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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
TL;DR: The data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells.
Abstract: Dioxygenases of the Ten-Eleven Translocation (TET) family are 5-methylcytosine oxidases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. We show that Tet1 and Tet2 have distinct roles in regulating 5hmC in mouse embryonic stem cells (mESC). Tet1 depletion diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is predominantly associated with decreased 5hmC in gene bodies. Enrichment of 5hmC is observed at the boundaries of exons that are highly expressed, and Tet2 depletion results in substantial loss of 5hmC at these boundaries. In contrast, at promoter/TSS regions, Tet2 depletion results in increased 5hmC, potentially because of the redundant activity of Tet1. Together, the data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells.

225 citations

Journal ArticleDOI
TL;DR: The epigenetic control of pluripotency and changes associated with pluripotent stem cell differentiation is reviewed, focusing on DNA methylation, DNA demethylation, and common histone tail modifications.
Abstract: The precise, temporal order of gene expression during development is critical to ensure proper lineage commitment, cell fate determination, and ultimately, organogenesis. Epigenetic regulation of chromatin structure is fundamental to the activation or repression of genes during embryonic development. In recent years, there has been an explosion of research relating to various modes of epigenetic regulation, such as DNA methylation, post-translational histone tail modifications, noncoding RNA control of chromatin structure, and nucleosome remodeling. Technological advances in genome-wide epigenetic profiling and pluripotent stem cell differentiation have been primary drivers for elucidating the epigenetic control of cellular identity during development and nuclear reprogramming. Not only do epigenetic mechanisms regulate transcriptional states in a cell-type-specific manner but also they establish higher order genomic topology and nuclear architecture. Here, we review the epigenetic control of pluripotency and changes associated with pluripotent stem cell differentiation. We focus on DNA methylation, DNA demethylation, and common histone tail modifications. Finally, we briefly discuss epigenetic heterogeneity among pluripotent stem cell lines and the influence of epigenetic patterns on genome topology.

220 citations

Journal ArticleDOI
TL;DR: A model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions is supported.
Abstract: Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.

212 citations

Journal ArticleDOI
TL;DR: The distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers ofDNA methylation are discussed.
Abstract: DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.

209 citations

Journal ArticleDOI
TL;DR: A review of recent advances in the understanding of the cytosine methylation cycle and its implication in cellular transformation, with an emphasis on TET enzymes and 5-hmC, is presented in this paper.
Abstract: The discovery that ten-eleven translocation (TET) proteins are α-ketoglutarate-dependent dioxygenases involved in the conversion of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxycytosine has revealed new pathways in the cytosine methylation and demethylation process. The description of inactivating mutations in TET2 suggests that cellular transformation is in part caused by the deregulation of this 5-mC conversion. The direct and indirect deregulation of methylation control through mutations in DNA methyltransferase and isocitrate dehydrogenase (IDH) genes, respectively, along with the importance of cytosine methylation in the control of normal and malignant cellular differentiation have provided a conceptual framework for understanding the early steps in cancer development. Here, we review recent advances in our understanding of the cytosine methylation cycle and its implication in cellular transformation, with an emphasis on TET enzymes and 5-hmC. Ongoing clinical trials targeting the activity of mutated IDH enzymes provide a proof of principle that DNA methylation is targetable, and will trigger further therapeutic applications aimed at controlling both early and late stages of cancer development.

208 citations


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

  • ...MethylCpG binding proteins were thought to be unable to bind 5-hmC [51] until recently [43], although the ability of MBD3 to specifically bind 5-hmC [45] is still controversial [44]....

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  • ...TET proteins participate in the regulation of gene transcription through the controlled generation of 5-hmC, 5-fC and 5-caC and their subsequent recognition by protein complexes involved in modulating chromatin structure or DNA repair [42-46]....

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References
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Journal ArticleDOI
TL;DR: A method based on the negative binomial distribution, with variance and mean linked by local regression, is proposed and an implementation, DESeq, as an R/Bioconductor package is presented.
Abstract: High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.

13,356 citations

Journal ArticleDOI
TL;DR: Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors.
Abstract: We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

12,293 citations


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

  • ...Transcript abundance was measured in fragments per kilobase of exon per million fragments mapped (FPKM) similarly to RPKM used in (Mortazavi et al., 2008)....

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Journal ArticleDOI
TL;DR: Details of the aims and methods of Bioconductor, the collaborative creation of extensible software for computational biology and bioinformatics, and current challenges are described.
Abstract: The Bioconductor project is an initiative for the collaborative creation of extensible software for computational biology and bioinformatics. The goals of the project include: fostering collaborative development and widespread use of innovative software, reducing barriers to entry into interdisciplinary scientific research, and promoting the achievement of remote reproducibility of research results. We describe details of our aims and methods, identify current challenges, compare Bioconductor to other open bioinformatics projects, and provide working examples.

12,142 citations


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

  • ...Finally, differentially expressed genes were identified by performing a negative binomial test using the DESeq package (Anders and Huber, 2010) of R/Bioconductor (Gentleman et al., 2004)....

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Journal ArticleDOI
15 May 2009-Science
TL;DR: It is shown here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro.
Abstract: DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

5,155 citations


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

  • ...This is expected because hydroxylation of 5mC results in 5hmC (Tahiliani et al., 2009), and both of these marks cannot exist on one base....

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Journal ArticleDOI
TL;DR: This study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.
Abstract: Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

4,503 citations


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

  • ...…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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