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Rett syndrome is a neurodevelopmental disorder with severe mental retardation caused by mutations in the MECP2 gene. Mutations in the MECP2 gene are also associated with other genetic disorders, including X linked mental retardation in males. Missense mutations identified so far are present primarily in the methyl CpG binding domain (MBD) of MECP2. Here, the functional significance of 28 MBD missense mutations identified in patients were analysed by transient expression of the mutant proteins in cultured cells. The effects of mutations were evaluated by analysis of the affinity of MeCP2 to pericentromeric heterochromatin in mouse L929 cells and on transcriptional repressive activity of MeCP2 in Drosophila SL2 cells. These analyses showed that approximately one-third (9/28) of MBD missense mutations showed strong impairment of MeCP2 function. The mutation of the R111 residue, which directly interacts with the methyl group of methyl cytosine, completely abolished MeCP2 function and mutations affecting ([beta]-s heets and a hairpin loop have substantial functional consequences. In contrast, mutatians that showed marginal or mild impairment of the function fell in unstructured regions with no DNA interaction. Since each of these mutations is known to be pathogenic, the mutations may indicate residues that are important for specific functions of MeCP2 in neurones.
Rett syndrome (MIM 312750), an X linked dominant neurodevelopmental disorder, is one of the most common causes of mental retardation in young females, occurring with an incidence of 1/10 000 to 1/15 000. (1) Rett syndrome is characterised by severe neurological dysfunction such as dementia, autistic features, loss of acquired motor skills and speech, stereotypical hand movements, and seizures. The gene responsible for this disorder was identified as MECP2, (2) which encodes a 486 amino acid protein that binds to methylated DNA. (3) DNA methylation at cytosine residues in CpG dinucleotides plays an important role in X chromosome inactivation, genomic imprinting, developmental regulation of gene expression, inactivation of retroposons, and repression of transcriptional noise in mammals. (3) MeCP2 protein possesses a highly conserved 70 amino acid methyl CpG binding domain (MBD) that can bind to DNA containing a single methylated CpG base pair. (3) Nuclear magnetic resonance (NMR) solution analysis of the MBD sh owed a novel wedge shaped structure with four antiparallel [beta]-sheets and a C-terminal helical region. (4,5) MeCP2 also contains a transcriptional repression domain (TRD) of approximately 100 amino acid residues, which interacts with the Sin3A histone deacetylase complex. (6,7) Therefore MeCP2 facilitates the formation of transcriptionally inactive, condensed chromatin by binding to methylated CpG through its MBD and recruiting histone deacetylase through the TRD.
Accumulating data indicate that approximately 80% of Rett syndrome patients carry an identifiable MECP2 mutation. (8) Association of MeCP2 with Rett syndrome is supported by the observation that mice carrying targeted mutations in the mecp2 gene develop symptoms characteristic of Rett syndrome patients. (9-11) Moreover, these rodent models indicate that the phenotypic presentation of MeCP2 dysfunction is influenced by genetic background and residual function of the mutant protein as well as by the relative levels of expression of the mutant protein, based on the more severe phenotype in male mice. (9-11) Perhaps not surprisingly based on these observations, mutations in MECP2 have also been identified in patients with disorders phenotypically distinct from Rett syndrome, including severe neonatal onset encephalopathy and X linked recessive mental retardation (for a recent review, see Hammer et al (12)). Molecular dissection of the mechanisms underlying the phenotypic variability in humans is not a simple tas k, particularly in females where individual variation in X chromosome inactivation patterns obscures detection of direct correlation between phenotypic expression of specific mutations. In males harbouring MECP2 mutations, however, the disparate phenotypes suggest that different mutations in MECP2 are not functionally equivalent. In order to investigate the functional significance of missense mutations seen in patients with MeCP2 related disorders, we have developed cell culture based assays that allow examination of chromatin binding and transcriptional repression. We have used these assays to characterise several common MBD mutations that cause Rett syndrome (13) and two mutations that have been identified in males with forms of X linked recessive mental retardation. (14) In this study, we analysed 22 additional missense mutations in the MBD using both assay systems, and compared them to the four most common missense mutations in the MBD in Rett syndrome and two mutations identified in patients with X linke d mental retardation characterised previously. (13,14) Based on the results of these functional assays and the recently reported solution structure of MBD complexed with DNA, (15) we describe the structure-function relationship of the MBD of MeCP2. In addition, we discuss the relationship between the functional significance of the mutations and the phenotypic variability of patients with MeCP2 related mental disorders.
MATERIALS AND METHODS
Cells and culture conditions
L929 cells (mouse fibroblast cell line, American Tissue Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/l glutamine, 500 U of penicillin per ml, and 100 [micro]g of streptomycin per ml in a humidified 5% [CO.sub.2] atmosphere. The Drosophila Schneider cell line 2 (SL2) derived from Drosophila embryos (kindly provided by Dr R M Evans) was grown in Schneider's Drosophila medium (Invitrogen) with the same supplements as described above.
Expression plasmids encoding MeCP2 mutants bearing MBD missense mutations identified in patients (2 16-32) were constructed as follows. MeCP2 mutant cDNAs were generated by site directed mutagenesis using PCR with mismatched primers and a full length MeCP2 cDNA template as described previously. (13) Synthesised mutant DNA was cloned into the enhanced fluorescence vector pEGFP-C1 (Clontech) and the Drosophila expression vector pAc5.1/V5-His (Invitrogen). All constructs …