By hook or by crook: multifaceted DNA-binding properties of MeCP2.
Ontology highlight
ABSTRACT:
SUBMITTER: Shin J
PROVIDER: S-EPMC4119793 | biostudies-literature | 2013 Feb
REPOSITORIES: biostudies-literature
Publications
By hook or by crook: multifaceted DNA-binding properties of MeCP2.
Shin Jaehoon J Ming Guo-Li GL Song Hongjun H
Cell 20130201 5
Two new studies reveal novel DNA-binding properties of MeCP2, mutations of which cause Rett syndrome. Baker et al. report critical roles for the AT-hook domain of MeCP2 in chromatin organization and clinical features of Rett syndrome. Mellén et al. find the methyl-CpG-binding domain of MeCP2 interacts with hydroxymethyl-CpG. ...[more]
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Project description:MeCP2 is a chromatin-associated protein that is mutated in Rett syndrome. Its methyl-CpG-binding domain interacts with DNA containing methylated cytosine, but other modes of recruitment to the genome have also been proposed. Here, we use in vitro and in vivo assays to investigate the DNA binding specificity of two AT-hook motifs in MeCP2. One exhibits robust sequence-specific DNA binding, whereas the other is a much weaker AT-hook. Our data indicate that these motifs are secondary contributors to DNA binding by MeCP2, and this view is supported by the absence of disease-causing missense mutations at these sites.
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Project description:Methyl-CpG-binding protein 2 (MeCP2) is a classic methylated-DNA-binding protein, dysfunctions of which lead to various neurodevelopmental disorders such as Rett syndrome and autism spectrum disorder. Initially recognized as a transcriptional repressor, MeCP2 has been studied extensively and its functions have been expanded dramatically in the past two decades. Recently, it was found to be involved in gene regulation at the post-transcriptional level. MeCP2 represses nuclear microRNA processing by interacting directly with the Drosha/DGCR8 complex. In addition to its multifaceted functions, MeCP2 is remarkably modulated by posttranslational modifications such as phosphorylation, SUMOylation, and acetylation, providing more regulatory dimensions to its functions. The role of MeCP2 in the central nervous system has been studied extensively, from neurons to glia. Future investigations combining molecular, cellular, and physiological methods are necessary for defining the roles of MeCP2 in the brain and developing efficient treatments for MeCP2-related brain disorders.
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Project description:Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that guides neuronal development through its binding to DNA, recognition of sites of methyl-CpG (mCpG) DNA modification, and interaction with other regulatory proteins. Our study explores the relationship between mCpG and hydroxymethyl-CpG (hmCpG) recognition mediated by its mCpG binding domain (MBD) and binding cooperativity mediated by its C-terminal polypeptide. Previous study of the isolated MBD of MeCP2 documented an unusual mechanism by which ion uptake is required for discrimination of mCpG and hmCpG from CpG. MeCP2 binding cooperativity suppresses discrimination of modified DNA and is highly sensitive to both the total ion concentration and the type of counterions. Higher than physiological total ion concentrations completely suppress MeCP2 binding cooperativity, indicating a dominant electrostatic component to the interaction. Substitution of SO4(2-) for Cl(-) at physiological total ion concentrations also suppresses MeCP2 binding cooperativity, This effect is of particular note as the intracellular Cl(-) concentration changes during neuronal development. A related effect is that the protein-stabilizing solutes, TMAO and glutamate, reduce MeCP2 (but not isolated MBD) binding affinity by 2 orders of magnitude without affecting the apparent binding cooperativity. These observations suggest that polypeptide flexibility facilitates DNA binding by MeCP2. Consistent with this view, nuclear magnetic resonance (NMR) analyses show that ions have discrete effects on the structure of MeCP2, both MBD and the C-terminal domains. Notably, anion substitution results in changes in the NMR chemical shifts of residues, including some whose mutation causes the autism spectrum disorder Rett syndrome. Binding cooperativity makes MeCP2 an effective competitor with histone H1 for accessible DNA sites. The relationship between MeCP2 binding specificity and cooperativity is discussed in the context of chromatin binding, neuronal function, and neuronal development.
