BACKGROUND
methyl-cpg-binding protein  <dig>  is one of the proteins associated with epigenetic regulation, and mutations of this gene have been identified in the majority of patients with a severe neurodevelopmental disorder, rett syndrome , characterized by seizures, ataxic gait, language dysfunction, and autistic behavior  <cit> .
mecp2-null mice exhibit neurological abnormalities strikingly similar to those of rtt, supporting the hypothesis that classical rtt is due to a loss of mecp <dig> function  <cit> , and that mecp <dig> is essential for neuronal development, maturation, synaptic activity, learning and memory  <cit> .
mecp <dig> has been thought to be a transcriptional repressor that acts by binding to a number of methylated-cpg dinucleotides in the mammalian genome.
however, its deficiency does not result in the significant deregulation of the expression of a subset of genes as determined by a comparative expression microarray analyses between mecp2-null mice and wild-type mice  <cit> , but induces global changes in neuronal chromatin structure  <cit> .
these findings indicate that mecp <dig> may be a global gene silencer.
furthermore, mecp <dig> deficiency affects the expression levels of a large number of genes as determined by a comparative expression microarray analyses between mecp2-knock-in mice and mecp2-duplication mice  <cit> , indicating that mecp <dig> target genes are numerous.
however, it is still worthwhile to identify mecp2-target genes which that are centrally involved in rtt pathogenesis, since mecp <dig> functions cell-autonomously in neuronal maturation and dendritic arborization and discrete subsets of genes regulated by mecp <dig> may be essential for mature neuronal function  <cit> .
so far several genes associated with neuronal function have been reported as mecp <dig> targets, such as brain-derived neurotrophic factor   <cit> , glucocorticoid-regulated genes  <cit> , interleukin- <dig> receptor-associated kinase  <dig>   <cit> , insulin-growth factor binding protein  <dig>   <cit> , a transmembrane modulator of na+, k+-atpase activity   <cit> , and cyclin-dependent kinase-like  <dig>   <cit> .
however, it is likely that there are more unidentified neuronal mecp2-targets associated with the neurological features of rtt.
in this study, we used a genome-microarray based approach  <cit>  rather than a standard expression-microarray approach, to identify genomic regions that are epigenetically regulated by mecp <dig>
RESULTS
screening for bacs containing mecp <dig> binding sites with epigenetic modification
we assumed that mecp <dig> was bound to multiple sites in human genome.
in order to clarify these sites, we first performed chip on chip assay using our in-house bac array  with an anti-mecp <dig> antibody in human oral cancer cell lines .
as a result, we obtained  <dig> "positive" bac clones, which were suggestive of having mecp <dig> binding sites, out of the  <dig>  clones on the array .
we next screened bac clones encompassing hypermethylation site and repressive histone modification sites based on dna methylation using the bamca and chip-on-bac array assay with an anti-histone h3k9-2me antibody in the same cell lines  <cit> .
we identified  <dig> "triple positive" bac out of the  <dig> "mecp <dig> positive" bac clones, which contain mecp2-binding site, hypermethylation site, and repressive histone modification site.
although "triple positive" did not necessary mean these three epigenetic modifications existed at the same genomic site in a genomic region  in a bac, we considered that the genomic regions in these  <dig> bac clones potentially contained the site regulated by mecp <dig>  at this step, we used oral cancer cell lines because they had previously screened to identify mecp <dig> targets in carcinogenesis , and used this screening data in this study.
search for neuronal genes
we next searched for neuronal genes in the genomic regions within these  <dig> bac clones using a genome database , and identified the following four genes: apbb <dig>  and sra <dig>  in bac rp11-115i <dig> , pcdhb <dig>  in bac rp11-79k <dig> , and pcdh <dig>  in rp11-205n <dig> .
among these genes, the consensus mecp2-binding sequence with a/t bases  within 1- <dig> or 6- <dig> base pairs from a cpg di-nucleotide  <cit>  was identified in the 5' flanking region  in apbp <dig>  pcdhb <dig> and pcdh <dig>  but not in sra <dig> .
