methyl-CpG-binding Protein 2 Expression Research Articles

MeCP2 modulates gene expression pathways in astrocytes

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Mi2β-Mediated Silencing of the Fetal γ-Globin Gene in Adult Erythroid Cells

Abstract 825Epigenetics has emerged as a key regulator of the fetal to adult hemoglobin switch during development. Understanding the mechanisms of fetal γ-globin gene silencing offers the promise of effective targeted therapy of β- thalassemia and sickle cell anemia. Previous studies carried out by our group using adult erythroid cells from mice transgenic for a yeast artificial chromosome containing the entire human β-globin locus (β-YAC) and primary human erythroid cells have shown that methyl-CpG binding protein 2 (MBD2) is critical for full silencing of the fetal γ-globin gene. MBD2 binds to methylated CpG rich promoters and silences the associated target genes by recruiting the Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. Absent or ≥75% decreased expression of MBD2 has been shown to have a 10–50 fold stimulatory effect on the expression of the γ-globin gene in adult β-YAC transgenic mice and a 5–6 fold effect in CD34+ progenitor derived human erythroid cells in culture. Mi2, a major component of the NuRD complex, is an ATP-dependent chromatin remodeler consisting of two isoforms, Mi2a (also known as CHD3) and Mi2β (also known as CHD4). We have observed that 80% knockdown of Mi2β leads to a significantly higher expression of the γ-globin gene in βYAC containing murine adult hematopoietic CID cells (∼100 fold) when compared to equivalent knockdown of other components of the MBD2/NuRD complex (∼5-10 fold), including MBD2. Remarkably, in CD34+ progenitor derived adult human erythroid cells, as little as 40% knockdown of Mi2β resulted in a ∼30 fold increase in γ/γ + β mRNA levels. Moreover, simultaneous knockdown of MBD2 and Mi2β resulted in no greater γ-globin gene expression than Mi2β knockdown alone. This suggests that Mi2β is acting independently of as well as through its role in the MBD2-NuRD complex to exert its silencing effect on the γ-globin gene. Complete conditional knockout of Mi2β in transgenic mouse hematopoietic cells has been shown by others to result in arrest of erythroid differentiation. We observed that in human CD34+ progenitor cells ≥80% Mi2β knockdown also altered differentiation. In contrast, 40–50% knockdown of Mi2β does not affect erythroid differentiation compared to scramble shRNA controls and resulted in 25 – 40% γ/γ + β globin RNA expression versus 2–3% in scramble shRNA controls. To determine possible mechanisms of Mi2β-mediated silencing outside of its role in the MBD2-NuRD complex, we examined the effect of Mi2β knockdown on two key mediators of γ-globin gene silencing, Bcl11A and KLF-1 (EKLF). Our results showed that RNA and protein levels of these proteins are diminished by 40% and 70% respectively in cells with 40–50% Mi2β knockdown. Knockdown of MBD2 caused no decrease in either Bcl11A or KLF-1 levels. Thus at least part of the effect of Mi2β on γ-globin gene silencing is through its positive effects on KLF-1 and Bcl11A expression. In summary, we have shown that less than a 50% decrease in Mi2β expression in primary human adult erythroid cells resulted in high levels of γ/γ + β gene expression without altering erythroid differentiation. The silencing effects of Mi2β occur both through the MBD2-NuRD repressor complex and through positive regulation of the KLF-1 and Bcl11A genes. These results also suggest that partial inhibition of Mi2β function could be of therapeutic benefit in the treatment of β- thalassemia and sickle cell anemia. Disclosures:No relevant conflicts of interest to declare.

Overexpression of cyclic GMP-dependent protein kinase reduces MeCP2 and HDAC2 expression.

Nitric oxide (NO) and the C-type natriuretic peptide (CNP) exert their action via stimulation of the cyclic GMP (cGMP)-signaling pathway, which includes the activation of cGMP-dependent protein kinases (PKG). The present report shows that the activation of PKG by local application of 8-bromo-cGMP in the caudate–putamen reduced the expression of the epigenetic markers, methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2), in dopaminergic projection areas of cocaine-treated rats. An effect of lesser amplitude was observed when rats were not injected with cocaine. We also studied the effect of PKG overexpression by injecting a plasmid vector containing the human PKG-Iα cDNA in either the caudate–putamen or the ventral tegmental area. Injection in the caudate–putamen reduced the epigenetic parameters with higher amplitude than the cGMP analog. The effect was abolished by the injection of a selective PKG inhibitor, confirming that it was due to PKG-dependent phosphorylation. As MeCP2 and HDAC2 modulate dynamic functions in the adult brain such as memory formation and synaptic plasticity, the downregulation of expression by PKG suggests that the cGMP pathway affects cognitive processes through a mechanism that comprises the MeCP2/HDAC2 complex and the subsequent control of gene silencing.

