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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TL;DR: This study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG and Alterations in genome-wide binding of Me CP2 provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.
Abstract: Methyl-CpG-binding protein 2 (MeCP2), a protein with affinity for methylated cytosines, is crucial for neuronal development and function. MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. We observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury. The functional implication of increased MeCP2 is largely unknown. To identify regions of the genome bound by MeCP2 in the DRG and the changes induced by nerve injury, a chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) was performed 4 weeks after spared nerve injury (SNI). While the number of binding sites across the genome remained similar in the SNI model and sham control, SNI induced the redistribution of MeCP2 to transcriptionally relevant regions. To determine how differential binding of MeCP2 can affect gene expression in the DRG, we investigated mmu-miR-126, a microRNA locus that had enriched MeCP2 binding in the SNI model. Enriched MeCP2 binding to miR-126 locus after nerve injury repressed miR-126 expression, and this was not mediated by alterations in methylation pattern at the miR-126 locus. Downregulation of miR-126 resulted in the upregulation of its two target genes Dnmt1 and Vegfa in Neuro 2A cells and in SNI model compared to control. These target genes were significantly downregulated in Mecp2-null mice compared to wild-type littermates, indicating a regulatory role for MeCP2 in activating Dnmt1 and Vegfa expression. Intrathecal delivery of miR-126 was not sufficient to reverse nerve injury-induced mechanical and thermal hypersensitivity, but decreased Dnmt1 and Vegfa expression in the DRG. Our study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.
15 citations
Cites background from "MeCP2 binds to 5hmc enriched within..."
...The occurrence of hmC within gene bodies, however, has been associated with active gene expression in neurons [34, 36]....
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...It is now known that MeCP2 can bind two alternatively methylated forms of DNA including methylated cytosine followed by a nucleotide other than guanine (mCH, where H = A, C or T) and hydroxymethylcytosine (hmC) [34, 35]....
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TL;DR: Inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment, and two drugs, AG-490 and Jaki, are capable of reactivating Me CP2 from the inactive X chromosome, in different cellular contexts.
Abstract: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. While MeCP2 mutations are lethal in most males, females survive birth but show severe neurological defects. Because X-chromosome inactivation (XCI) is a random process, approximately 50% of the cells silence the wild-type (WT) copy of the MeCP2 gene. Thus, reactivating the silent WT copy of MeCP2 could provide therapeutic intervention for RTT. Toward this goal, we screened ~ 28,000 small-molecule compounds from several libraries using a MeCP2-luciferase reporter cell line and cortical neurons from a MeCP2-EGFP mouse model. We used gain/increase of luminescence or fluorescence as a readout of MeCP2 reactivation and tested the efficacy of these drugs under different drug regimens, conditions, and cellular contexts. We identified inhibitors of the JAK/STAT pathway as XCI-reactivating agents, both by in vitro and ex vivo assays. In particular, we show that AG-490, a Janus Kinase 2 (JAK2) kinase inhibitor, and Jaki, a pan JAK/STAT inhibitor, are capable of reactivating MeCP2 from the inactive X chromosome, in different cellular contexts. Our results suggest that inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment.
15 citations
Cites background from "MeCP2 binds to 5hmc enriched within..."
...Mutations affecting the nuclear localization signal region of MeCP2 or early truncating mutations are responsible for a severe phenotype in comparison to missense mutations, whereas C-terminal mutations are associated with milder phenotypes [9, 10]....
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TL;DR: A novel, supervised, integrative learning framework to perform whole‐genome methylation and hydroxymethylation predictions in CpG dinucleotides and a novel, beam‐search driven feature selection algorithm to identify the most discriminative predictor variables, and a platform for performing integrative analysis and reconstruction of the epigenome are developed.
Abstract: Motivation 5-Methylcytosine and 5-Hydroxymethylcytosine in DNA are major epigenetic modifications known to significantly alter mammalian gene expression. High-throughput assays to detect these modifications are expensive, labor-intensive, unfeasible in some contexts and leave a portion of the genome unqueried. Hence, we devised a novel, supervised, integrative learning framework to perform whole-genome methylation and hydroxymethylation predictions in CpG dinucleotides. Our framework can also perform imputation of missing or low quality data in existing sequencing datasets. Additionally, we developed infrastructure to perform in silico, high-throughput hypotheses testing on such predicted methylation or hydroxymethylation maps. Results We test our approach on H1 human embryonic stem cells and H1-derived neural progenitor cells. Our predictive model is comparable in accuracy to other state-of-the-art DNA methylation prediction algorithms. We are the first to predict hydroxymethylation in silico with high whole-genome accuracy, paving the way for large-scale reconstruction of hydroxymethylation maps in mammalian model systems. We designed a novel, beam-search driven feature selection algorithm to identify the most discriminative predictor variables, and developed a platform for performing integrative analysis and reconstruction of the epigenome. Our toolkit DIRECTION provides predictions at single nucleotide resolution and identifies relevant features based on resource availability. This offers enhanced biological interpretability of results potentially leading to a better understanding of epigenetic gene regulation. Availability and implementation http://www.pradiptaray.com/direction, under CC-by-SA license. Contacts pradiptaray@gmail.com or mchen@utdallas.edu or michael.zhang@utdallas.edu. Supplementary information Supplementary data are available at Bioinformatics online.
