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      The Dynamics of Genome-wide DNA Methylation Reprogramming in Mouse Primordial Germ Cells

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          Summary

          Genome-wide DNA methylation reprogramming occurs in mouse primordial germ cells (PGCs) and preimplantation embryos, but the precise dynamics and biological outcomes are largely unknown. We have carried out whole-genome bisulfite sequencing (BS-Seq) and RNA-Seq across key stages from E6.5 epiblast to E16.5 PGCs. Global loss of methylation takes place during PGC expansion and migration with evidence for passive demethylation, but sequences that carry long-term epigenetic memory (imprints, CpG islands on the X chromosome, germline-specific genes) only become demethylated upon entry of PGCs into the gonads. The transcriptional profile of PGCs is tightly controlled despite global hypomethylation, with transient expression of the pluripotency network, suggesting that reprogramming and pluripotency are inextricably linked. Our results provide a framework for the understanding of the epigenetic ground state of pluripotency in the germline.

          Highlights

          ► DNA demethylation in PGCs occurs in two phases ► Global loss of methylation reveals evidence for a passive demethylation mechanism ► Global methylation erasure coincides with expression of the pluripotency network ► VECs (variably erased CGIs) may act as carriers of transgenerational inheritance

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          Most cited references39

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          Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

          RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.
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            Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

            The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Epigenetic inheritance at the agouti locus in the mouse.

              Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.
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                Author and article information

                Journal
                Mol Cell
                Mol. Cell
                Molecular Cell
                Cell Press
                1097-2765
                1097-4164
                28 December 2012
                28 December 2012
                : 48
                : 6
                : 849-862
                Affiliations
                [1 ]Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK
                [2 ]Bioinformatics Group, The Babraham Institute, Cambridge CB22 3AT, UK
                [3 ]Department of Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland, Campus Saarbrücken, 66123 Saarbrücken, Germany
                [4 ]Laboratory of Translational Genetics, Vesalius Research Center, VIB and KULeuven, 3000 Leuven, Belgium
                [5 ]Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK
                [6 ]Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK
                Author notes
                []Corresponding author wolf.reik@ 123456babraham.ac.uk
                Article
                MOLCEL4496
                10.1016/j.molcel.2012.11.001
                3533687
                23219530
                fe215d02-9af6-42da-b4ec-63cb7a788269
                © 2012 ELL & Excerpta Medica.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 22 August 2012
                : 4 October 2012
                : 1 November 2012
                Categories
                Article

                Molecular biology
                Molecular biology

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