressive histone modification, although the reported genes are located at sites where MeCP2 is bound. A newly-developed ChIP-sequencing approach using a next-generation sequencer, which is a more quantitative method to assess methylation , will shed light on the identification of new MeCP2 target genes.
It has been thought that MeCP2 represses transcription by binding specifically to methylated DNA. However, it was recently reported that MeCP2 is also bound to unmethylated DNA [26, 27]. In this context, our data supported this notion, because MeCP2 repressed transcriptional activity of PCDHB1 and PCDH7 genes either via methylated or unmethylated promoter constructs. However, the transcriptional activity was more effectively repressed via the methylated promoter constructs than via the unmethylated promoter constructs, which are consist with a report showing that the affinity of MeCP2 for methylated DNA is ~3-fold greater than unmethylated DNA .
The protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system . They are divided into two groups (the clustered and nonclustered Pcdh families) based on their genomic structure. PCDHB1 and PCDH7 are belonged to the clusterd and nonclustered families, respectively. Genome association studies have shown that single-nucleotide polymorphisms and deletions in Pcdh genes, such as PCDH10, PCDH11Y and PCDH12, are associated with bipolar disorder, schizophrenia and autism, respectively [[29–33]].
The clustered Pcdh family is subdivided into three distinct gene groups in mammals (Pcdh-α, Pcdh-β, and Pcdh-γ). Pcdh-α expression is down regulated by myelination during neuronal maturation [34, 35], and Pcdh-β, namely PCDHB16, is expressed in dendritic spines and plays an important role in synaptogenesis . Since the expression of PCDHB1 is not detectable in normal brains during development , the presence of PCDHB1 in the brains of RTT patients and the up-regulation of PCDHB1 in Mecp2-null mice may be associated with the neurological findings in RTT brains, such as decreased neuronal size, increased cell density and reduced dendritic arborization [[5, 37–39]]. Furthermore, since our results indicate that PCDHB1 is epigenetically regulated by MeCP2, the Pcdh-β gene cluster may be epigenetically regulated similar to the Pcdh-α gene cluster in which epigenetic regulation produces isoforms in neurons .
PCDH7 is predominantly expressed in the somatosensory and visual cortices in the cerebral cortex, external granule cell layer in the cerebellar cortex, and the brainstem starting from embryonic day 17, and PCDH7 exhibits a critical period for the establishment of specific synaptic connections [41, 42]. PCDH7 is also expressed in the ganglion cell layer of the retina , and its over-expression leads to a morphological change and Ca2+-dependent cell adhesion in mouse fibroblast L cells . Therefore, the up-regulation of PCDH7, observed in the brains of Mecp2-null mice and neuroblastoma cells following MECP2-siRNA treatment, could potentially alter synaptic connections. However, no up-regulation was found in the brain tissues of RTT patients, and this may be due to the area of the brain examined (prefrontal cortex). Another finding in our study, in which the upstream region of PCDH7 was unexpectedly unmethylated despite its transcriptional respression by MeCP2 binding to its promoter, was consistent with the recent report that MeCP2 can bind to less methylated regions of genes and repress their expression .
Several lines of evidence suggest that MeCP2 acts as (1) a promoter of neuronal differentiation [46, 47], (2) an effecter of dendritic arborization [[3, 5, 38, 39]], (3) a modulator of synapses in postmitotic neurons), (4) an essential factor for the maturation of NMDA receptors  and (5) a controller of the balance between excitatory and inhibitory synaptic transmission through the maintenance of density between glutamate and GABA receptors [[49–54]]. Here we show that MeCP2 also regulates protocadherins, including PCDH7 that is potentially associated with synaptogenesis. Therefore, our findings may help to clarify the pathogenesis of RTT with synaptic dysfunction.