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      Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers

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          Abstract

          Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.

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          Most cited references39

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          A unique chromatin signature uncovers early developmental enhancers in humans.

          Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.
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            Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

            CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.
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              Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.

              In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                September 2012
                September 2012
                21 June 2012
                21 June 2012
                : 40
                : 17
                : 8255-8265
                Affiliations
                1Université de Rennes 1, 2CNRS UMR6290, Team SP@RTE, Campus de Beaulieu, Rennes F-35042, 3Université Lille Nord de France, Lille F-59000, 4INSERM UMR1011, Lille F-59000, 5UDSL, Lille F-59000, 6Institut Pasteur de Lille, Lille F-59019, 7INSERM UMR1085, Campus de Beaulieu, Rennes F-35042 and 8CNRS UMR8199, Lille F-59000, France
                Author notes
                *To whom correspondence should be addressed. Tel: +33 2 23 23 61 31; Fax: +33 2 23 23 67 94; Email: gilles.salbert@ 123456univ-rennes1.fr
                Correspondence may also be addressed to Jérôme Eeckhoute. Tel: +33 3 20 97 42 19; Fax: +33 3 20 97 42 01; Email: jerome.eeckhoute@ 123456inserm.fr .

                Present address: Aurélien A. Sérandour, Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK.

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                gks595
                10.1093/nar/gks595
                3458548
                22730288
                9104e5a8-3f1e-4b3b-b349-23aaa7afcf6b
                © The Author(s) 2012. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 April 2012
                : 23 May 2012
                : 25 May 2012
                Page count
                Pages: 11
                Categories
                Gene Regulation, Chromatin and Epigenetics

                Genetics
                Genetics

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