Methyl-CpG-binding Protein 2 Overexpression Research Articles

CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to prevent DNA hypermethylation and ensure normal transcription in growing oocytes

The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.

MiR-488-3p facilitates wound healing through CYP1B1-mediated Wnt/β-catenin signaling pathway by targeting MeCP2.

Diabetic wounds are difficult to heal, but the pathogenesis is unknown. MicroRNAs (miRNAs) are thought to play important roles in wound healing. The effect of miR-488-3p in wound healing was studied in this article. The gene methylation was measured by methylation specific PCR (MSP) assay. A dual-luciferase reporter assay was adopted to analyze the interaction between miR-488-3p and MeCP2. Cytochrome P450 1B1 (CYP1B1) is a monooxygenase belonging to the cytochrome P450 family that aids in wound healing. Our findings showed that the miR-488-3p and CYP1B1 expression levels were much lower in wound tissues of diabetics with skin defects, but the methyl-CpG-binding protein 2 (MeCP2) level was significantly higher than that in control skin tissues. MiR-488-3p overexpression increased cell proliferation and migration, as well as HUVEC angiogenesis, while inhibiting apoptosis, according to function experiments. In vitro, MeCP2 inhibited wound healing by acting as a target of miR-488-3p. We later discovered that MeCP2 inhibited CYP1B1 expression by enhancing its methylation state. In addition, CYP1B1 knockdown inhibited wound healing. Furthermore, MeCP2 overexpression abolished the promoting effect of miR-488-3p on wound healing. It also turned out that CYP1B1 promoted wound healing by activating the Wnt4/β-catenin pathway. Animal experiments also showed that miR-488-3p overexpression could accelerate wound healing in diabetic male SD rats. MiR-488-3p is a potential therapeutic target for diabetic wound healing since it improved wound healing by activating the CYP1B1-mediated Wnt4/-catenin signaling cascade via MeCP2.

Methylated Nucleotide-Based Proteolysis-Targeting Chimera Enables Targeted Degradation of Methyl-CpG-Binding Protein 2.

Methyl-CpG-binding protein 2 (MeCP2), a reader of DNA methylation, has been extensively investigated for its function in neurological and neurodevelopmental disorders. Emerging evidence indicates that MeCP2 exerts an oncogenic function in cancer; however, the endeavor to develop a MeCP2-targeted therapy remains a challenge. This work attempts to address it by introducing a methylated nucleotide-based targeting chimera termed methyl-proteolysis-targeting chimera (methyl-PROTAC). The methyl-PROTAC incorporates a methylated cytosine into an oligodeoxynucleotide moiety to recruit MeCP2 for targeted degradation in a von Hippel-Lindau- and proteasome-dependent manner, thus displaying antiproliferative effects in cancer cells reliant on MeCP2 overexpression. This selective cytotoxicity endows methyl-PROTAC with the capacity to selectively eliminate cancer cells that are addicted to the overexpression of the MeCP2 oncoprotein. Furthermore, methyl-PROTAC-mediated MeCP2 degradation induces apoptosis in cancer cells. These findings underscore the therapeutic potential of methyl-PROTAC to degrade undruggable epigenetic regulatory proteins. In summary, the development of methyl-PROTAC introduces an innovative strategy by designing a modified nucleotide-based degradation approach for manipulating epigenetic factors, thereby representing a promising avenue for the advancement of PROTAC-based therapeutics.

Circular RNA CircDHRS3 Aggravates IL-1β-induced ECM Degradation, Apoptosis, and Inflammatory Response via Mediating MECP2 Expression.

Previous studies have reported that circular RNA hsa_circ_0010024 (circDHRS3), microRNA (miR)-193a-3p, and Methyl CpG binding protein 2 (MECP2) are unconventionally expressed in osteoarthritis (OA) cartilage samples. However, the regulatory mechanisms among circDHRS3, miR-193a-3p, and MECP2 in OA pathogenesis are unclear. Changes of circDHRS3, miR-193a-3p, and MECP2 mRNA were detected by qRT-PCR. Several protein levels were evaluated using western blotting. Cell proliferation was analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) and cell counting assays. Cell apoptosis was determined by flow cytometry assay. Detection of pro-inflammatory cytokines was conducted using ELISA. The relationship between circDHRS3 or MECP2 and miR-193a-3p was validated by dual-luciferase reporter assay. We verified that circDHRS3 and MECP2 were overexpressed in OA cartilage samples, whereas miR-193a-3p was downregulated. CircDHRS3 silencing weakened IL-1β-induced chondrocyte cartilage extracellular matrix (ECM) degradation, apoptosis, and inflammatory response. CircDHRS3 adsorbed miR-193a-3p to modulate MECP2 expression. Also, silencing of miR-193a-3p impaired circDHRS3 silencing-mediated suppression on IL-1β-induced chondrocyte injury. Also, MECP2 overexpression alleviated miR-193a-3p mimic-mediated inhibition on IL-1β-prompted chondrocyte injury. CircDHRS3 silencing reduced MECP2 expression via sponging miR-193a-3p, thereby weakening IL-1β-induced chondrocyte ECM degradation, apoptosis, and inflammatory response.

