Publications by authors named "Miguel R Branco"

41 Publications

Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol.

Methods Mol Biol 2021 ;2198:333-348

Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.

Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.
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http://dx.doi.org/10.1007/978-1-0716-0876-0_26DOI Listing
January 2021

Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia.

Nat Commun 2020 07 14;11(1):3506. Epub 2020 Jul 14.

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, E1 2AT, UK.

Acute myeloid leukemia (AML) is characterised by a series of genetic and epigenetic alterations that result in deregulation of transcriptional networks. One understudied source of transcriptional regulators are transposable elements (TEs), whose aberrant usage could contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to AML pathogenesis remain unexplored. Here we identify six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppresses cell growth by inducing apoptosis in leukemia cell lines. This work reveals that ERVs are a previously unappreciated source of AML enhancers that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.
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http://dx.doi.org/10.1038/s41467-020-17206-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360734PMC
July 2020

TET1 and 5-Hydroxymethylation Preserve the Stem Cell State of Mouse Trophoblast.

Stem Cell Reports 2020 12 21;15(6):1301-1316. Epub 2020 May 21.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 3EG, UK; Departments of Biochemistry & Molecular Biology and Medical Genetics, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada; Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada. Electronic address:

The ten-eleven translocation factor TET1 and its conferred epigenetic modification 5-hydroxymethylcytosine (5hmC) have important roles in maintaining the pluripotent state of embryonic stem cells (ESCs). We previously showed that TET1 is also essential to maintain the stem cell state of trophoblast stem cells (TSCs). Here, we establish an integrated panel of absolute 5hmC levels, genome-wide DNA methylation and hydroxymethylation patterns, transcriptomes, and TET1 chromatin occupancy in TSCs and differentiated trophoblast cells. We show that the combined presence of 5-methylcytosine (5mC) and 5hmC correlates with transcriptional activity of associated genes. Hypoxia can slow down the global loss of 5hmC that occurs upon differentiation of TSCs. Notably, unlike in ESCs and epiblast cells, most TET1-bound regions overlap with active chromatin marks and TFAP2C binding sites and demarcate putative trophoblast enhancer regions. These chromatin modification and occupancy patterns are highly informative to identify novel candidate regulators of the TSC state.
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http://dx.doi.org/10.1016/j.stemcr.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724466PMC
December 2020

Tet3 ablation in adult brain neurons increases anxiety-like behavior and regulates cognitive function in mice.

Mol Psychiatry 2020 Feb 26. Epub 2020 Feb 26.

Department of Genetics, Faculty of Medicine, University of Porto (FMUP), 4200-319, Porto, Portugal.

TET3 is a member of the ten-eleven translocation (TET) family of enzymes which oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Tet3 is highly expressed in the brain, where 5hmC levels are most abundant. In adult mice, we observed that TET3 is present in mature neurons and oligodendrocytes but is absent in astrocytes. To investigate the function of TET3 in adult postmitotic neurons, we crossed Tet3 floxed mice with a neuronal Cre-expressing mouse line, Camk2a-CreERT2, obtaining a Tet3 conditional KO (cKO) mouse line. Ablation of Tet3 in adult mature neurons resulted in increased anxiety-like behavior with concomitant hypercorticalism, and impaired hippocampal-dependent spatial orientation. Transcriptome and gene-specific expression analysis of the hippocampus showed dysregulation of genes involved in glucocorticoid signaling pathway (HPA axis) in the ventral hippocampus, whereas upregulation of immediate early genes was observed in both dorsal and ventral hippocampal areas. In addition, Tet3 cKO mice exhibit increased dendritic spine maturation in the ventral CA1 hippocampal subregion. Based on these observations, we suggest that TET3 is involved in molecular alterations that govern hippocampal-dependent functions. These results reveal a critical role for epigenetic modifications in modulating brain functions, opening new insights into the molecular basis of neurological disorders.
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http://dx.doi.org/10.1038/s41380-020-0695-7DOI Listing
February 2020

Crossroads between transposons and gene regulation.

Philos Trans R Soc Lond B Biol Sci 2020 03 10;375(1795):20190330. Epub 2020 Feb 10.

BioFrontiers Institute, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA.