mecp2-binding status in the upstream regions of the neuronal genes in sh-sy5y human neuroblastoma cells
since the observed epigenetic alterations were found in oral cancer cell lines, we confirmed whether mecp <dig> was bound to the apbp <dig>  pcdhb <dig> and pcdh <dig> genes using the chip-pcr assay in human neuroblastoma sh-sy5y cells as neuronal origin cells.
within the 5' flanking regions of these genes, we found that mecp <dig> was bound to the regions of pcdh <dig>  pcdhb <dig> genes, but not to the apbp <dig> gene .
in this assay, we confirmed that mecp <dig> was bound to the promoter region of snurf/snrpn gene  but not bound to the promoter region of the gapdh gene  in sh-sy5y cells  <cit> .
methylation status in the upstream regions of the two neuronal genes in sh-sy5y human neuroblastoma cells
we examined the methylation status in the upstream regions of the pcdhb <dig> and pcdh <dig> genes, in order to determine whether the cpg sites were hypermethylated for mecp <dig> binding .
the pcdhb <dig> up-stream region was highly methylated as expected.
however, the pcdh <dig> up-stream region was unexpectedly less methylated, and the result was nonetheless consistent with a recent report, in which mecp <dig> not only binds to highly methylated regions, but also binds to less methylated regions  <cit> .
suppression of pcdhb <dig> and pcdh <dig> genes by mecp <dig> in sh-sy5y human neuroblastoma cells
to examine whether the expression of the pcdhb <dig> and pcdh <dig> genes was controlled by mecp <dig>  we evaluated the effects of wild-type or mbd-deleted mecp <dig> on either unmethylated or methylated promoter by the luciferase assay in sh-sy5y cells.
methylation status of these constructs was confirmed by bisulfite sequencing .
luciferase fusion plasmids containing  <dig>  kb of upstream sequences of the pcdhb <dig> or pcdh <dig> transcription start site were methylated by methylase sssi in vitro.
these luciferase reporter plasmids were co-transfected in combination with the mecp2-expressing plasmid into sh-sy5y cells.
the transcriptional activity of the unmethylated pcdhb <dig> promoter was suppressed by wild-type mecp <dig>  but not mbd-deleted mutant mecp <dig> .
likewise, the transcriptional activity of the methylated pcdhb <dig> promoter was suppressed by wild-type mecp <dig>  but not mbd-deleted mutant mecp <dig> .
approximately 70% reduction in promoter activity was found in methylated promoters, compared with unmethylated promoters, by mock , indicating that endogenous mecp <dig> preferentially suppresses pcdhb <dig> methylated promoter .
similar to pcdhb <dig>  the transcriptional activity of both the unmethylated and methylated pcdh <dig> promoter was suppressed by wild-type mecp <dig>  but not mbd-deleted mutant mecp <dig> .
we did not find down-regulation of the sv <dig> promoter region by wild-type mecp <dig>  suggesting that the sv <dig> gene is not a mecp2-target gene .
taken together, these results indicate that not only methylated but also unmethylated promoter of the pcdhb <dig> and pcdh <dig> genes are regulated by mecp <dig> in sh-sy5y cells.
knock-down effect on the two neuronal genes with mecp2-sirna in sh-sy5y human neuroblastoma cells
if pcdhb <dig> and pcdh <dig> genes are controlled by mecp <dig>  their expression from these genes should be increased under the mecp <dig> deficient condition.
to assess this, we performed knockdown experiments using mecp2-sirna.
we first confirmed the transfection efficiency of sirna in sh-sy5y cells, and the fluorescent labeled sirna showed that the efficiency of sirna delivery was approximately 50~60%.
as a result, the expression level of mecp <dig> mrna decreased by 85% by following treatment with mecp2-sirna compared with scramble-sirna .
under this condition, the expression levels of pcdhb <dig> and pcdh <dig> mrna were significantly increased by mecp2-sirna compared with scramble-sirna  .
expression levels of the two neuronal genes in mecp2-null mice and rtt brain tissue samples
we examined the expression of the two genes in brain tissue samples from mecp2-null mice   <cit> .
we compared the mrna expression level in the frontal cortex of 14-day-old mecp2-null mice with those of wild-type male mice.
as a result, we found that pcdhb <dig> and pcdh <dig> mrna were significantly increased in mecp2-null mice compared with wild-type mice  .
we further investigated the expression of these genes in postmortem rtt brain tissues.