Methyl-CpG Binding Protein 2 as a Potential Diagnostic and Prognostic Marker Facilitates Glioma Progression Through Activation of Wnt/β-Catenin Pathway

Glioma is the primary malignant tumor in the central nervous system and has high malignancy, mortality, and recurrence rates. Because of its heterogeneity and drug resistance, the blood-brain barrier, and other factors, the treatment of glioma has mainly been surgical resection combined with traditional radiotherapy and chemotherapy. However, the therapeutic effect has not been satisfactory. Methyl-CpG binding protein 2 (MeCP2) is an epigenetic regulator that has been reported to regulate the initiation and progression of glioma. However, the underlying mechanism in glioma has remained unclear. The gene expression of MeCp2, miR-138-5p, the epithelial-mesenchymal transition, the apoptosis-related gene, and the Wnt/β-Catenin pathway-related gene and proliferation were detected by reverse transcription-quantitative polymerase chain reaction or Western blot. The cell proliferation and apoptosis of the glioma cell was assessed using the CCK-8 assay and flow cytometry assay. The relationship between miR-138-5p and MeCp2 was measured using the dual luciferase reporter assay. The effect of MeCp2 in U87 cells was examined in a xenograft tumorigenesis model in vivo. In our study, we found that MeCP2 was upregulated in glioma tissues and cell lines and that MeCP2 knockdown repressed cell proliferation and epithelial-mesenchymal transition but boosted cell apoptosis in glioma. Furthermore, MeCP2 knockdown attenuated in vivo glioma growth in a mice model. Mechanistically, miR-138-5p hindered the expression of MeCP2 by target MeCP2 and then inactivated the Wnt/β-catenin signaling pathway. In addition, subsequent rescue assays disclosed that miR-138-5p repressed the glioma malignant phenotype and MeCP2 overexpression reversed the inhibitory effect of miR-138-5p upregulation. Consistently, overexpression of MeCP2 elevated glioma development. However, inhibition of the Wnt/β-catenin signaling pathway with XAV-939 rescued the facilitation effect by overexpressing miR-138-5p. Our results have revealed that miR-138-5p/MeCP2/Wnt/β-catenin signaling might be a new target axis for glioma treatment strategies.

Targeted RNA editing in brainstem alleviates respiratory dysfunction in a mouse model of Rett syndrome

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editasewt) and either Mecp2-targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2-targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2-targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

MeCP2 inhibits ischemic neuronal injury by enhancing methylation of the FOXO3a promoter to repress the SPRY2-ZEB1 axis

Methyl CpG binding protein 2 (MeCP2) is involved in nerve regeneration following ischemic stroke, but the related mechanism remains unclear. Here, we found low MeCP2 expression in hippocampal tissues. Using functional analysis, we demonstrated that MeCP2 accelerated FOXO3a methylation and subsequently inhibited its expression, thus repressing the apoptosis of neuronal cells. Mechanistically, FOXO3a could bind to the promoter region of SPRY2, consequently inducing its transcription and promoting the expression of the downstream target gene ZEB1. Altogether, our study revealed that overexpression of MeCP2 can protect mice against ischemic brain injury via disruption of the FOXO3a/SPRY2/ZEB1 signaling axis. Our results identify ectopic expression of MeCP2 as a therapeutic target in ischemic stroke.

The role of MeCP2 and the BDNF/TrkB signaling pathway in the stress resilience of mice subjected to CSDS.

