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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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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: The findings indicate the MeCP2 is not an obligate component of the Sin3a corepressor complex and may therefore engage a more diverse range of cofactors for repressive function.

91 citations


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

  • ...To identify these factors, nuclear extracts prepared from rodent brain (Klose and Bird, 2004) were incubated with magnetic beads coated with DNA containing unmodified C, 5mC, or 5hmC DNA in the presence of excess of nonspecific DNA competitor, followed by isolation of the beads and visualization of proteins after elution and SDSPAGE....

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  • ...To identify these factors, nuclear extracts prepared from rodent brain (Klose and Bird, 2004) were incubated with magnetic beads coated with DNA containing unmodified C, 5mC, or 5hmC DNA in the presence of excess of nonspecific DNA competitor, followed by isolation of the beads and visualization of…...

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Journal ArticleDOI
TL;DR: The present review attempts to match Me CP2 structural domains, or lack thereof, and specific chromatin features needed for the proper recruitment of MeCP2 to its multiple functions as either activator or repressor in Rett syndrome.
Abstract: Despite a vast body of literature linking chromatin structure to regulation of gene expression, the role of architectural proteins in higher order chromatin transitions required for transcription activation and repression has remained an under-studied field. To demonstrate the current knowledge of the role of such proteins, we have focused our attention on the methylated DNA binding and chromatin-associated protein MeCP2. Structural studies using chromatin assembled in vitro have revealed that MeCP2 can associate with nucleosomes in an N-terminus dependent manner and efficiently condense nucleosome arrays. The present review attempts to match MeCP2 structural domains, or lack thereof, and specific chromatin features needed for the proper recruitment of MeCP2 to its multiple functions as either activator or repressor. We specifically focused on MeCP2's role in Rett syndrome, a neurological disorder associated with specific MeCP2 mutations.

89 citations


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

  • ...…been shown to occur in response to a variety 1424 Cell 151, 1417–1430, December 21, 2012 ª2012 Elsevier Inc. of stimuli (Chen et al., 2003; Tao and Wu, 2009; Rutlin and Nelson, 2011; Adkins and Georgel, 2011; Gonzales et al., 2012) could alter its substrate specificity and downstream functions....

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  • ...…proteins that bind them vary in each cell type, the phenotypic consequences of changes in the function of MeCP2, whether as a result of mutation (Adkins and Georgel, 2011; Tao andWu, 2009; Amir et al., 1999) or posttranslational modification (Rutlin and Nelson, 2011; Gonzales et al., 2012),…...

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  • ...Because the levels of 5hmC and 5mC and the proteins that bind them vary in each cell type, the phenotypic consequences of changes in the function of MeCP2, whether as a result of mutation (Adkins and Georgel, 2011; Tao andWu, 2009; Amir et al., 1999) or posttranslational modification (Rutlin and Nelson, 2011; Gonzales et al....

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  • ...of stimuli (Chen et al., 2003; Tao and Wu, 2009; Rutlin and Nelson, 2011; Adkins and Georgel, 2011; Gonzales et al., 2012) could alter its substrate specificity and downstream functions....

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Journal ArticleDOI
TL;DR: Phosphorylation is one of several factors that are important for interpreting the complexities of MeCP2 transcriptional modulation, including phosphorylation, acetylation, and ubiquitylation.
Abstract: Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) lead to disrupted neuronal function and can cause the neurodevelopmental disorder Rett syndrome. MeCP2 is a transcriptional regulator that binds to methylated DNA and is most abundant in neuronal nuclei. The mechanisms by which MeCP2 regulates gene expression remain ambiguous, as it has been reported to function as a transcriptional silencer or activator and to execute these activities through both gene-specific and genome-wide mechanisms. We hypothesized that posttranslational modifications of MeCP2 may be important for reconciling these apparently contradictory functions. Our results demonstrate that MeCP2 contains multiple posttranslational modifications, including phosphorylation, acetylation, and ubiquitylation. Phosphorylation of MeCP2 at S229 or S80 influenced selective in vivo interactions with the chromatin factors HP1 and SMC3 and the cofactors Sin3A and YB-1. pS229 MeCP2 was specifically enriched at the RET promoter, and phosphorylation of MeCP2 was necessary for differentiation-induced activation and repression of the MeCP2 target genes RET and EGR2. These results demonstrate that phosphorylation is one of several factors that are important for interpreting the complexities of MeCP2 transcriptional modulation.

89 citations


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

  • ..., 1999) or posttranslational modification (Rutlin and Nelson, 2011; Gonzales et al., 2012), will be cell type and circuit specific....

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  • ...…been shown to occur in response to a variety 1424 Cell 151, 1417–1430, December 21, 2012 ª2012 Elsevier Inc. of stimuli (Chen et al., 2003; Tao and Wu, 2009; Rutlin and Nelson, 2011; Adkins and Georgel, 2011; Gonzales et al., 2012) could alter its substrate specificity and downstream functions....

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  • ...…each cell type, the phenotypic consequences of changes in the function of MeCP2, whether as a result of mutation (Adkins and Georgel, 2011; Tao andWu, 2009; Amir et al., 1999) or posttranslational modification (Rutlin and Nelson, 2011; Gonzales et al., 2012), will be cell type and circuit specific....

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  • ...of stimuli (Chen et al., 2003; Tao and Wu, 2009; Rutlin and Nelson, 2011; Adkins and Georgel, 2011; Gonzales et al., 2012) could alter its substrate specificity and downstream functions....

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Journal ArticleDOI
TL;DR: To understand the methylation‐dependent chromatin structure, AFM studies of fibers isolated from cultured cells containing normal or elevated levels of m5C suggested that DNA meth‐ylation induced fiber compaction only in the presence of linker histones.
Abstract: Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA methylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.

70 citations


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

  • ...This is consistent with the analysis of individual genes shown in Figure 7A, and confirms previous studies demonstrating that the 5mC-enriched DNA is present MNase-resistant compact structures (Karymov et al., 2001)....

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Journal ArticleDOI
TL;DR: It is postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.
Abstract: Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.

59 citations


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

  • ...However, the observations that in brain nuclei a large fraction of MeCP2 is localized within highly nuclease accessible regions (Thambirajah et al., 2012), that loss of MeCP2 can lead to downregulation of expressed genes (Ben-Shachar et al....

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  • ...Given the high abundance of MeCP2 in the brain (Figure S4B) (Guy et al., 2001; Skene et al., 2010; Thambirajah et al., 2012), andourdemonstration thatMeCP2bindsavidly to 5hmCcontainingDNA in vitro, wewere next interested in assessing its potential role in global regulation of chromatin…...

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  • ...For example, it has recently been shown that two populations of MeCP2 are present in the brain: one in chromatin regions that are enriched in nucleosomes, and the other that is loosely bound to highly accessible chromatin domains (Thambirajah et al., 2012)....

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  • ...Given the high abundance of MeCP2 in the brain (Figure S4B) (Guy et al., 2001; Skene et al., 2010; Thambirajah et al., 2012), andourdemonstration thatMeCP2bindsavidly to 5hmCcontainingDNA in vitro, wewere next interested in assessing its potential role in global regulation of chromatin accessibility....

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  • ...However, the observations that in brain nuclei a large fraction of MeCP2 is localized within highly nuclease accessible regions (Thambirajah et al., 2012), that loss of MeCP2 can lead to downregulation of expressed genes (Ben-Shachar et al., 2009; this study), and that 5hmC is enriched in the gene…...

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