Bioinformatics analysis of prognostic value and immunological role of MeCP2 in pan-cancer.

Methyl-CpG-binding protein 2(MeCP2) is an important epigenetic regulatory factor that promotes many tumor developments, such as liver cancer, breast cancer, and colorectal cancer. So far, no pan-cancer analysis has been reported. Therefore, this study aims to explore pan-cancer's prognostic value, immune infiltration pattern, and biological function. We used bioinformatics methods to analyze the expression and prognostic significance of MeCP2, and the relationship between MeCP2 and clinicopathological parameters, genetic variation, methylation, phosphorylation, immune cell infiltration, and biological function in pan-cancer from using a public database. The results showed that expression of MeCP2 was up-regulated in 8 cancers and down-regulated in 2 cancers, which was remarkably correlated with the prognosis, pathological stage, grade and subtype of cancers. The promoter methylation level of MeCP2 DNA was decreased in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), liver hepatocellular carcinoma (LIHC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), testicular germ cell tumors (TGCT), and stomach adenocarcinoma (STAD);decreased phosphorylation of S25, S90, S92, S241, S286, S325 and S435 was found in MeCP2, such as UCEC, lung adenocarcinoma (LUAD), ovarian serous cystadenocarcinoma (OV), colon adenocarcinoma (COAD), and kidney renal clear cell carcinoma (KIRC). Furthermore, MeCP2 expression was significantly associated with multiple immunomodulators and immune cell infiltration levels across most tumors. Therefore, our pan-cancer explored the prognostic markers and immunotherapeutic value of MeCP2 in different cancers.

Zinc finger protein 483 (ZNF483) regulates neuronal differentiation and methyl-CpG-binding protein 2 (MeCP2) intracellular localization.

Rett syndrome (OMIM #312750) is a developmental neurological disorder that is caused by a mutation in methyl-CpG-binding protein 2 (MeCP2). MeCP2 localizes to the nucleus, binds to methylated DNA, and regulates gene expression during neuronal development. MeCP2 assembles multiple protein complexes and its functions are controlled by interactions with its binding partners. Therefore, functional analysis of MeCP2 binding proteins is important. Previously, we proposed nine MeCP2-binding candidates in the cerebral cortex. In this study, we characterized and examined the function of the MeCP2 binding protein zinc finger protein 483 (ZNF483) to determine the significance of the MeCP2-ZNF483 interaction in neuronal development. Phylogenetic profiling revealed that the ZNF483 protein is broadly conserved in metazoans. In contrast, MeCP2 was obtained during evolution to chordates. To investigate ZNF483 functions, ZNF483-knockout P19 cell lines were established using the CRISPR-Cas9 system. These cell lines showed decreased cell proliferation, altered aggregate formation, decreased neuronal marker NeuN expression, and altered MeCP2 phosphorylation patterns. Notably, cytosolic localization of MeCP2 was enhanced by ZNF483-overexpression. Taken together, we propose that ZNF483 might be involved in the promotion of neuronal differentiation by regulating the subcellular localization of MeCP2 in P19 cells.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

GLP inhibits heterochromatin clustering and myogenic differentiation by repressing MeCP2.

Myogenic differentiation is accompanied by alterations in the chromatin states, which permit or restrict the transcriptional machinery and thus impact distinctive gene expression profiles. The mechanisms by which higher-order chromatin remodeling is associated with gene activation and silencing during differentiation is not fully understood. In this study, we provide evidence that the euchromatic lysine methyltransferase GLP regulates heterochromatin organization and myogenic differentiation. Interestingly, GLP represses expression of the methyl-binding protein MeCP2 that induces heterochromatin clustering during differentiation. Consequently, MeCP2 and HP1γ localization at major satellites are altered upon modulation of GLP expression. In GLP knockdown cells, depletion of MeCP2 restored both chromatin organization and myogenic differentiation. These results identify a novel regulatory axis between a histone methylation writer and DNA methylation reader, which is important for heterochromatin organization during differentiation.

NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation.

The brain is sensitive to the dose of MeCP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is known about its regulation. In this study, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3' UTR, resulting in an enrichment of inefficiently translated long mRNA isoforms. Furthermore, normalization of NUDT21 via siRNA-mediated knockdown in duplication patient lymphoblasts restores MeCP2 to normal levels. Ultimately, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation.

MeCP2 dependent heterochromatin reorganization during neural differentiation of a novel Mecp2-deficient embryonic stem cell reporter line.

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2(-/y)) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.

The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas.

BACKGROUND: Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)). METHODS: We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry. RESULTS: Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression. CONCLUSIONS: Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.

Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

MeCP2 preferentially binds to methylated linker DNA in the absence of the terminal tail of histone H3 and independently of histone acetylation.