as a result, the aberrant expression of the pcdhb <dig> gene was found in three of four rtt patients  compared with control individuals .
however, there was no apparent difference in the expression of pcdh <dig> gene in the brain tissue of controls and rtt individuals.
discussion
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 mecp <dig>  and directly contribute to its neuropathogenesis.
to address this question, several attempts to identify mecp <dig> 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  <cit> , indicating that the accumulation of subtle changes affect brain function and that brains are less tolerant of background transcriptional noise than other organs  <cit> .
to date, several neuronal molecules regulated by mecp <dig>  such as bdnf, igfbp <dig> and crmp <dig> have been identified by candidate gene approaches or mecp <dig> target screenings using expression microarrays  <cit> .
to our knowledge, this is the first attempt to identify mecp <dig> target genes using chip-on-bac array approach using a genome microarray.
we identified two genes pcdhb <dig> and pcdh <dig> that encode molecules associated with neuronal function.
however, we did not detect the previously reported mecp <dig> 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 mecp <dig> binding, dna methylation and repressive histone modification, although the reported genes are located at sites where mecp <dig> is bound.
a newly-developed chip-sequencing approach using a next-generation sequencer, which is a more quantitative method to assess methylation  <cit> , will shed light on the identification of new mecp <dig> target genes.
it has been thought that mecp <dig> represses transcription by binding specifically to methylated dna.
however, it was recently reported that mecp <dig> is also bound to unmethylated dna  <cit> .
in this context, our data supported this notion, because mecp <dig> repressed transcriptional activity of pcdhb <dig> and pcdh <dig> 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 mecp <dig> for methylated dna is ~3-fold greater than unmethylated dna  <cit> .
the protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system  <cit> .
they are divided into two groups  based on their genomic structure.
pcdhb <dig> and pcdh <dig> 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 pcdh <dig>  pcdh11y and pcdh <dig>  are associated with bipolar disorder, schizophrenia and autism, respectively  <cit> .
the clustered pcdh family is subdivided into three distinct gene groups in mammals .
pcdh-α expression is down regulated by myelination during neuronal maturation  <cit> , and pcdh-β, namely pcdhb <dig>  is expressed in dendritic spines and plays an important role in synaptogenesis  <cit> .
since the expression of pcdhb <dig> is not detectable in normal brains during development  <cit> , the presence of pcdhb <dig> in the brains of rtt patients and the up-regulation of pcdhb <dig> 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  <cit> .
furthermore, since our results indicate that pcdhb <dig> is epigenetically regulated by mecp <dig>  the pcdh-β gene cluster may be epigenetically regulated similar to the pcdh-α gene cluster in which epigenetic regulation produces isoforms in neurons  <cit> .
pcdh <dig> 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  <dig>  and pcdh <dig> exhibits a critical period for the establishment of specific synaptic connections  <cit> .
pcdh <dig> is also expressed in the ganglion cell layer of the retina  <cit> , and its over-expression leads to a morphological change and ca2+-dependent cell adhesion in mouse fibroblast l cells  <cit> .
therefore, the up-regulation of pcdh <dig>  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 .
another finding in our study, in which the upstream region of pcdh <dig> was unexpectedly unmethylated despite its transcriptional respression by mecp <dig> binding to its promoter, was consistent with the recent report that mecp <dig> can bind to less methylated regions of genes and repress their expression  <cit> .
several lines of evidence suggest that mecp <dig> acts as  a promoter of neuronal differentiation  <cit> ,  an effecter of dendritic arborization  <cit> ,  a modulator of synapses in postmitotic neurons),  an essential factor for the maturation of nmda receptors  <cit>  and  a controller of the balance between excitatory and inhibitory synaptic transmission through the maintenance of density between glutamate and gaba receptors  <cit> .
here we show that mecp <dig> also regulates protocadherins, including pcdh <dig> that is potentially associated with synaptogenesis.
therefore, our findings may help to clarify the pathogenesis of rtt with synaptic dysfunction.
CONCLUSIONS
in this study we identified two novel direct mecp <dig> target genes by chip and reporter assay.
expression of pcdhb <dig> and pcdh <dig> were regulated by mecp <dig> in human neuroblastoma cells and brain tissue.
on the basis of the previous findings of the nature of protocadherins, dysregulation of these molecules are potentially associated with the neuronal and synaptic dysfunction observed in the brains of rtt patients.

