Methylation gets SMRT. Functional insights into Rett syndrome

Rett syndrome, a neurodevelopmental disorder, is caused by mutations in the methyl-CpG binding protein MeCP2. A recent report demonstrates that MeCP2 cooperates with the SMRT corepressor complex to inhibit expression of a hairy-related repressor during primary neurogenesis in Xenopus, and that this can be modulated by Notch signaling. Rett syndrome mutations that disrupt interaction with the SMRT corepressor complex also prevent regulation of MeCP2 by activated Notch."Well-timed silence hath more eloquence than speech."-Martin Farquhar Tupper (1810-1889)     

Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1

DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated CpG after DNA replication to maintain methylation patterns. Although the N-terminal region of Dnmt1 is known to interact with various proteins, such as methyl-CpG-binding protein 2 (MeCP2), the associations of protein kinases with this region have not been reported. In the present study, we found that a 110-kDa protein kinase in mouse brain could bind to the N-terminal domain of Dnmt1. This 110-kDa kinase was identified as cyclin-dependent kinase-like 5 (CDKL5) by LC-MS/MS analysis. CDKL5 and Dnmt1 were found to colocalize in nuclei and appeared to interact with each other. Catalytically active CDKL5, CDKL5(1-352), phosphorylated the N-terminal region of Dnmt1 in the presence of DNA. Considering that defects in the MeCP2 or CDKL5 genes cause Rett syndrome, we propose that the interaction between Dnmt1 and CDKL5 may contribute to the pathogenic processes of Rett syndrome.     

Solution structure of the matrix attachment region-binding domain of chicken MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel beta-sheet and an alpha-helix alpha1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, alpha2/alpha3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein.DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.     

Rett syndrome-causing mutations in human MeCP2 result in diverse structural changes that impact folding and DNA interactions

Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in alpha-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms.     

Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

Background  

In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.     

MeCP2-chromatin interactions include the formation of chromatosome-like structures and are altered in mutations causing Rett syndrome

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the approximately 146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.     

MeCP2与神经发育性疾病

作为一种转录抑制因子,甲基化CpG结合蛋白2（MeCP2）含有结合甲基化DNA和转录抑制两个特征性的结构域,具有调节转录激活、调节染色体构象、参与RNA剪切等多种功能,在神经发育过程中起着重要的作用。近来的研究表明,MeCP2基因突变与Rett综合征、孤独症等多种神经发育性疾病相关,已成为研究基因型与人类神经发育性疾病关系的一个热点。就MeCP2在Rett综合征、孤独症及药物成瘾方面的进展作一综述。     

SIRT1-mediated deacetylation of MeCP2 contributes to BDNF expression

Methyl-CpG binding protein 2 (MeCP2) binds methylated cytosines at CpG sites on DNA and it is thought to function as a critical epigenetic regulator. Mutations in the MeCP2 gene have been associated to Rett syndrome, a human neurodevelopmental disorder. Here we show that MeCP2 is acetylated by p300 and that SIRT1 mediates its deacetylation. SIRT1, the mammalian homologue of Sir2 in yeast, is a nicotinamide-adenine dinucleotide (NAD⁺)-dependent histone deacetylase that belongs to the family of HDAC class III sirtuins. Importantly, SIRT1 has been shown to play a critical role in synaptic plasticity and memory formation. This study reveals a functional interplay between two critical epigenetic regulators, MeCP2 and SIRT1, which controls MeCP2 binding activity to the brain-derived neurotrophic factor (BDNF) promoter in a specific region of the brain.     

SIRT1-mediated deacetylation of MeCP2 contributes to BDNF expression

Methyl-CpG binding protein 2 (MeCP2) binds methylated cytosines at CpG sites on DNA and it is thought to function as a critical epigenetic regulator. Mutations in the MeCP2 gene have been associated to Rett syndrome, a human neurodevelopmental disorder. Here we show that MeCP2 is acetylated by p300 and that SIRT1 mediates its deacetylation. SIRT1, the mammalian homologue of Sir2 in yeast, is a nicotinamide-adenine dinucleotide (NAD⁺)-dependent histone deacetylase that belongs to the family of HDAC class III sirtuins. Importantly, SIRT1 has been shown to play a critical role in synaptic plasticity and memory formation. This study reveals a functional interplay between two critical epigenetic regulators, MeCP2 and SIRT1, which controls MeCP2 binding activity to the brain-derived neurotrophic factor (BDNF) promoter in a specific region of the brain.