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Journal ArticleDOI

MeCP2 binds to 5hmc enriched within active genes and accessible chromatin in the nervous system

TL;DR: In this paper, a quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo is presented.
Abstract: SUMMARY The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.

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Citations
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Journal ArticleDOI
09 Aug 2013-Science
TL;DR: The results extend the knowledge of the unique role of DNA methylation in brain development and function, and offer a new framework for testing the role of the epigenome in healthy function and in pathological disruptions of neural circuits.
Abstract: DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity.

1,629 citations


Cites background or result from "MeCP2 binds to 5hmc enriched within..."

  • ...hmC accumulates during early postnatal brain development in mice (31, 32), becoming enriched in highly expressed genes (33)....

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  • ...Overall, transcriptional activity is associated with intragenic hmCG enrichment, as reported (33), with in utero establishment of adult hmCG patterns for cell type–specific genes and loss of hmC enrichment associated with developmentally coupled transcriptional down-regulation....

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  • ...These constitutively highly expressed genes enriched for neuronal function also show extensive intragenic mCH hypomethylation in neurons in contrast to glia (box 3), and they are enriched for hmCG (box 4) as previously described (32, 33)....

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  • ...Our base-resolution analysis of hmC using TAB-Seq revealed that intragenic and global hmCG levels are largely equivalent between chromosomes, whereas hmCG/CG is 22% lower on the male ChrX, consistent with previous reports from enrichment based detection of hmC (32, 33) (Fig....

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Journal ArticleDOI
TL;DR: This Review synthesizes recent data from human and rodent studies from which emerges a circuit-level framework for understanding reward deficits in depression, and discusses some of the molecular and cellular underpinnings of this framework, ranging from adaptations in glutamatergic synapses and neurotrophic factors to transcriptional and epigenetic mechanisms.
Abstract: Mood disorders are common and debilitating conditions characterized in part by profound deficits in reward-related behavioural domains. A recent literature has identified important structural and functional alterations within the brain's reward circuitry--particularly in the ventral tegmental area-nucleus accumbens pathway--that are associated with symptoms such as anhedonia and aberrant reward-associated perception and memory. This Review synthesizes recent data from human and rodent studies from which emerges a circuit-level framework for understanding reward deficits in depression. We also discuss some of the molecular and cellular underpinnings of this framework, ranging from adaptations in glutamatergic synapses and neurotrophic factors to transcriptional and epigenetic mechanisms.

1,365 citations

Journal ArticleDOI
24 Oct 2013-Nature
TL;DR: Methylation, oxidation and repair now offer a model for a complete cycle of dynamic cytosine modification, with mounting evidence for its significance in the biological processes known to involve active demethylation.
Abstract: DNA methylation has a profound impact on genome stability, transcription and development. Although enzymes that catalyse DNA methylation have been well characterized, those that are involved in methyl group removal have remained elusive, until recently. The transformative discovery that ten-eleven translocation (TET) family enzymes can oxidize 5-methylcytosine has greatly advanced our understanding of DNA demethylation. 5-Hydroxymethylcytosine is a key nexus in demethylation that can either be passively depleted through DNA replication or actively reverted to cytosine through iterative oxidation and thymine DNA glycosylase (TDG)-mediated base excision repair. Methylation, oxidation and repair now offer a model for a complete cycle of dynamic cytosine modification, with mounting evidence for its significance in the biological processes known to involve active demethylation.

1,336 citations

Journal ArticleDOI
TL;DR: Recent advances in biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation and many regulatory mechanisms of this process have been identified.
Abstract: A key mode of regulating DNA methylation is through active demethylation driven by TET-mediated oxidation of 5-methylcytosine (5mC). This Review discusses our latest understanding of the mechanisms and regulation of active DNA demethylation, and the roles of active demethylation (and the oxidized 5mC intermediates) in gene regulation, genome stability, development and disease.

1,012 citations

Journal ArticleDOI
28 Feb 2013-Cell
TL;DR: Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

897 citations


Cites background from "MeCP2 binds to 5hmc enriched within..."

  • ...…binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation....