Elevated IKKα Accelerates the Differentiation of Human Neuronal Progenitor Cells and Induces MeCP2-Dependent BDNF Expression

The IκB kinase α (IKKα) is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs). We find that expression of an extra copy of IKKα (IKKα+) blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF), which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95), syntaxin, and methyl-CpG binding protein 2 (MeCP2). Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF) promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression.

The Impact of MeCP2 Loss- or Gain-of-Function on Synaptic Plasticity

Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional regulator of gene expression that is an important epigenetic factor in the maintenance and development of the central nervous system. The neurodevelopmental disorders Rett syndrome and MECP2 duplication syndrome arise from loss-of-function and gain-of-function alterations in MeCP2 expression, respectively. Several animal models have been developed to recapitulate the symptoms of Rett syndrome and MECP2 duplication syndrome. Cell morphology, neurotransmission, and cellular processes that support learning and memory are compromised as a result of MeCP2 loss- or gain-of-function. Interestingly, loss-of-MeCP2 function and MeCP2 overexpression trigger diametrically opposite changes in synaptic transmission. These findings indicate that the precise regulation of MeCP2 expression is a key requirement for the maintenance of synaptic and neuronal homeostasis and underscore its importance in central nervous system function. This review highlights the functional role of MeCP2 in the brain as a regulator of synaptic and neuronal plasticity as well as its etiological role in the development of Rett syndrome and MECP2 duplication syndrome.

Up-Regulated Methyl CpG Binding Protein-2 in Intractable Temporal Lobe Epilepsy Patients and a Rat Model

Methyl CpG binding protein-2 (MeCP2) is a multifunctional nuclear protein, and regulates dendritic morphology, synaptic transmission, spontaneous neurotransmission, and short-term synaptic plasticity in the central nervous system. This study was designed to investigate the expression of MeCP2 mRNA and protein in intractable temporal lobe epilepsy (TLE) patients and an experimental animal model. MeCP2 expression was detected in 35 temporal neocortex tissue samples from patients with intractable TLE and 14histologically normal temporal lobe tissue samples from trauma patients without epilepsy by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and double-label immunofluorescence. In addition, the timing of MeCP2 expression was evaluated in the hippocampus and adjacent cortex of lithium chloride/pilocarpine-induced TLE rats and uninduced controls. MeCP2 was found to be expressed mainly in the nuclei of neurons, and not expressed in astrocytes. MeCP2 expression was significantly higher in the TLE patients and rats than in the control groups. Following seizures in the rat model, MeCP2 expression gradually increased in the hippocampus and adjacent cortex during the acute period (days 1 and 2) and the latent period (days 7 and 14), but decreased during the chronic period (days 30 and 60). Up-regulated expression of MeCP2 in intractable TLE patients and experimental animals suggested that MeCP2 may be involved in the pathogenesis of TLE.

Microglia — major players in Rett syndrome?

Recent studies have uncovered a role for astrocytes in the pathophysiology of Rett syndrome, most cases of which are linked to mutations in methyl-CpG-binding protein 2 (MECP2). A new study shows that microglial dysfunction may also have an important role in this disorder. Transplantation of wild-type murine bone marrow into male Mecp2−/− mice that had undergone postnatal irradiation led to engraftment of MECP2-expressing microglia-like cells in the brain parenchyma and a cessation of disease progression. Moreover, mice in which MECP2 expression was restricted to myeloid cells, including microglia, showed a similar phenotype. Finally, pharmacological blockade of microglial phagocytic activity in these animals attenuated disease improvement.

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.

BackgroundClaudin-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)).MethodsWe 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.ResultsClaudin-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.ConclusionsOur 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 carcinomaVirtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452

Cell-Autonomous Alterations in Dendritic Arbor Morphology and Connectivity Induced by Overexpression of MeCP2 in Xenopus Central Neurons In Vivo

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes.

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the stria terminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.

Drosophila as a model for MECP2 gain of function in neurons.

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model.