 methyl-cpg-binding protein is one of the proteins associated with epigenetic regulation, and mutations of this gene have been identified in the majority of patients with a severe neurodevelopmental disorder, rett syndrome, characterized by seizures, ataxic gait, language dysfunction, and autistic behavior. mecp2-null mice exhibit neurological abnormalities strikingly similar to those of rtt, supporting the hypothesis that classical rtt is due to a loss of mecp function, and that mecp is essential for neuronal development, maturation, synaptic activity, learning and memory. mecp has been thought to be a transcriptional repressor that acts by binding to a number of methylated-cpg dinucleotides in the mammalian genome. however, its deficiency does not result in the significant deregulation of the expression of a subset of genes as determined by a comparative expression microarray analyses between mecp2-null mice and wild-type mice, but induces global changes in neuronal chromatin structure. these findings indicate that mecp may be a global gene silencer. furthermore, mecp deficiency affects the expression levels of a large number of genes as determined by a comparative expression microarray analyses between mecp2-knock-in mice and mecp2-duplication mice, indicating that mecp target genes are numerous. however, it is still worthwhile to identify mecp2-target genes which that are centrally involved in rtt pathogenesis, since mecp functions cell-autonomously in neuronal maturation and dendritic arborization and discrete subsets of genes regulated by mecp may be essential for mature neuronal function. so far several genes associated with neuronal function have been reported as mecp targets, such as brain-derived neurotrophic factor, glucocorticoid-regulated genes, interleukin- receptor-associated kinase, insulin-growth factor binding protein, a transmembrane modulator of na+, k+-atpase activity, and cyclin-dependent kinase-like. however, it is likely that there are more unidentified neuronal mecp2-targets associated with the neurological features of rtt. in this study, we used a genome-microarray based approach rather than a standard expression-microarray approach, to identify genomic regions that are epigenetically regulated by mecp  screening for bacs containing mecp binding sites with epigenetic modification we assumed that mecp was bound to multiple sites in human genome. in order to clarify these sites, we first performed chip on chip assay using our in-house bac array with an anti-mecp antibody in human oral cancer cell lines. as a result, we obtained "positive" bac clones, which were suggestive of having mecp binding sites, out of the clones on the array. we next screened bac clones encompassing hypermethylation site and repressive histone modification sites based on dna methylation using the bamca and chip-on-bac array assay with an anti-histone h3k9-2me antibody in the same cell lines. we identified "triple positive" bac out of the "mecp positive" bac clones, which contain mecp2-binding site, hypermethylation site, and repressive histone modification site. although "triple positive" did not necessary mean these three epigenetic modifications existed at the same genomic site in a genomic region in a bac, we considered that the genomic regions in these bac clones potentially contained the site regulated by mecp at this step, we used oral cancer cell lines because they had previously screened to identify mecp targets in carcinogenesis, and used this screening data in this study. search for neuronal genes we next searched for neuronal genes in the genomic regions within these bac clones using a genome database, and identified the following four genes: apbb and sra in bac rp11-115i, pcdhb in bac rp11-79k, and pcdh in rp11-205n. among these genes, the consensus mecp2-binding sequence with a/t bases within 1- or 6- base pairs from a cpg di-nucleotide was identified in the 5' flanking region in apbp pcdhb and pcdh but not in sra. mecp2-binding status in the upstream regions of the neuronal genes in sh-sy5y human neuroblastoma cells since the observed epigenetic alterations were found in oral cancer cell lines, we confirmed whether mecp was bound to the apbp pcdhb and pcdh genes using the chip-pcr assay in human neuroblastoma sh-sy5y cells as neuronal origin cells. within the 5' flanking regions of these genes, we found that mecp was bound to the regions of pcdh pcdhb genes, but not to the apbp gene. in this assay, we confirmed that mecp was bound to the promoter region of snurf/snrpn gene but not bound to the promoter region of the gapdh gene in sh-sy5y cells. methylation status in the upstream regions of the two neuronal genes in sh-sy5y human neuroblastoma cells we examined the methylation status in the upstream regions of the pcdhb and pcdh genes, in order to determine whether the cpg sites were hypermethylated for mecp binding. the pcdhb up-stream region was highly methylated as expected. however, the pcdh up-stream region was unexpectedly less methylated, and