Publications by authors named "Sebastien A Smallwood"

20 Publications

  • Page 1 of 1

A genome-scale map of DNA methylation turnover identifies site-specific dependencies of DNMT and TET activity.

Nat Commun 2020 05 29;11(1):2680. Epub 2020 May 29.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.
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http://dx.doi.org/10.1038/s41467-020-16354-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260214PMC
May 2020

DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.

Nat Struct Mol Biol 2019 06 27;26(6):471-480. Epub 2019 May 27.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Current understanding of chromosome folding is largely reliant on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo, quantitatively and without crosslinking and ligation, we implemented a modified version of DNA adenine methyltransferase identification (DamID) named DamC, which combines DNA methylation-based detection of chromosomal interactions with next-generation sequencing and biophysical modeling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.
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http://dx.doi.org/10.1038/s41594-019-0231-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561777PMC
June 2019

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3.

Cell Stem Cell 2019 02 27;24(2):257-270.e8. Epub 2018 Dec 27.

Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland. Electronic address:

Self-renewal and differentiation of pluripotent murine embryonic stem cells (ESCs) is regulated by extrinsic signaling pathways. It is less clear whether cellular metabolism instructs developmental progression. In an unbiased genome-wide CRISPR/Cas9 screen, we identified components of a conserved amino-acid-sensing pathway as critical drivers of ESC differentiation. Functional analysis revealed that lysosome activity, the Ragulator protein complex, and the tumor-suppressor protein Folliculin enable the Rag GTPases C and D to bind and seclude the bHLH transcription factor Tfe3 in the cytoplasm. In contrast, ectopic nuclear Tfe3 represses specific developmental and metabolic transcriptional programs that are associated with peri-implantation development. We show differentiation-specific and non-canonical regulation of Rag GTPase in ESCs and, importantly, identify point mutations in a Tfe3 domain required for cytoplasmic inactivation as potentially causal for a human developmental disorder. Our work reveals an instructive and biomedically relevant role of metabolic signaling in licensing embryonic cell fate transitions.
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http://dx.doi.org/10.1016/j.stem.2018.11.021DOI Listing
February 2019

Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency.

Cell Syst 2018 07;7(1):63-76.e12

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK; Epigenetics Programme, Babraham Institute, Cambridge, UK; Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge, UK. Electronic address:

Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.
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http://dx.doi.org/10.1016/j.cels.2018.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066359PMC
July 2018

Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach.

Methods Mol Biol 2018 ;1708:161-169

Epigenetics Programme, Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

The epigenetic mark 5-methylcytosine confers heritable regulation of gene expression that can be dynamically modulated during transitions in cell fate. With the development of high-throughput sequencing technologies, it is now possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape, but the application of these techniques to limited material remains challenging. Here, we present an optimized protocol to perform whole-genome bisulfite sequencing on low inputs (100-5000 somatic cells) using a post-bisulfite adapter tagging approach. In this strategy, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then sequencing adapters are added by complementary strand synthesis using random tetramer priming, and libraries are subsequently amplified by PCR.
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http://dx.doi.org/10.1007/978-1-4939-7481-8_9DOI Listing
July 2018

Genome-Wide Analysis of DNA Methylation in Single Cells Using a Post-bisulfite Adapter Tagging Approach.

Methods Mol Biol 2018 ;1712:87-95

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058, Basel, Switzerland.

DNA methylation is an epigenetic mark implicated in the regulation of key biological processes. Using high-throughput sequencing technologies and bisulfite-based approaches, it is possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape with a single-nucleotide resolution and absolute quantification. However, these methods were only applicable to bulk populations of cells. Here, we present a protocol to perform whole-genome bisulfite sequencing on single cells (scBS-Seq) using a post-bisulfite adapter tagging approach. In this method, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then DNA fragments are pre-amplified with concomitant integration of the sequencing adapters, and libraries are subsequently amplified and indexed by PCR. Using scBS-Seq we can accurately measure DNA methylation at up to 50% of individual CpG sites and 70% of CpG islands.
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http://dx.doi.org/10.1007/978-1-4939-7514-3_7DOI Listing
July 2018

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

Epigenetics Chromatin 2017 12;10:25. Epub 2017 May 12.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT UK.

Background: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci.

Results: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis.

Conclusions: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.
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http://dx.doi.org/10.1186/s13072-017-0133-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429541PMC
July 2017

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

Nat Protoc 2017 03 9;12(3):534-547. Epub 2017 Feb 9.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
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http://dx.doi.org/10.1038/nprot.2016.187DOI Listing
March 2017

Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

Genome Biol 2016 Apr 18;17:72. Epub 2016 Apr 18.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK.

Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.
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http://dx.doi.org/10.1186/s13059-016-0944-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834828PMC
April 2016

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.

Genes Dev 2015 Dec 19;29(23):2449-62. Epub 2015 Nov 19.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, United Kingdom; Centre for Trophoblast Research, University of Cambridge CB2 3EG, Cambridge, United Kingdom.

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.
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http://dx.doi.org/10.1101/gad.271353.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691949PMC
December 2015

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.

Mol Cell 2015 Nov 5;60(4):611-25. Epub 2015 Nov 5.

Medical Research Council Clinical Sciences Centre (MRC CSC), Faculty of Medicine, Imperial College London, London W12 0NN, UK. Electronic address:

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.
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http://dx.doi.org/10.1016/j.molcel.2015.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672152PMC
November 2015

Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape.

Genome Biol 2015 Sep 25;16:209. Epub 2015 Sep 25.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Background: Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome.