There is accumulating evidence to support the idea that brain-derived neurotrophic factor (BDNF) is involved in stress resilience. However, the precise molecular mechanisms underlying resilience in major depressive disorder (MDD) remain unknown. The objective of this study was to explore the role of methyl CpG binding protein 2 (MeCP2) and the BDNF/tropomyosin-receptor-kinase B (TrkB) signaling pathway in the stress resilience to chronic social defeat stress (CSDS) in mice. We found that the overexpression of MeCP2 inhibited BDNF transcription, resulting in BDNF mRNA and protein downregulation in neuro-2a cells. The overexpression of MeCP2 increased S80-MeCP2 and decreased S421-MeCP2, BDNF, the ratio of S133-cyclic AMP response element binding protein (CREB)/CREB and p-TrkB/TrkB expression in neuro-2a cells. In addition, using the CSDS mouse model, we found that MeCP2 mRNA levels were decreased in the medial prefrontal cortex (mPFC) of resilient mice and increased in the hippocampus of susceptible mice. BDNF exon IV promoter and BDNF mRNA levels were decreased in the mPFC and hippocampus of susceptible mice. Finally, MeCP2 and S80-MeCP2 protein levels were increased in the mPFC and hippocampus of susceptible mice, whereas the protein expression of S421-MeCP2 and BDNF, the ratio of S133-CREB/CREB, and the levels of p-TrkB/TrkB were decreased in susceptible mice. These data suggest that the overexpression of MeCP2 inhibits BDNF transcription in neuro-2a cells. The inhibition of MeCP2 expression and activation of the BDNF/TrkB signaling pathway may confer stress resilience in CSDS mice.

Increase in Cisplatin Resistance by MeCP2 in Human Gastric Cancer through the Activation of the AKT Pathway by Facilitating PDK-1 Transcription.

Increasing evidence indicates that an imbalance in oncogenes is implicated in cancer chemotherapy resistance. Methyl-CpG binding protein 2 (MeCP2), which acts as a major epigenetic regulator of the expression of various genes, is involved in the carcinogenesis and progression of gastric cancer. However, is it not known whether the role of MeCP2 is vital in acquired cisplatin resistance in gastric cancer. This study aimed to determine whether inhibition of MeCP2 expression could sensitize DDP-resistant GC cells to DDP and elucidate the underlying molecular mechanism. qRT-PCR and western blotting were used to evaluate MeCP2 expression in DDP-resistant GC cells. Subsequently, cell viability, colony formation, cell cycle, apoptosis, and tumorigenicity assays were performed to explore the in vitro and in vivo roles of MeCP2. Chromatin immunoprecipitation- qPCR and luciferase reporter assays were used to identify whether 3-phosphoinositide-dependent protein kinase 1 (PDK-1) was a direct target gene of MeCP2. MeCP2 was upregulated in malignant DDP-resistant cells compared to non-DDP-resistant GC cells or normal gastric epithelial cells. MeCP2 knockdown increased the sensitivity of DDP-resistant GC cells to DDP, resulting in reduced cell growth, G0/G1 phase arrest, and increased apoptosis, whereas MeCP2 overexpression attenuated DDP sensitivity of DDP-resistant GC cells. In addition, MeCP2 knockdown enhanced DDP sensitivity in vivo. MeCP2 elevated PDK-1 expression by binding to CpG sites in promoter regions, and inhibition of PDK-1 reversed the inductive effect of MeCP2 overexpression on DDP resistance in GC cells. These findings indicate that silencing of MeCP2 may potentiate DDP-induced cell death, thereby providing a promising therapeutic strategy for GC.

Impact of MeCP2 Overexpression on Myelination Defects Found in Pitt‐Hopkins Syndrome Model Mice