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Project description:Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ?10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.
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Project description:The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to investigate the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.
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Project description:The AT-hook is a small DNA-binding protein motif which was first described in the high mobility group non-histone chromosomal protein HMG-I(Y). Since its discovery, this motif has been observed in other DNA-binding proteins from a wide range of organisms. Using pattern searches and position-dependent matrices, we have extracted the AT-hook motifs present in a non-redundant protein sequence database. We have classified these motifs into three types according to their sequence similarity and have found that they are prevalent in many eukaryotic nuclear proteins in single or multiple copies. Furthermore, AT-hook motifs are frequently associated with known functional domains seen in chromatin proteins and in DNA-binding proteins (e.g. histone folds, homeodomains and zinc fingers). In general, it appears that the AT-hook motif is an auxiliary protein motif cooperating with other DNA-binding activities and facilitating changes in the structure of the DNA either as a polypeptide on its own [e.g. HMG-I(Y)] or as part of a multidomain protein [e.g. Swi2p in Saccharomyces cerevisiae or HRX (ALL-1) in Homo sapiens]. It is most interesting that this motif seems to be quite specific to known or predicted chromosomal/DNA-binding proteins, suggesting that it may act as a versatile minor groove tether.
| S-EPMC147871 | biostudies-other
Project description:The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed.
| S-EPMC4045320 | biostudies-literature
Project description:Mutations in the MECP2 gene cause the autism spectrum disorder Rett syndrome (RTT). One of the most common MeCP2 mutations associated with RTT occurs at threonine 158, converting it to methionine (T158M) or alanine (T158A). To understand the role of T158 mutations in the pathogenesis of RTT, we generated knockin mice that recapitulate the MeCP2 T158A mutation. We found a causal role for T158A mutation in the development of RTT-like phenotypes, including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those present in Mecp2 null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. The age-dependent development of event-related neuronal responses was disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials (ERPs) may serve as a biomarker for RTT and treatment evaluation.
| S-EPMC3267879 | biostudies-literature
Project description:Mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome (RTT), a neurological disorder affecting cognitive development, respiration, and motor function. Genetic restoration of MeCP2 expression reverses RTT-like phenotypes in mice, highlighting the need to search for therapeutic approaches. Here, we have developed knockin mice recapitulating the most common RTT-associated missense mutation, MeCP2 T158M. We found that the T158M mutation impaired MECP2 binding to methylated DNA and destabilized MeCP2 protein in an age-dependent manner, leading to the development of RTT-like phenotypes in these mice. Genetic elevation of MeCP2 T158M expression ameliorated multiple RTT-like features, including motor dysfunction and breathing irregularities, in both male and female mice. These improvements were accompanied by increased binding of MeCP2 T158M to DNA. Further, we found that the ubiquitin/proteasome pathway was responsible for MeCP2 T158M degradation and that proteasome inhibition increased MeCP2 T158M levels. Together, these findings demonstrate that increasing MeCP2 T158M protein expression is sufficient to mitigate RTT-like phenotypes and support the targeting of MeCP2 T158M expression or stability as an alternative therapeutic approach.
| S-EPMC5409785 | biostudies-literature
Project description:Mutations in the X-linked MECP2 cause Rett syndrome, a devastating neurological disorder typified by a period of apparently normal development followed by loss of cognitive and psychomotor skills. Data from rare male patients suggest symptom onset and severity can be influenced by the location of the mutation, with amino acids 270 and 273 marking the difference between neonatal encephalopathy and death, on the one hand, and survival with deficits on the other. We therefore generated two mouse models expressing either MeCP2-R270X or MeCP2-G273X. The mice developed phenotypes at strikingly different rates and showed differential ATRX nuclear localization within the nervous system, over time, coinciding with phenotypic progression. We discovered that MeCP2 contains three AT-hook-like domains over a stretch of 250 amino acids, like HMGA DNA-bending proteins; one conserved AT-hook is disrupted in MeCP2-R270X, lending further support to the notion that one of MeCP2's key functions is to alter chromatin structure.
| S-EPMC3641682 | biostudies-literature