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Analysis of the minimal functional unit for MeCP2 protein shows that its function is to recruit the NCoR/SMRT co-repressor complex to methylated sites on chromatin, which may have use in designing strategies for gene therapy of Rett syndrome. MeCP2 to the Rett-scue Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene, which tend to be clustered in two discrete regions of the protein (MeCP2). In this report, the authors parse the minimal form of MeCP2 that is required to retain its functionality, and interrogate which of its many proposed roles is relevant for Rett syndrome progression. The identification of a minimal functional unit for MeCP2 could be helpful in the design of therapeutic strategies for gene therapy for Rett syndrome. Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome 1 . The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine 2 , 3 . Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners 4 , including transcriptional co-repressors (for example, the NCoR/SMRT complex 5 ), transcriptional activators 6 , RNA 7 , chromatin remodellers 8 , 9 , microRNA-processing proteins 10 and splicing factors 11 . Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons 12 . At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain 13 and the NCoR/SMRT interaction domain 5 . Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.

Background Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects girls, is characterised by a period of apparently normal development until 6–18 months of age when motor and communication abilities regress. More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. Surprisingly, although the disorder is caused by mutations in a single gene, disease severity in affected individuals can be quite variable. To explore the source of this phenotypic variability, we propose that specific MECP2 mutations lead to different degrees of disease severity. Methods Using a database of 1052 participants assessed over 4940 unique visits, the largest cohort of both typical and atypical RTT patients studied to date, we examined the relationship between MECP2 mutation status and various phenotypic measures over time. Results In general agreement with previous studies, we found that particular mutations, such as p.Arg133Cys, p.Arg294X, p.Arg306Cys, 3° truncations and other point mutations, were relatively less severe in both typical and atypical RTT. In contrast, p.Arg106Trp, p.Arg168X, p.Arg255X, p.Arg270X, splice sites, deletions, insertions and deletions were significantly more severe. We also demonstrated that, for most mutation types, clinical severity increases with age. Furthermore, of the clinical features of RTT, ambulation, hand use and age at onset of stereotypies are strongly linked to overall disease severity. Conclusions We have confirmed that MECP2 mutation type is a strong predictor of disease severity. These data also indicate that clinical severity continues to become progressively worse regardless of initial severity. These findings will allow clinicians and families to anticipate and prepare better for the needs of individuals with RTT.

Key Points Methyl-CpG-binding protein 2 (MeCP2) functions throughout the brain. Inactivation of MeCP2 in various brain regions and neuronal subtypes has defined the role of MeCP2 in these areas. MeCP2 is a protein that associates with chromatin. The methyl-CpG-binding domain (MBD) is the primary determinant of DNA binding by MeCP2, but other DNA-binding modules are also reported in the molecule. There is evidence that MeCP2 can positively and negatively regulate gene expression at transcriptional and post-transcriptional levels. Mutations in patients with Rett syndrome (RTT) highlight critical regions of MeCP2 (the MBD, an AT-hook and the NCOR–SMRT interaction domain (NID) that determine the presence and severity of RTT pathology. Different models of MeCP2 function (chromatin compaction or recruitment of nuclear receptor co-repressor (NCOR)–SMRT (silencing mediator of retinoic acid and thyroid hormone receptor)) might be consistent with the RTT mutation spectrum. Rett syndrome is a neurological disorder associated with mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). This Review details emerging insights into the link between the functions of MeCP2 and the pathogenesis of Rett syndrome. Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Two decades of research have fostered the view that MeCP2 is a multifunctional chromatin protein that integrates diverse aspects of neuronal biology. More recently, studies have focused on specific RTT-associated mutations within the protein. This work has yielded molecular insights into the critical functions of MeCP2 that promise to simplify our understanding of RTT pathology.

Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2), cause Rett syndrome and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia and encephalopathy with early lethality. Rett syndrome is characterized by apparently normal early development followed by regression, motor abnormalities, seizures and features of autism, especially stereotyped behaviours. The mechanisms mediating these features are poorly understood. Here we show that mice lacking Mecp2 from GABA (γ-aminobutyric acid)-releasing neurons recapitulate numerous Rett syndrome and autistic features, including repetitive behaviours. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of Rett syndrome. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size, consistent with a presynaptic reduction in glutamic acid decarboxylase 1 ( Gad1 ) and glutamic acid decarboxylase 2 ( Gad2 ) levels, and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal function of GABA-releasing neurons and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes. The GABAergic system in Rett syndrome Rett syndrome, a neurodevelopmental disorder with autistic features, is caused by mutations in the methyl-CpG-binding protein 2 gene ( MECP2 ). A number of mouse models with full and cell-type specific deletions of Mecp2 have been generated, but show only a subset of the signs of Rett syndrome. Now Huda Zoghbi and colleagues report that mice with selective deletion of MeCP2 in GABAergic neurons show not only impaired GABAergic function, but capitulate many of the key features of Rett syndrome. The finding that disturbance of inhibitory neurons causes a variety of neuropsychiatric phenotypes suggests that the GABAergic system may be a promising target for therapeutic intervention. Mutations in the methyl-CpG-binding protein 2 (MeCP2) gene cause Rett syndrome, a neurodevelopmental disorder with features of autism. Multiple mouse models of MeCP2 have been generated, but show only a subset of the symptoms of Rett syndrome. These authors find that mice with selective deletion of MeCP2 in GABA-mediated neurons show not only impaired GABA-mediated function, but capitulate multiple key features of Rett, further suggesting a role of inhibitory function in neuropsychiatric disease.