IntroductionStudies examining neurodevelopmental disorders (NDDs) have emphasized a relationship between Pitt Hopkins syndrome (PTHS), which is caused by mutations in the Transcription Factor 4(TCF4) gene, and myelination defects that suggest novel molecular phenotypes in NDDs (Phan et al., 2020). Known phenotypic overlap between PTHS and Rett syndrome (RTT) suggests that there are possible common signaling pathways between the two NDDs. We have found that increasing expression of Methyl‐CpG‐Binding Protein 2 (MeCP2), the causative protein in most cases of RTT, decreases hyperactivity and corrects impairments in learning and memory in a mouse model of PTHS (Tcf4+/‐).AimThe current study sought to understand if the positive effects of MeCP2 overexpression on behavioral phenotypes in the Tcf4+/‐ model were due to a reversal of myelination deficits.Methods/ResultsWe generated four distinct animal genotypes (WT, MECP2Tg1/o, Tcf4+/‐, MECP2Tg1/o; Tcf4+/‐) and performed RNA‐sequencing from the hippocampus and striatum followed by quantitative RT‐PCR, revealing that myelin‐associated genes are differentially expressed in MECP2Tg1/o; Tcf4+/‐ mice in both brain regions. However, Western blotting revealed decreased expression for the myelinating oligodendrocytes (OLs) protein Myelin Oligodendrocyte Glycoprotein (MOG) in mice of both the Tcf4+/‐ and MECP2Tg1/o; Tcf4+/‐ genotypes without changes in the OL precursor cell marker Chondroitin Sulfate Proteoglycan (NG2). We are currently quantifying potential changes in OL morphology and number (mature and immature cells), and myelin synthesis via immunohistochemistry of brain sections.ConclusionsAltogether, our preliminary results indicate that behavioral phenotypic rescue in MECP2Tg1/o; Tcf4+/‐ mice may not result from a correction of myelination deficits characteristic of Tcf4+/‐animals.

MeCP2 confers 5-fluorouracil resistance in gastric cancer via upregulating the NOX4/PKM2 pathway

BackgroundIncreasing evidence suggests that aberrant methylation is involved in 5-fluorouracil (5-FU) resistance in gastric cancer (GC). Our previous work has identified that Methyl-CpG binding protein 2 (MeCP2) promotes GC progression by binding to the methylation sites of promoter regions of specific genes to affect the downstream signaling pathways. However, the function and molecular mechanisms of MeCP2 in GC 5-FU resistance remain unclear.MethodsWe detected the expression of MeCP2 in 5-FU-resistant GC cells and examined cell behaviors when MeCP2 was silenced. The molecular mechanisms were explored through chromatin immunoprecipitation (ChIP)-qRT-PCR, luciferase reporter assay, clinical tissue samples analysis, and in vivo tumorigenicity assay.ResultsMeCP2 was up-regulated in 5-FU-resistant GC cells. Knockdown of MeCP2 enhanced the sensitivity of the cells to 5-FU. Moreover, MeCP2 promoted NOX4 transcription in the cells by binding to the promoter of NOX4. Silencing NOX4 rescued the inductive effect of MeCP2 overexpression on 5-FU sensitivity of GC cells and reduced the expression of NOX4 and PKM2 in MeCP2 overexpressed 5-FU-resistant GC cells. In addition, our in vivo experiments demonstrated that MeCP2 knockdown enhanced 5-FU sensitivity in tumors.ConclusionMeCP2 confers 5-FU resistance in GC cells via upregulating the NOX4/PKM2 pathway, which may lead to a promising therapeutic strategy for GC.

Macrophage-targeted delivery of siRNA to silence Mecp2 gene expression attenuates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by the infiltration of macrophages in the fibrotic region. Currently, no therapeutic strategies effectively control disease progression, and the 5‐year mortality of patients after diagnosis is unacceptably high. Thus, developing an effective and safe treatment for IPF is urgently needed. The present study illustrated that methyl‐CpG‐binding protein 2 (MECP2), a protein responsible for the interpretation of DNA methylome‐encoded information, was abnormally expressed in lung and bronchoalveolar lavage fluid samples of IPF patients and mice with onset of pulmonary fibrosis. And further studies verified that the overexpression of MECP2 occurred mainly in macrophages. Inhibition of Mecp2 expression in macrophages robustly abrogated alternatively activated macrophage (M2) polarization by regulating interferon regulatory factor 4 expression. Accordingly, cationic liposomes loading Mecp2 small interfering RNA (siRNA) were raised for the treatment of pulmonary fibrosis. It was noted that the liposomes accumulated in the fibrotic region after intratracheal injection, especially in macrophages. In addition, intratracheal administration of Mecp2 siRNA‐loaded liposomes significantly reversed the established pulmonary fibrosis with few side‐effects and high safety coefficients. Collectively, these results are essential not only for further understanding the DNA methylation in pathogenesis of IPF but also for providing a potent therapeutic strategy for IPF treatment in the clinic practice.

MeCP2 prevents age-associated cognitive decline via restoring synaptic plasticity in a senescence-accelerated mouse model.