Results: We perform deep RNA-Seq and de novo transcriptome assembly at different stages of mouse oogenesis. This reveals thousands of novel non-annotated genes, as well as alternative promoters, for approximately 10 % of reference genes expressed in oocytes. In addition, a large fraction of novel promoters coincide with MaLR and ERVK transposable elements. Integration with our transcriptome assembly reveals that transcription correlates accurately with DNA methylation and accounts for approximately 85-90 % of the methylome. We generate a mouse model in which transcription across the Zac1/Plagl1 locus is abrogated in oocytes, resulting in failure of DNA methylation establishment at all CpGs of this locus. ChIP analysis in oocytes reveals H3K4me2 enrichment at the Zac1 imprinted control region when transcription is ablated, establishing a connection between transcription and chromatin remodeling at CpG islands by histone demethylases.

Conclusions: By precisely defining the mouse oocyte transcriptome, this work not only highlights transcription as a cornerstone of DNA methylation establishment in female germ cells, but also provides an important resource for developmental biology research.
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http://dx.doi.org/10.1186/s13059-015-0769-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582738PMC
September 2015

Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation.

Cell Rep 2014 Dec 12;9(6):1990-2000. Epub 2014 Dec 12.

Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK. Electronic address:

Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup.
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http://dx.doi.org/10.1016/j.celrep.2014.11.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542306PMC
December 2014

Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.

Nat Methods 2014 Aug 20;11(8):817-820. Epub 2014 Jul 20.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.
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http://dx.doi.org/10.1038/nmeth.3035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117646PMC
August 2014

Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.

Stem Cell Reports 2014 Apr 3;2(4):520-33. Epub 2014 Apr 3.

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen, 9713AV Groningen, the Netherlands.

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.
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http://dx.doi.org/10.1016/j.stemcr.2014.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986662PMC
April 2014

Genome-wide analysis of DNA methylation in low cell numbers by reduced representation bisulfite sequencing.

Methods Mol Biol 2012 ;925:187-97

Epigenetics Programme, The Babraham Institute, Cambridge, UK.

Development of high-throughput sequencing technologies now enables genome-wide analysis of DNA methylation of mammalian cells and tissues. Here, we present a protocol for Reduced Representation Bisulfite Sequencing (RRBS) applicable to low amounts of starting material (from 200 to 5,000 cells). RRBS is a cost-effective and powerful technique offering the advantages of absolute DNA methylation quantification and single nucleotide resolution while covering mainly CpG islands. Typically one sequencing experiment using the Illumina Genome Analyser IIx platform provides information on the DNA methylation status of more than half of the CpG islands of the mouse genome.
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http://dx.doi.org/10.1007/978-1-62703-011-3_12DOI Listing
December 2012

De novo DNA methylation: a germ cell perspective.

Trends Genet 2012 Jan 21;28(1):33-42. Epub 2011 Oct 21.

Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK.

DNA methylation is a fundamentally important epigenetic modification of the mammalian genome that has widespread influences on gene expression. During germ-cell specification and maturation, epigenetic reprogramming occurs and the DNA methylation landscape is profoundly remodelled. Defects in this process have major consequences for embryonic development and are associated with several genetic disorders. In this review we report our current understanding of the molecular mechanisms associated with de novo DNA methylation in germ cells. We discuss recent discoveries connecting histone modifications, transcription and the DNA methylation machinery, and consider how these new findings could lead to a model for methylation establishment. Elucidating how DNA methylation marks are established in the germline has been a challenge for nearly 20 years, but represents a key step towards a full understanding of several biological processes including genomic imprinting, epigenetic reprogramming and the establishment of the pluripotent state in early embryos.
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http://dx.doi.org/10.1016/j.tig.2011.09.004DOI Listing
January 2012

Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.

Nat Genet 2011 Jun 26;43(8):811-4. Epub 2011 Jun 26.

Epigenetics Programme, The Babraham Institute, Cambridge, UK.

Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming. Here we present, to our knowledge, the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L but are not distinct at the sequence level, including in CpG periodicity. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilization reprogramming but, notably, the majority show incomplete demethylation in embryonic day (E) 3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.
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http://dx.doi.org/10.1038/ng.864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146050PMC
June 2011

Transcription is required for establishment of germline methylation marks at imprinted genes.

Genes Dev 2009 Jan;23(1):105-17

Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB22 3AT, United Kingdom.

Genomic imprinting requires the differential marking by DNA methylation of genes in male and female gametes. In the female germline, acquisition of methylation imprint marks depends upon the de novo methyltransferase Dnmt3a and its cofactor Dnmt3L, but the reasons why specific sequences are targets for Dnmt3a and Dnmt3L are still poorly understood. Here, we investigate the role of transcription in establishing maternal germline methylation marks. We show that at the Gnas locus, truncating transcripts from the furthest upstream Nesp promoter disrupts oocyte-derived methylation of the differentially methylated regions (DMRs). Transcription through DMRs in oocytes is not restricted to this locus but occurs across the prospective DMRs at many other maternally marked imprinted domains, suggesting a common requirement for transcription events. The transcripts implicated here in gametic methylation are protein-coding, in contrast to the noncoding antisense transcripts involved in the monoallelic silencing of imprinted genes in somatic tissues, although they often initiate from alternative promoters in oocytes. We propose that transcription is a third essential component of the de novo methylation system, which includes optimal CpG spacing and histone modifications, and may be required to create or maintain open chromatin domains to allow the methylation complex access to its preferred targets.
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http://dx.doi.org/10.1101/gad.495809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632167PMC
January 2009