Epigenetic mechanisms, such as DNA methylation, regulate transcriptional programs to afford the genome flexibility in responding to developmental and environmental cues in health and disease. A prime example involving epigenetic dysfunction is the postnatal neurodevelopmental disorder Rett syndrome (RTT), which is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). Despite decades of research, it remains unclear how MeCP2 regulates transcription or why RTT features appear 6–18 months after birth. Here we report integrated analyses of genomic binding of MeCP2, gene-expression data, and patterns of DNA methylation. In addition to the expected high-affinity binding to methylated cytosine in the CG context (mCG), we find a distinct epigenetic pattern of substantial MeCP2 binding to methylated cytosine in the non-CG context (mCH, where H = A, C, or T) in the adult brain. Unexpectedly, we discovered that genes that acquire elevated mCH after birth become preferentially misregulated in mouse models of MeCP2 disorders, suggesting that MeCP2 binding at mCH loci is key for regulating neuronal gene expression in vivo. This pattern is unique to the maturing and adult nervous system, as it requires the increase in mCH after birth to guide differential MeCP2 binding among mCG, mCH, and nonmethylated DNA elements. Notably, MeCP2 binds mCH with higher affinity than nonmethylated identical DNA sequences to influence the level of Bdnf , a gene implicated in the pathophysiology of RTT. This study thus provides insight into the molecular mechanism governing MeCP2 targeting and sheds light on the delayed onset of RTT symptoms. Significance Decades of research have not deciphered the mechanism by which methyl-CpG binding protein 2 (MeCP2) regulates transcription and why Rett symptoms manifest 1 to 2 y after birth. We hypothesized that the temporal dynamics of MeCP2 binding might provide an answer. We developed mice with an EGFP-tagged MeCP2 allele to identify high-resolution MeCP2 binding profiles in the adult mouse brain. Using genomic binding profiles, methylation maps, and mRNA deep-sequencing data, we found MeCP2 binds to non-CG methylation (mCH, not mCG) to regulate expression of genes altered in mouse models of MeCP2 disorders. These data and the parallel timing of mCH and MeCP2 postnatal accumulation suggest MeCP2 binds mCH as neurons mature to regulate gene expression, offering an explanation for the delayed onset of Rett.

In this study, the authors show that MeCP2 interacts with the NCoR/SMRT co-repressor complex and that a discrete cluster of Rett syndrome–causing mutations in the C-terminal domain of MeCP2 disrupts this interaction, impairing transcriptional repression. Knock-in mice expressing one of these MeCP2 missense mutations exhibit severe motor phenotypes. Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Almost two decades of research into RTT have greatly advanced our understanding of the function and regulation of the multifunctional protein MeCP2. Here, we review recent advances in understanding how loss of MeCP2 impacts different stages of brain development, discuss recent findings demonstrating the molecular role of MeCP2 as a transcriptional repressor, assess primary and secondary effects of MeCP2 loss and examine how loss of MeCP2 can result in an imbalance of neuronal excitation and inhibition at the circuit level along with dysregulation of activity-dependent mechanisms. These factors present challenges to the search for mechanism-based therapeutics for RTT and suggest specific approaches that may be more effective than others.

BackgroundThe aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach.Methods and resultsWe studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT.ConclusionsNetwork analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes.

Transplanting bone marrow from wild-type mice into MECP2-lacking mice results in wild-type microglial engraftment, extends lifespan and reduces symptoms of disease such as breathing and locomotor abnormalities, implicating microglia in the pathophysiology of Rett syndrome. Marrow implants in Rett syndrome The X-linked autism spectrum disorder known as Rett syndrome is predominantly linked to mutations in the MECP2 gene. It is typically associated with neuronal dysfunction, almost exclusively in girls, but new evidence suggests that restoring MECP2 function in other cell types may also arrest disease development. Here, the authors show in a mouse model that transplanting bone marrow from wild-type mice into mice lacking Mecp2 results in an invasion of donor-derived microglial cells into the brain, accompanied by increased lifespan and reduced signs of disease, including improved breathing and locomotion. The donor cells expressed normal MECP2 and high levels of the neurotrophic factor IGF-1. These results point to a crucial role for microglia in Rett syndrome, and open the possibility that bone-marrow implants might be of therapeutic benefit. Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein 1 , 2 , 3 , 4 , 5 . Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction 6 . However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2 -null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm cre on an Mecp2 -null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2 -expressing microglia within the context of an Mecp2 -null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2 +/− females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.

Rett syndrome is caused by mutation of the MECP2 gene that codes for a protein that binds methylated DNA; this study reveals that MeCP2 affects the expression of long genes, which often serve neuronal functions. Role of MECP2 in Rett syndrome Autism-related Rett syndrome is caused by disruption of the MECP2 gene, which codes for a methyl-DNA binding protein, but how MECP2 may control transcription of other genes has remained unclear. Now Michael Greenberg and colleagues show that disruption of the Mecp2 gene in a mouse model and in human Rett syndrome leads to preferential upregulation of longer genes, and that these often serve neuronal functions. Further data indicate that decreasing the expression of long genes, via hypomethylation of the dinucleotide CA, attenuates Rett-related dysfunctions in cultured neurons lacking MECP2 . Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism 1 . MECP2 encodes a methyl-DNA-binding protein 2 that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription 3 , 4 , 5 , 6 , 7 , 8 , 9 . Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.