Age‐related cognitive decline in neurodegenerative diseases, such as Alzheimer's disease (AD), is associated with the deficits of synaptic plasticity. Therefore, exploring promising targets to enhance synaptic plasticity in neurodegenerative disorders is crucial. It has been demonstrated that methyl‐CpG binding protein 2 (MeCP2) plays a vital role in neuronal development and MeCP2 malfunction causes various neurodevelopmental disorders. However, the role of MeCP2 in neurodegenerative diseases has been less reported. In the study, we found that MeCP2 expression in the hippocampus was reduced in the hippocampus of senescence‐accelerated mice P8 (SAMP8) mice. Overexpression of hippocampal MeCP2 could elevate synaptic plasticity and cognitive function in SAMP8 mice, while knockdown of MeCP2 impaired synaptic plasticity and cognitive function in senescence accelerated‐resistant 1 (SAMR1) mice. MeCP2‐mediated regulation of synaptic plasticity may be associated with CREB1 pathway. These results suggest that MeCP2 plays a vital role in age‐related cognitive decline by regulating synaptic plasticity and indicate that MeCP2 may be promising targets for the treatment of age‐related cognitive decline in neurodegenerative diseases.

MECP2 Increases the Pro-Inflammatory Response of Microglial Cells and Phosphorylation at Serine 423 Regulates Neuronal Gene Expression upon Neuroinflammation.

Methyl-CpG-binding protein 2 (MECP2) is a critical transcriptional regulator for synaptic function. Dysfunction of synapses, as well as microglia-mediated neuroinflammation, represent the earliest pathological events in Alzheimer’s disease (AD). Here, expression, protein levels, and activity-related phosphorylation changes of MECP2 were analyzed in post-mortem human temporal cortex. The effects of wild type and phosphorylation-deficient MECP2 variants at serine 423 (S423) or S80 on microglial and neuronal function were assessed utilizing BV2 microglial monocultures and co-cultures with mouse cortical neurons under inflammatory stress conditions. MECP2 phosphorylation at the functionally relevant S423 site nominally decreased in the early stages of AD-related neurofibrillary pathology in the human temporal cortex. Overexpression of wild type MECP2 enhanced the pro-inflammatory response in BV2 cells upon treatment with lipopolysaccharide (LPS) and interferon-γ (IFNγ) and decreased BV2 cell phagocytic activity. The expression of the phosphorylation-deficient MECP2-S423A variant, but not S80A, further increased the pro-inflammatory response of BV2 cells. In neurons co-cultured with BV2 cells, the MECP2-S423A variant increased the expression of several genes, which are important for the maintenance and protection of neurons and synapses upon inflammatory stress. Collectively, functional analyses in different cellular models suggest that MECP2 may influence the inflammatory response in microglia independently of S423 and S80 phosphorylation, while the S423 phosphorylation might play a role in the activation of neuronal gene expression, which conveys neuroprotection under neuroinflammation-related stress.

Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons.

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.SIGNIFICANCE STATEMENT Neuronal function is critically dependent on methyl-CpG binding protein 2 (MeCP2), a nuclear protein abundantly expressed in neurons. The importance of MeCP2 is underscored by the severe childhood neurologic disorders, Rett syndrome (RTT) and MECP2 multiplication disorders, which are caused by depletion and overabundance of MeCP2, respectively. To clarify the molecular function of MeCP2 and to understand the pathogenesis of MECP2-related disorders, we performed detailed structural analyses of neuronal nuclei by using mouse models and high-resolution microscopy. We show that the level of MeCP2 critically regulates 3D structure of heterochromatic foci, and this is mediated in part by the AT-hook 2 domain of MeCP2. Our results demonstrate that one primary function of MeCP2 is to regulate chromatin structure.

Role of miR-132/methyl-CpG-binding protein 2 in the regulation of neural stem cell differentiation.

Methyl-CpG-binding protein 2 (MeCP2) is a well-known transcription repressor, and mutations in MECP2 cause serious neurological disorders. Many studies have suggested that MeCP2 is involved in neural maturation only, and have not reported its role in neural stem cell differentiation. In the present study, we investigated this possible role of MeCP2 in neural stem cells. We used two different differentiation methods to explore how MeCP2 influences neural stem cell differentiation. When we transfected MeCP2-overexpressing lentivirus into neural stem cells, astrocytic differentiation was impaired. This impaired astrocytic differentiation occurred even in conditions of 20% fetal bovine serum, which favored astrocytic differentiation. In addition, miR-132 had the largest expression change after differentiation among several central nervous system related miRNAs. A luciferase assay confirmed that miR-132 directly targeted MeCP2, and that miR-132 was able to reduce MeCP2 expression at both the RNA and protein levels. The upregulation of miR-132 by miRNA mimics promoted astrocytic differentiation, which was fully recovered by MeCP2 overexpression. These results indicate that miR-132 regulates cell lineage differentiation by reducing MeCP2. The study was approved by the Ethics Committee of Shanghai Tenth People's Hospital of TongJi University, China (approval No. SHDSYY-2018-4748) on March 10, 2018.