the result was nonetheless consistent with a recent report, in which mecp not only binds to highly methylated regions, but also binds to less methylated regions. suppression of pcdhb and pcdh genes by mecp in sh-sy5y human neuroblastoma cells to examine whether the expression of the pcdhb and pcdh genes was controlled by mecp we evaluated the effects of wild-type or mbd-deleted mecp on either unmethylated or methylated promoter by the luciferase assay in sh-sy5y cells. methylation status of these constructs was confirmed by bisulfite sequencing. luciferase fusion plasmids containing kb of upstream sequences of the pcdhb or pcdh transcription start site were methylated by methylase sssi in vitro. these luciferase reporter plasmids were co-transfected in combination with the mecp2-expressing plasmid into sh-sy5y cells. the transcriptional activity of the unmethylated pcdhb promoter was suppressed by wild-type mecp but not mbd-deleted mutant mecp. likewise, the transcriptional activity of the methylated pcdhb promoter was suppressed by wild-type mecp but not mbd-deleted mutant mecp. approximately 70% reduction in promoter activity was found in methylated promoters, compared with unmethylated promoters, by mock, indicating that endogenous mecp preferentially suppresses pcdhb methylated promoter. similar to pcdhb the transcriptional activity of both the unmethylated and methylated pcdh promoter was suppressed by wild-type mecp but not mbd-deleted mutant mecp. we did not find down-regulation of the sv promoter region by wild-type mecp suggesting that the sv gene is not a mecp2-target gene. taken together, these results indicate that not only methylated but also unmethylated promoter of the pcdhb and pcdh genes are regulated by mecp in sh-sy5y cells. knock-down effect on the two neuronal genes with mecp2-sirna in sh-sy5y human neuroblastoma cells if pcdhb and pcdh genes are controlled by mecp their expression from these genes should be increased under the mecp deficient condition. to assess this, we performed knockdown experiments using mecp2-sirna. we first confirmed the transfection efficiency of sirna in sh-sy5y cells, and the fluorescent labeled sirna showed that the efficiency of sirna delivery was approximately 50~60%. as a result, the expression level of mecp mrna decreased by 85% by following treatment with mecp2-sirna compared with scramble-sirna. under this condition, the expression levels of pcdhb and pcdh mrna were significantly increased by mecp2-sirna compared with scramble-sirna. expression levels of the two neuronal genes in mecp2-null mice and rtt brain tissue samples we examined the expression of the two genes in brain tissue samples from mecp2-null mice. we compared the mrna expression level in the frontal cortex of 14-day-old mecp2-null mice with those of wild-type male mice. as a result, we found that pcdhb and pcdh mrna were significantly increased in mecp2-null mice compared with wild-type mice. we further investigated the expression of these genes in postmortem rtt brain tissues. as a result, the aberrant expression of the pcdhb gene was found in three of four rtt patients compared with control individuals. however, there was no apparent difference in the expression of pcdh gene in the brain tissue of controls and rtt individuals. discussion 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 mecp and directly contribute to its neuropathogenesis. to address this question, several attempts to identify mecp 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 mecp such as bdnf, igfbp and crmp have been identified by candidate gene approaches or mecp target screenings using expression microarrays. to our knowledge, this is the first attempt to identify mecp target genes using chip-on-bac array approach using a genome microarray. we identified two genes pcdhb and pcdh that encode molecules associated with neuronal function. however, we did not detect the previously reported mecp 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 mecp binding, dna methylation and repressive histone modification, although the reported genes are located at sites where mecp 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 mecp target genes. it has been thought that mecp represses transcription by binding specifically to methylated dna. however, it was recently reported that mecp is also bound to unmethylated dna. in this context, our data supported this notion, because mecp repressed transcriptional activity of pcdhb and pcdh 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 mecp 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 based on their genomic structure. pcdhb and pcdh 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 pcdh pcdh11y and pcdh are associated with bipolar disorder, schizophrenia and autism, respectively. the clustered pcdh family is subdivided into three distinct gene groups in mammals. pcdh-α expression is down regulated by myelination during neuronal maturation, and pcdh-β, namely pcdhb is expressed in dendritic spines