Methyl CpG binding protein 2 (MeCP2) is a basic protein that contains a DNA methyl binding domain. The mechanism by which the highly positive charge of MeCP2 and its ability to bind methylated DNA contribute to the specificity of its binding to chromatin has long remained elusive. In this paper, we show that MeCP2 binds to nucleosomes in a very similar way to linker histones both in vitro and in vivo. However, its binding specificity strongly depends on DNA methylation. We also observed that as with linker histones, this binding is independent of the core histone H3 N-terminal tail and is not affected by histone acetylation.

Targeted delivery of an Mecp2 transgene to forebrain neurons improves the behavior of female Mecp2-deficient mice.

Rett syndrome is an X-linked neurological condition affecting almost exclusively girls that is caused by mutations of the MECP2 gene. Recent studies have shown that transgenic delivery of MeCP2 function to Mecp2-deficient male mice can improve their Rett-like behavior. However, as the brain of a Rett girl contains a mosaic of MeCP2 expressing and non-expressing neurons, and the over-expression of MeCP2 in neurons can induce a severe progressive neurological phenotype, testing whether functional rescue can be achieved by gene re-introduction strategies in a female model of Rett syndrome is warranted. To address this, we generated transgenic mice expressing an epitope-tagged Mecp2 transgene in forebrain neurons. These mice over-express MeCP2 protein at about 1.6 times normal levels in cortex and develop impaired motor behavior by 9-12 months of age. To test whether forebrain-targeted MeCP2 restoration would improve behavior in female Mecp2(-/+) mice, we crossed these transgenics with Mecp2(-/+) mice and examined the behavioral properties of the female rescue mice for 1 year. These assessments revealed that the diminished rearing activity, impaired mobility and the diminished locomotive activity of female Mecp2(-/+) mice were restored to wild-type levels in the rescue mice. These results show that improvement of Rett-like behavior can be achieved in Mecp2(-/+) females by targeted gene re-introduction without inducing deficits relating to MeCP2 over-expression.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain.

Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons.

A segment of the Mecp2 promoter is sufficient to drive expression in neurons.

Rett syndrome (RTT) is caused by mutations in the gene encoding methyl CpG-binding protein 2 (MeCP2). Although MeCP2 shows widespread expression in both neuronal and non-neuronal tissues, the symptoms of RTT are largely neurological. Herein, we have identified the regulatory region of the mouse Mecp2 gene that is sufficient for its restricted expression in neurons. A segment of the Mecp2 gene (-677/+56) exhibited strong promoter activity in neuronal cell lines and cortical neurons, but was inactive in non-neuronal cells and glia. The region necessary for neuronal-specific promoter activity was located within a 19 bp region (-63/-45). Several nuclear factors were found to bind to this region and some of these factors were enriched in nuclear extracts prepared from the brain. To examine the activity of the Mecp2 promoter in vivo, we generated transgenic mice expressing the LacZ reporter driven by the -677/+56 region of the Mecp2 gene. The transgene was expressed in the mesencephalon as early as embryonic day 10 and in the hindbrain and spinal cord by E12. Interestingly, a marked induction of transgene expression was observed postnatally throughout the brain, similar to that of endogenous MeCP2. However, expression of the transgene was absent in non-neuronal tissues that are known to express Mecp2. Taken together, these data indicate that the -677/+56 region of the Mecp2 promoter partially recapitulates the native expression pattern of the Mecp2 gene, which possesses restricted expression in neurons of the central nervous system.

Epigenetic basis for the transcriptional hyporesponsiveness of the human inducible nitric oxide synthase gene in vascular endothelial cells.

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome.

We have identified a girl with characteristic features of Rett syndrome (RTT) who carries a de novo balanced translocation involving chromosomes 1 and 7. Both breakpoints were mapped by fluorescence in situ hybridization with selected genomic clones from the regions of interest. Southern blot hybridisations, utilizing probes derived from breakpoint spanning BACs, detected several aberrant fragments specific for the patient. Sequence analysis of the cloned junction fragment indicated that on chromosome 1 the predominantly brain-expressed Netrin G1 (NTNG1) gene is disrupted, whereas on chromosome 7 there was no indication for a truncated gene. The chromosome 1 breakpoint lies within the 3' part of NTNG1 and affects alternatively spliced transcripts, suggesting that the phenotype in this patient is the result of disturbed NTNG1 expression. In silico translation of the NTNG1 splice variants predicted protein isoforms with different C-termini: one membrane bound through a glycosylphosphatidylinositol anchor and the other soluble. The membrane-bound protein isoform would be affected by the breakpoint, whereas the soluble form would remain intact. Our results suggest that the central nervous system is sensitive to NTNG1 expression levels and that NTNG1 is a novel candidate disease gene for RTT.