Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process

Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2

MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target \beta-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease

Molecular basis of R133C Rett syndrome

Rett syndrome is a debilitating autistic spectrum disorder affecting one in ten thousand girls. Patients develop normally for up to eighteen months before a period of regression involving stagnation in head growth, loss of speech, hand use and mobility. It is almost exclusively caused by mutation in Methyl CpG binding Protein 2 (MeCP2). MeCP2 has traditionally been thought of as a transcriptional repressor, although its exact function remains unknown and it has recently been shown that the protein can also bind to hydroxymethylation and non-CpG methylation, which occurs predominantly at CAC sites in the mature nervous system. Genotype-phenotype studies of the most common Rett-causing mutations in affected patients revealed that a missense mutation, R133C results in a milder form of Rett syndrome. The reasons for this are unclear, as the mutation lies right in the heart of the methylated DNA binding domain. Previous in vitro studies of R133C showed a severe deficit in binding to methylated cytosine. A subsequent study found that R133C binding to hydroxymethylated cytosine was specifically impaired, whereas binding to methylated cytosine was indistinguishable from wildtype. Defining the DNA binding impairment of MeCP2R133C would yield important insights into Rett disease pathophysiology and provide an explanation for the phenotypic spectrum seen in patients. To shed light on these matters, a novel mouse model of the R133C mutation was created. The R133C mouse had a phenotype that was less severe than other missense mutant mice, in terms of survival, growth, Rett-like phenotypic score and some behavioural paradigms thus recapitulating the patient data. At the molecular level in adult mouse brain, MeCP2R133C protein abundance was reduced. Immunohistochemistry showed that MeCP2R133C had an abnormal pattern of localisation in the nucleus of neurons. In vitro electrophoretic mobility shift assays suggested that MeCP2R133C binding to (hydroxy)methyl-CAC may be reduced to a greater extent than binding to mCpG. Chromatin immunoprecipitation experiments confirmed the deficit in binding to methylated sites and supported a disproportionate reduction in binding to methylation in a CAC sequence context. Analysis of adult mouse cerebellar gene expression revealed a subtle upregulation of long genes and downregulation of short genes. Based on these data, it is proposed that Rett syndrome caused by the R133C mutation results from a combination of protein instability and defective binding to methylated DNA. Methyl-CAC binding is potentially abolished. The downstream biological consequence of this is a length-dependent deregulation of gene expression in the brain

Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA

MBD1, a member of the methyl-CpG-binding domain family of proteins, has been reported to repress transcription of methylated and unmethylated promoters. As some MBD1 isoforms contain two DNA-binding domains—an MBD, which recognizes methylated DNA; and a CXXC3 zinc finger, which binds unmethylated CpG—it is unclear whether these two domains function independently of each other or if they cooperate in facilitating recruitment of MBD1 to particular genomic loci. In this report we investigate DNA-binding specificity of MBD and CXXC3 domains in vitro and in vivo. We find that the methyl-CpG-binding domain of MBD1 binds more efficiently to methylated DNA within a specific sequence context. We identify genes that are targeted by MBD1 in human cells and demonstrate that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to specific sites at these loci, while DNA binding by the CXXC3 motif is largely dispensable. In summary, the binding preferences of MBD1, although dependent upon the presence of methylated DNA, are clearly distinct from those of other methyl-CpG-binding proteins, MBD2 and MeCP2

Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome

The possibility that alterations in DNA methylation are mechanistic drivers of development, aging and susceptibility to disease is widely acknowledged, but evidence remains patchy or inconclusive. Of partic-ular interest in this regard is the brain, where it has been reported that DNA methylation impacts on neu-ronal activity, learning and memory, drug addiction and neurodegeneration. Until recently, however, lit-tle was known about the ‘landscape ’ of the human brain methylome. Here we assay 1.9 million CpGs in each of 43 brain samples representing different individuals and brain regions. The cerebellum was a consistent outlier compared to all other regions, and showed over 16 000 differentially methylated re-gions (DMRs). Unexpectedly, the sequence charac-teristics of hypo- and hypermethylated domains in cerebellum were distinct. In contrast, very few DMRs distinguished regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable in these regions. These results lead to the surprising conclusion that, with the exception of cerebellum, DNA methylation patterns are more ho-mogeneous between different brain regions from the same individual, than they are for a single brain re-gion between different individuals. This finding sug-gests that DNA sequence composition, not develop-mental status, is the principal determinant of the hu-man brain DNA methylome

LSH and G9a/GLP complex are required for developmentally programmed DNA methylation

LSH, a member of the SNF2 family of chromatin remodeling ATPases encoded by the Hells gene, is essential for normal levels of DNA methylation in the mammalian genome. While the role of LSH in the methylation of repetitive DNA sequences is well characterized, its contribution to the regulation of DNA methylation and the expression of protein-coding genes has not been studied in detail. In this report we investigate genome-wide patterns of DNA methylation at gene promoters in Hells−/− mouse embryonic fibroblasts (MEFs). We find that in the absence of LSH, DNA methylation is lost or significantly reduced at ∼20% of all normally methylated promoter sequences. As a consequence, a large number of genes are misexpressed in Hells−/− MEFs. Comparison of Hells−/− MEFs with wild-type MEFs and embryonic stem (ES) cells suggests that LSH is important for de novo DNA methylation events that accompany the establishment and differentiation of embryonic lineage cells. We further show that the generation of normal DNA methylation patterns and stable gene silencing at specific promoters require cooperation between LSH and the G9a/GLP complex of histone methylases. At such loci, G9a recruitment is compromised when LSH is absent or greatly reduced. Taken together, our data suggest a mechanism whereby LSH promotes binding of DNA methyltransferases and the G9a/GLP complex to specific loci and facilitates developmentally programmed DNA methylation and stable gene silencing during lineage commitment and differentiation

Morphological and functional reversal of phenotypes in a mouse model of Rett syndrome

Rett syndrome is a neurological disorder caused by mutation of the X-linked MECP2 gene. Mice lacking functional Mecp2 display a spectrum of Rett syndrome-like signs, including disturbances in motor function and abnormal patterns of breathing, accompanied by structural defects in central motor areas and the brainstem. Although routinely classified as a neurodevelopmental disorder, many aspects of the mouse phenotype can be effectively reversed by activation of a quiescent Mecp2 gene in adults. This suggests that absence of Mecp2 during brain development does not irreversibly compromise brain function. It is conceivable, however, that deep-seated neurological defects persist in mice rescued by late activation of Mecp2. To test this possibility, we have quantitatively analysed structural and functional plasticity of the rescued adult male mouse brain. Activation of Mecp2 in ∼70% of neurons reversed many morphological defects in the motor cortex, including neuronal size and dendritic complexity. Restoration of Mecp2 expression was also accompanied by a significant improvement in respiratory and sensory-motor functions, including breathing pattern, grip strength, balance beam and rotarod performance. Our findings sustain the view that MeCP2 does not play a pivotal role in brain development, but may instead be required to maintain full neurological function once development is complete