and plays an important role in synaptogenesis. since the expression of pcdhb is not detectable in normal brains during development, the presence of pcdhb in the brains of rtt patients and the up-regulation of pcdhb 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. furthermore, since our results indicate that pcdhb is epigenetically regulated by mecp the pcdh-β gene cluster may be epigenetically regulated similar to the pcdh-α gene cluster in which epigenetic regulation produces isoforms in neurons. pcdh 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 and pcdh exhibits a critical period for the establishment of specific synaptic connections. pcdh 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 pcdh 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. another finding in our study, in which the upstream region of pcdh was unexpectedly unmethylated despite its transcriptional respression by mecp binding to its promoter, was consistent with the recent report that mecp can bind to less methylated regions of genes and repress their expression. several lines of evidence suggest that mecp acts as a promoter of neuronal differentiation, an effecter of dendritic arborization, a modulator of synapses in postmitotic neurons), an essential factor for the maturation of nmda receptors and a controller of the balance between excitatory and inhibitory synaptic transmission through the maintenance of density between glutamate and gaba receptors. here we show that mecp also regulates protocadherins, including pcdh that is potentially associated with synaptogenesis. therefore, our findings may help to clarify the pathogenesis of rtt with synaptic dysfunction.  in this study we identified two novel direct mecp target genes by chip and reporter assay. expression of pcdhb and pcdh were regulated by mecp in human neuroblastoma cells and brain tissue. on the basis of the previous findings of the nature of protocadherins, dysregulation of these molecules are potentially associated with the neuronal and synaptic dysfunction observed in the brains of rtt patients. 
within these regions, chromatin immunoprecipitation analysis revealed that mecp binds to the upstream regions of the protocadherin genes pcdhb and pcdh in human neuroblastoma sh-sy5y cells. pcdhb and pcdh promoter activities were down-regulated by mecp but not by mbd-deleted mecp these gene expression were up-regulated following mecp reduction with sirna in sh-sy5y cells and in the brains of mecp2-null mice. using a genome-microarray approach, we found genomic regions that contain sites potentially regulated by mecp based on the features of mecp binding, dna methylation, and repressive histone modification in human cell lines. we identified mecp target genes that encode neuronal adhesion molecules using chip-on-bac array approach. however, it is likely that there are more unidentified neuronal mecp2-targets associated with the neurological features of rtt. rett syndrome is a neurodevelopmental and autistic disease caused by mutations of methyl-cpg-binding protein gene. mecp protein is mainly expressed in neurons and binds to methylated gene promoters to suppress their expression, indicating that rett syndrome is caused by the deregulation of target genes in neurons. furthermore, pcdhb was up-regulated in postmortem brains from rett syndrome patients. since these protocadherin genes are generally essential for brain development, aberrant regulation of these molecules may contribute to the pathogenesis of the neurological features observed in rett syndrome.
 rett syndrome is a neurodevelopmental and autistic disease caused by mutations of methyl-cpg-binding protein gene. mecp protein is mainly expressed in neurons and binds to methylated gene promoters to suppress their expression, indicating that rett syndrome is caused by the deregulation of target genes in neurons. however, it is likely that there are more unidentified neuronal mecp2-targets associated with the neurological features of rtt.  using a genome-microarray approach, we found genomic regions that contain sites potentially regulated by mecp based on the features of mecp binding, dna methylation, and repressive histone modification in human cell lines. within these regions, chromatin immunoprecipitation analysis revealed that mecp binds to the upstream regions of the protocadherin genes pcdhb and pcdh in human neuroblastoma sh-sy5y cells. pcdhb and pcdh promoter activities were down-regulated by mecp but not by mbd-deleted mecp these gene expression were up-regulated following mecp reduction with sirna in sh-sy5y cells and in the brains of mecp2-null mice. furthermore, pcdhb was up-regulated in postmortem brains from rett syndrome patients.  we identified mecp target genes that encode neuronal adhesion molecules using chip-on-bac array approach. since these protocadherin genes are generally essential for brain development, aberrant regulation of these molecules may contribute to the pathogenesis of the neurological features observed in rett syndrome. 