The Expression of Spinal Methyl-CpG-Binding Protein 2, DNA Methyltransferases and Histone Deacetylases is Modulated in Persistent Pain States

BackgroundDNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2) and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated.ResultsUsing immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury.ConclusionOur results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

Early intervention with psychostimulants or antidepressants to increase methyl-CpG-binding protein 2 (MeCP2) expressions: A potential therapy for Rett syndrome

SummaryRett syndrome (RTT) is a severe X-linked postnatal neurodevelopmental disorder. The syndrome is caused primarily by mutations in the methyl CpG binding protein 2 (MeCP2) gene on Xq28. Most individuals with RTT are female, and female RTT is normally heterozygous for mutations in MeCP2. Patients with RTT display a normal period of development prior to the onset of symptoms, at which point they undergo a period of regression. Currently, no effective medication is available for this disorder, although animal studies have suggested that RTT symptoms are potentially reversible. For females with RTT, the severity of symptoms and progression of the disease varies a great deal, despite its homogenous genetic origin. These differences could be attributed to differences in the mutation points of MeCP2 and the skew caused by X-chromosome inactivation. Thus, the increased expression in the normal MeCP2 gene could decrease the severity of the disease. Based on findings from studies on animals indicating that fluoxetine (an antidepressant) and cocaine (a psychostimulant) can increase MeCP2 expression in the brain, it is suggested that early intervention with antidepressants or psychostimulants could increase the normal MeCP2 expression in females with RTT, who are normally heterozygous. This therapeutic hypothesis could be tested in an RTT animal model. Following the identification of the antidepressants or psychostimulants with the greatest influence on MeCP2 expression, a combination of early detection of the disorder with early intervention may result in improved therapeutic outcomes. Furthermore, a trial investigating the effects of antidepressants or psychostimulants on MeCP2 expression in lymphocyte culture from patients with RTT is suggested for clinical therapeutic prediction.

X-Chromosome Inactivation in Rett Syndrome Human Induced Pluripotent Stem Cells

Rett syndrome (RTT) is a neurodevelopmental disorder that affects girls due primarily to heterozygous mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2). Random X-chromosome inactivation (XCI) results in cellular mosaicism in which some cells express wild-type (WT) MECP2 while other cells express mutant MECP2. The generation of patient-specific human induced pluripotent stem cells (hiPSCs) facilitates the production of RTT-hiPSC-derived neurons in vitro to investigate disease mechanisms and identify novel drug treatments. The generation of RTT-hiPSCs has been reported by many laboratories, however, the XCI status of RTT-hiPSCs has been inconsistent. Some report RTT-hiPSCs retain the inactive X-chromosome (post-XCI) of the founder somatic cell allowing isogenic RTT-hiPSCs that express only the WT or mutant MECP2 allele to be isolated from the same patient. Post-XCI RTT-hiPSCs-derived neurons retain this allele-specific expression pattern of WT or mutant MECP2. Conversely, others report RTT-hiPSCs in which the inactive X-chromosome of the founder somatic cell reactivates (pre-XCI) upon reprogramming into RTT-hiPSCs. Pre-XCI RTT-hiPSC-derived neurons exhibit random XCI resulting in cellular mosaicism with respect to WT and mutant MECP2 expression. Here we review and attempt to interpret the inconsistencies in XCI status of RTT-hiPSCs generated to date by comparison to other pluripotent systems in vitro and in vivo and the methods used to analyze XCI. Finally, we discuss the relative strengths and weaknesses of post- and pre-XCI hiPSCs in the context of RTT, and other X-linked and autosomal disorders for translational medicine.

The Chromatin-binding Protein HMGN1 Regulates the Expression of Methyl CpG-binding Protein 2 (MECP2) and Affects the Behavior of Mice

High mobility group N1 protein (HMGN1), a nucleosomal-binding protein that affects the structure and function of chromatin, is encoded by a gene located on chromosome 21 and is overexpressed in Down syndrome, one of the most prevalent genomic disorders. Misexpression of HMGN1 affects the cellular transcription profile; however, the biological function of this protein is still not fully understood. We report that HMGN1 modulates the expression of methyl CpG-binding protein 2 (MeCP2), a DNA-binding protein known to affect neurological functions including autism spectrum disorders, and whose alterations in HMGN1 levels affect the behavior of mice. Quantitative PCR and Western analyses of cell lines and brain tissues from mice that either overexpress or lack HMGN1 indicate that HMGN1 is a negative regulator of MeCP2 expression. Alterations in HMGN1 levels lead to changes in chromatin structure and histone modifications in the MeCP2 promoter. Behavior analyses by open field test, elevated plus maze, Reciprocal Social Interaction, and automated sociability test link changes in HMGN1 levels to abnormalities in activity and anxiety and to social deficits in mice. Targeted analysis of the Autism Genetic Resource Exchange genotype collection reveals a non-random distribution of genotypes within 500 kbp of HMGN1 in a region affecting its expression in families predisposed to autism spectrum disorders. Our results reveal that HMGN1 affects the behavior of mice and suggest that epigenetic changes resulting from altered HMGN1 levels could play a role in the etiology of neurodevelopmental disorders.

Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury

The pathogenesis of neuropathic pain remains largely unknown. Epigenetic mechanisms may play a major role in regulating expression of pro- or antinociceptive genes. DNA methylation is a major epigenetic mechanism in vertebrates, and methyl- CpG-binding protein 2 (MeCP2) is directly involved in methylation-mediated gene silencing. To determine how changes in global DNA methylation and MeCP2 expression occur following chronic constriction injury (CCI) and how repression of DNA methylation affects these changes and attenuates neuropathic pain, we used intrathecal 5-azacytidine, a DNA methyltransferase inhibitor, in CCI rats. Rats received 0.9% saline or 5-azacytidine (10 μmol·d −1) via spinal injection once daily from day 3 to day 14 after CCI surgery. Global DNA methylation and MeCP2 expression increased in the spinal cord in CCI rats on day 14 after CCI surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine from day 5 to day 14 after CCI surgery. The increases in global DNA methylation and MeCP2 expression in the spinal cord in CCI rats were also significantly inhibited by intrathecal 5-azacytidine. These results demonstrate that increased global DNA methylation and MeCP2 expression in the spinal cord after nerve damage may play an important role in neuropathic pain. 5-azacytidine shows potential for treating neuropathic pain.

A role for glia in the progression of Rett’s syndrome

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system

The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2's function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post-transcriptional and post-translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals.

Essential Role of MeCP2 in the Regulation of Myofibroblast Differentiation during Pulmonary Fibrosis

DNA methylation is a key mechanism for repression of gene expression, including that of α-smooth muscle actin (α-SMA) gene expression in fibroblasts. However, the trans-acting factors that interact with the methylated α-SMA gene to regulate its expression have not been identified. Using gel shift and chromatin immunoprecipitation (ChIP) assays, methyl CpG binding protein 2 (MeCP2) was shown to bind to the α-SMA gene. Suppression of MeCP2 gene expression by siRNA or its deficiency in lung fibroblasts isolated from MeCP2 knockout mice caused significant reduction of α-SMA gene expression. In contrast, transient transfection of MeCP2 expression plasmid into fibroblasts enhanced α-SMA gene expression. Moreover, in vivo studies revealed that compared to their wild type littermates, MeCP2-deficient mice exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition, and myofibroblast differentiation in response to endotracheal injection of bleomycin. Thus, MeCP2 is essential for myofibroblast differentiation and pulmonary fibrosis.

IUGR differentially alters MeCP2 expression and H3K9Me3 of the PPARγ gene in male and female rat lungs during alveolarization

Intrauterine growth restriction (IUGR) increases the risk of postnatal lung disease, with males more affected. In rat lungs, IUGR impairs alveolarization in conjunction with altered expression of peroxisome proliferator-activated receptor gamma (PPARγ). In non-lung cells, PPARγ transcription is regulated in part by the epigenetic modifying enzyme, and the methyl CpG binding protein 2 (MeCP2). However, it is unknown if IUGR affects MeCP2 expression or its interaction with PPARγ in the rat lung during alveolarization. In this study, we hypothesized that the rat lung would be characterized by the presence of MeCP2 short and long mRNA transcripts, MeCP2 protein isoforms, and the MeCP2 regulatory micro RNA, miR132. We further hypothesized that IUGR would, in a gender-specific manner, alter the levels of MeCP2 components in association with changes in PPARγ mRNA, MeCP2 occupancy at the PPARγ promoters, and PPARγ histone 3 lysine 9 trimethylation (H3K9Me3). To test these hypotheses, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. We demonstrated the presence of MeCP2 mRNA, protein, and miR132 in the rat lung throughout alveolarization. We also demonstrated that IUGR alters MeCP2 expression and its interaction with PPARγ in a gender-divergent manner. We conclude that IUGR induces gender-specific alterations in the epigenetic milieu in the rat lung. We speculate that in the IUGR rat lung, this altered epigenetic milieu may predispose to gender-specific alterations in alveolarization.