A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects.

ATRX is a chromatin remodelling ATPase that is involved in transcriptional regulation, DNA damage repair and heterochromatin maintenance. It has been widely studied for its role in ALT-positive cancers, but its role in neurological function remains elusive. Hypomorphic mutations in the X-linked ATRX gene cause a rare form of intellectual disability combined with alpha-thalassemia called ATR-X syndrome in hemizygous males. Clinical features also include facial dysmorphism, microcephaly, short stature, musculoskeletal defects and genital abnormalities. As complete deletion of ATRX in mice results in early embryonic lethality, the field has largely relied on conditional knockout models to assess the role of ATRX in multiple tissues. Given that null alleles are not found in patients, a more patient-relevant model was needed. Here, we have produced and characterized the first patient mutation knock-in model of ATR-X syndrome, carrying the most common causative mutation, R246C. This is one of a cluster of missense mutations located in the chromatin-binding domain and disrupts its function. The knock-in mice recapitulate several aspects of the patient disorder, including craniofacial defects, microcephaly, reduced body size and impaired neurological function. They provide a powerful model for understanding the molecular mechanisms underlying ATR-X syndrome and testing potential therapeutic strategies.

Neuronal non-CG methylation is an essential target for MeCP2 function.

DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

The Molecular Basis of MeCP2 Function in the Brain.

MeCP2 is a reader of the DNA methylome that occupies a large proportion of the genome due to its high abundance and the frequency of its target sites. It has been the subject of extensive study because of its link with 'MECP2-related disorders', of which Rett syndrome is the most prevalent. This review integrates evidence from patient mutation data with results of experimental studies using mouse models, cell lines and in vitro systems to critically evaluate our understanding of MeCP2 protein function. Recent evidence challenges the idea that MeCP2 is a multifunctional hub that integrates diverse processes to underpin neuronal function, suggesting instead that its primary role is to recruit the NCoR1/2 co-repressor complex to methylated sites in the genome, leading to dampening of gene expression.

Toxicity of overexpressed MeCP2 is independent of HDAC3 activity.

Duplication of the X-linked MECP2 gene causes a severe neurological syndrome whose molecular basis is poorly understood. To determine the contribution of known functional domains to overexpression toxicity, we engineered a mouse model that expresses wild-type or mutated MeCP2 from the Mapt (Tau) locus in addition to the endogenous protein. Animals that expressed approximately four times the wild-type level of MeCP2 failed to survive to weaning. Strikingly, a single amino acid substitution that prevents MeCP2 from binding to the TBL1X(R1) subunit of nuclear receptor corepressor 1/2 (NCoR1/2) complexes, when expressed at equivalent high levels, was phenotypically indistinguishable from wild type, suggesting that excessive corepressor recruitment underlies toxicity. In contrast, mutations affecting the DNA-binding domain were toxic when overexpressed. As the NCoR1/2 corepressors are thought to act through histone deacetylation by histone deacetylase 3 (HDAC3), we asked whether mutations in NCoR1 and NCoR2 that drastically reduced their ability to activate this enzyme would relieve the MeCP2 overexpression phenotype. Surprisingly, severity was unaffected, indicating that the catalytic activity of HDAC3 is not the mediator of toxicity. Our findings shed light on the molecular mechanisms underlying MECP2 duplication syndrome and call for a re-evaluation of the precise biological role played by corepressor recruitment.

Radically truncated MeCP2 rescues Rett syndrome-like neurological defects.

Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome. The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine. Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners, including transcriptional co-repressors (for example, the NCoR/SMRT complex), transcriptional activators, RNA, chromatin remodellers, microRNA-processing proteins and splicing factors. Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons. 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 and the NCoR/SMRT interaction domain. 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.