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References
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Journal ArticleDOI
TL;DR: Observations provide a molecular mechanism by which MeCP2 can gain access to chromatin in order to target corepressor complexes that further modify chromatin structure.
Abstract: We have investigated the interactions of the methyl-CpG binding transcriptional repressor MeCP2 with nucleosomal DNA. We find that MeCP2 forms discrete complexes with nucleosomal DNA associating with methyl-CpGs exposed in the major groove via the methyl-CpG-binding domain (MBD). In addition to the MBD, the carboxyl-terminal segment of MeCP2 facilitates binding both to naked DNA and to the nucleosome core. These observations provide a molecular mechanism by which MeCP2 can gain access to chromatin in order to target corepressor complexes that further modify chromatin structure.

178 citations


"MeCP2 binds to 5hmc enriched within..." refers background in this paper

  • ...They are also consistent with the observations that MeCP2 stably associates with nucleosomes (Chandler et al., 1999), that it can compete with histone H1 for nucleosomebinding sites (Ghosh et al., 2010), and that the levels of MeCP2 and histone H1 are inversely correlated in neurons (Skene et al.,…...

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  • ...They are also consistent with the observations that MeCP2 stably associates with nucleosomes (Chandler et al., 1999), that it can compete with histone H1 for nucleosomebinding sites (Ghosh et al....

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Journal ArticleDOI
TL;DR: The number of cells per unit volume was determined in the cerebellar cortex of man and 19 other mammals and the ratio of Purkinje cells to granule cells varies remarkably among the mammals and is not correlated with brain weight.
Abstract: The number of cells per unit volume was determined in the cerebellar cortex of man and 19 other mammals. The cell density (i.e. the number of cells per unit volume) decreases from mammals with a low brain weight to those with a higher brain weight. This decrease in the number of cells is found to be proportional for all three layers of the cerebellar cortex. In addition, the ratio of Purkinje cells to granule cells was determined. In contrast to the decrease of all cell types with increasing brain weight, this ratio varies remarkably among the mammals and is not correlated with brain weight. In man, this ratio is 1∶2991, while it is lower in all other mammals investigated. These differences in the ratio of Purkinje cells to granule cells and the decrease in cell density with increasing brain weight are discussed in relation to brain evolution.

145 citations


"MeCP2 binds to 5hmc enriched within..." refers background in this paper

  • ...GCs are the smallest and most numerous neurons of the cerebellum, present at several hundred times the abundance of PCs (Palay and Chan-Palay, 1974; Lange, 1975)....

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Journal ArticleDOI
TL;DR: The results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.
Abstract: Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.

137 citations


"MeCP2 binds to 5hmc enriched within..." refers background in this paper

  • ...time to the presence ofmuchmore stable complexes established within that domain by binding of MeCP2 or other less abundant MBD family proteins to 5mC (Lopez-Serra et al., 2006)....

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  • ...See also Figure S7. time to the presence ofmuchmore stable complexes established within that domain by binding of MeCP2 or other less abundant MBD family proteins to 5mC (Lopez-Serra et al., 2006)....

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Journal ArticleDOI
TL;DR: It is shown that strong methylation-independent as well asmethylation-dependent binding by MeCP2 is influenced by DNA length, which suggests that chromatin binding byMeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.
Abstract: Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.

129 citations


"MeCP2 binds to 5hmc enriched within..." refers background in this paper

  • ...…also consistent with the observations that MeCP2 stably associates with nucleosomes (Chandler et al., 1999), that it can compete with histone H1 for nucleosomebinding sites (Ghosh et al., 2010), and that the levels of MeCP2 and histone H1 are inversely correlated in neurons (Skene et al., 2010)....

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  • ..., 1999), that it can compete with histone H1 for nucleosomebinding sites (Ghosh et al., 2010), and that the levels of MeCP2 and histone H1 are inversely correlated in neurons (Skene et al....

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Journal ArticleDOI
TL;DR: The mutation of the R111 residue, which directly interacts with the methyl group of methyl cytosine, completely abolished MeCP2 function and mutations affecting β-sheets and a hairpin loop have substantial functional consequences.
Abstract: 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 β-sheets and a hairpin loop have substantial functional consequences. In contrast, mutations 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.

91 citations


"MeCP2 binds to 5hmc enriched within..." refers background in this paper

  • ...To determine if this is the case, binding of MeCP2 MBDs (amino acids [aa] 1–205) carrying a variety of previously characterized RTT mutations were assayed (Kudo et al., 2003)....

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