15 citations
01 Jan 2016
TL;DR: This work reviews histone PTMs, histone variants and DNA modifications in the functioning of the nucleosome as an epigenetic signalling module, and discusses the impact of changes in DNA sequence on the epigenome.
Abstract: Epigenetics refer to processes such as histone post-translational modifications (PTMs), DNA methylation and RNA that regulate gene activity and expression but are not dependent on alterations in DNA sequence. Herein, we review histone PTMs, histone variants and DNA modifications in the functioning of the nucleosome as an epigenetic signalling module. The majority of the human genome is transcribed, with most of the genome producing non-coding RNA, some of which is a component of the nuclear matrix, a dynamic RNA protein nuclear substructure. Non-coding RNA and coding RNA are associated with epigenetic modifiers, architectural chromatin proteins, coactivators and corepressors. The impact of changes in DNA sequence (single nucleotide polymorphisms) on the epigenome is discussed. 1.1 Basic and Higher-Order Levels of Chromatin Structure In a mammalian nucleus, the bulk of genomic DNA is condensed into higher-order chromatin structures. The basic level of chromatin organization consists of a nucleosome array, the 11-nm ‘beads-on-a-string’ fibre. The nucleosome consists of a histone octamer, arranged as a (H3–H4)2 tetramer and two H2A–H2B dimers, around which a 147-bp stretch of DNA is wrapped in almost two left-handed superhelical turns. Nucleosomes are joined by a 20–50-bp stretch of DNA called linker DNA. Core histones are primarily globular, containing a histone-fold structure, except for their highly positively charged N-terminal tails, which emanate from the nucleosome in all directions to interact with linker DNA, nearby nucleosomes or other proteins (Luger et al. 1997; Luger and Richmond 1998). Linker histones, H1/ H5, bind to the DNA entry/exit points of nucleosomes and to the linker DNA region between nucleosomes and are crucial for the formation of higher-order chromatin structure (Felsenfeld and Groudine 2003; Li and Reinberg 2011). Except for H4, all histones have variants, some of which are expressed at the time of DNA synthesis (H3.1, H2A.1, replication-dependent) and others expressed throughout the cell cycle (H2A.Z, H3.3, replication-independent) (Maze et al. 2014; Law and Cheung 2013). The insertion of histone variants affects chromatin structure and function (Talbert and Henikoff 2010). H3.3 is mostly deposited during transcription and is enriched at active genes, promoters and regulatory elements. Accordingly, H3.3 is enriched in active marks. However, H3.3 is also present at telomeres where it is required for the transcriptional repression of telomeric repeats. Different histone chaperones are involved in the deposition of H3.3 at different genomic loci (Elsaesser et al. 2010; Goldberg et al. 2010; Szenker et al. 2011). Similarly, the outcome of H2A.Z incorporation is context-dependent (Altaf et al. 2009; Draker et al. 2012). H2A.Z stabilizes nucleosome structure and facilitates chromatin compaction (Li et al. 1993; Chen et al. 2013a, b). The abundance of the replacement histones (e.g. H3.3) increases in differentiated non-cycling cells (e.g. cardiomyocytes) and in cells as they age (Wunsch and Lough 1987; Rogakou and Sekeri-Pataryas 1999; Feser and Tyler 2011; Pina and Suau 1987). Of note, while G.P. Delcuve et al.
15 citations
TL;DR: It is demonstrated that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation, which are sustained in the commitment to form a new retina in mammals.
Abstract: Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectom...
15 citations
Cites background from "MeCP2 binds to 5hmc enriched within..."
...[95] MellenM, Ayata P, Dewell S, et al....
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...Another study showed the accumulation of 5hmC in post-mitotic mouse neurons associated with active transcription and in functional demethylation by decreasing the occupancy of methylcytosine binding protein MeCP2 in the transcript unit, and in consequence activating gene expression [95]....
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References
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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
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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Harvard University1, Brigham and Women's Hospital2, University of Wisconsin-Madison3, University of California, Berkeley4, Technical University of Denmark5, Icahn School of Medicine at Mount Sinai6, Vienna University of Technology7, University of Erlangen-Nuremberg8, German Cancer Research Center9, University of Milan10, Johns Hopkins University11, University of Washington12, Scripps Research Institute13, Walter and Eliza Hall Institute of Medical Research14, University of Iowa15
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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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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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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