MeCP2 mediates transgenerational transmission of chronic pain.

Pain symptoms can be transmitted across generations, but the mechanisms underlying these outcomes remain poorly understood. Here, we identified an essential role for primary somatosensory cortical (S1) glutamate neuronal DNA methyl-CpG binding protein 2 (MeCP2) in the transgenerational transmission of pain. In a female mouse chronic pain model, the offspring displayed significant pain sensitization. In these mice, MeCP2 expression was increased in S1 glutamate (GluS1) neurons, correlating with increased neuronal activity. Downregulation of GluS1 neuronal MeCP2 in maternal mice with pain abolished offspring pain sensitization, whereas overexpression of MeCP2 in naïve maternal mice induced pain sensitization in offspring. Notably, single-cell sequencing and chromatin immunoprecipitation analysis showed that the expression of a wide range of genes was changed in offspring and maternal GluS1 neurons, some of which were regulated by MeCP2. These results collectively demonstrate the putative importance of MeCP2 as a key regulator in pain transgenerational transmission through actions on GluS1 neuronal maladaptation.

Overexpression of MECP2 attenuates cigarette smoke extracts induced lung epithelial cell injury by promoting CYP1B1 methylation.

MECP2 (Methyl-CpG-binding protein 2) has been shown to have a critical role in regulating DNA methylation against smoke exposed lung injury. However, the biological function of MECP2 and the underlying molecular mechanism remains elusive. Human bronchial epithelial (16HBE) and alveolar type II epithelial cells (AECII) were exposed to increasing concentrations of cigarette smoke extracts (CSE) solution to establish CSE-induced lung epithelial cell injury models. Our findings revealed that MECP2 was down-regulated, while CYP1B1 was up-regulated in CSE-induced lung epithelial cell injury models by quantitative real time PCR, western blotting and immunofluorescence staining. Down-regulated CYP1B1 was ascribed to the demethylation of its promoter by methylation-specific PCR (MSP). The in vitro experiments further showed that MECP2 overexpression significantly attenuated CSE-triggered cell growth attenuation, cell cycle arrest, apoptosis and ROS generation in lung epithelial cells by CCK-8 and flow cytometry assays. In molecular level, we further demonstrated that MECP2 overexpression obviously suppressed the expression of CYP1B1 through enhancing DNA methylation. Therefore, our data suggest that MECP2 protects against CSE-induced lung epithelial cell injury possibly through down-regulating CYP1B1 expression via elevating its methylation status.

Atypical Response Properties of the Auditory Cortex of Awake MECP2-Overexpressing Mice.

Methyl-CpG binding protein 2 (MECP2) is a gene associated with DNA methylation and has been found to be important for maintaining brain function. In humans, overexpression of MECP2 can cause a severe developmental disorder known as MECP2 duplication syndrome. However, it is still unclear whether MECP2 overexpression also causes auditory abnormalities, which are common in people with autism. MECP2-TG is a mouse model of MECP2 duplication syndrome and has been widely used for research on social difficulty and other autism-like disorders. In this study, we used a combination of multiple electrophysiological techniques to document the response properties of the auditory cortex of awake MECP2-TG mice. Our results showed that while the auditory brainstem responses are similar, cortical activity patterns including local field potentials (LFPs), multiunit activity (MUA), and single-neuron responses differ between MECP2-TG and wild-type (WT) mice. At the single-neuron level, the spike waveform of fast-spiking (FS) neurons from MECP2-TG mice is different from that of WT mice, as reflected by reduced peak/trough ratios in the transgenic mice. Both regular-spiking (RS) and FS neurons exhibited atypical response properties in MECP2-TG mice compared with WT mice, such as prolonged latency and an elevated intensity threshold; furthermore, regarding the response strength to different stimuli, MECP2-TG mice exhibited stronger responses to noise than to pure tone, while this pattern was not observed in WT mice. Our findings suggest that MECP2 overexpression can cause the auditory cortex to have atypical response properties, an implication that could be helpful for further understanding the nature of auditory deficits in autism.