Transposons are mobile genetic elements that have made a large contribution to genome evolution in a largely species-specific manner. A wide variety of different transposons have invaded genomes throughout evolution, acting in a first instance as 'selfish' elements, whose success was determined by their ability to self-replicate and expand within the host genome. However, their coevolution with the host has created many crossroads between transposons and the regulation of host gene expression. Transposons are an abundant source of transcriptional modulatory elements, such as gene promoters and enhancers, splicing and termination sites, and regulatory non-coding RNAs. Moreover, transposons have driven the evolution of host defence mechanisms that have been repurposed for gene regulation. However, dissecting the potential functional roles of transposons remains challenging owing to their evolutionary path, as well as their repetitive nature, which requires the development of specialized analytical tools. In this special issue, we present a collection of articles that lay out current paradigms in the field and discuss a vision for future research. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
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http://dx.doi.org/10.1098/rstb.2019.0330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061990PMC
March 2020

Correction to: Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

Cell Mol Life Sci 2020 07;77(14):2885

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057, Braga, Portugal.

The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.
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http://dx.doi.org/10.1007/s00018-019-03443-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326887PMC
July 2020

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

Cell Mol Life Sci 2020 Jul 23;77(14):2871-2883. Epub 2019 Oct 23.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057, Braga, Portugal.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.
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http://dx.doi.org/10.1007/s00018-019-03335-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326798PMC
July 2020

Functional evaluation of transposable elements as enhancers in mouse embryonic and trophoblast stem cells.

Elife 2019 04 23;8. Epub 2019 Apr 23.

Centre for Genomic Health, Life Sciences Institute, Queen Mary University of London, London, United Kingdom.

Transposable elements (TEs) are thought to have helped establish gene regulatory networks. Both the embryonic and extraembryonic lineages of the early mouse embryo have seemingly co-opted TEs as enhancers, but there is little evidence that they play significant roles in gene regulation. Here we tested a set of long terminal repeat TE families for roles as enhancers in mouse embryonic and trophoblast stem cells. Epigenomic and transcriptomic data suggested that a large number of TEs helped to establish tissue-specific gene expression programmes. Genetic editing of individual TEs confirmed a subset of these regulatory relationships. However, a wider survey via CRISPR interference of RLTR13D6 elements in embryonic stem cells revealed that only a minority play significant roles in gene regulation. Our results suggest that a subset of TEs are important for gene regulation in early mouse development, and highlight the importance of functional experiments when evaluating gene regulatory roles of TEs.
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http://dx.doi.org/10.7554/eLife.44344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544436PMC
April 2019

Author Correction: Regulation of transposable elements by DNA modifications.

Nat Rev Genet 2019 07;20(7):432

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, UK.

The originally published article contained an error in Figure 2a: for the left side of the figure part (showing piRNA-directed DNA methylation of mouse transposable elements), DNMT3A/B should have been DNMT3C. The article has now been corrected online.
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http://dx.doi.org/10.1038/s41576-019-0117-3DOI Listing
July 2019

Regulation of transposable elements by DNA modifications.

Nat Rev Genet 2019 07;20(7):417-431

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, UK.

Maintenance of genome stability requires control over the expression of transposable elements (TEs), whose activity can have substantial deleterious effects on the host. Chemical modification of DNA is a commonly used strategy to achieve this, and it has long been argued that the emergence of 5-methylcytosine (5mC) in many species was driven by the requirement to silence TEs. Potential roles in TE regulation have also been suggested for other DNA modifications, such as N6-methyladenine and oxidation derivatives of 5mC, although the underlying mechanistic relationships are poorly understood. Here, we discuss current evidence implicating DNA modifications and DNA-modifying enzymes in TE regulation across different species.
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http://dx.doi.org/10.1038/s41576-019-0106-6DOI Listing
July 2019

Correction to: Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

Genome Biol 2019 02 22;20(1):43. Epub 2019 Feb 22.

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

Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published. The correct additional file is given below.
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http://dx.doi.org/10.1186/s13059-019-1656-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385436PMC
February 2019

Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages.

Nat Commun 2018 10 10;9(1):4189. Epub 2018 Oct 10.

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, E1 2AT, UK.

The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells to understand how chromatin conformation relates to cell-specific transcriptional programmes. We find that key TSC genes that are kept repressed in ESCs exhibit interactions between H3K27me3-marked regions in ESCs that depend on Polycomb repressive complex 1. Interactions that are prominent in TSCs are enriched for enhancer-gene contacts involving key TSC transcription factors, as well as TET1, which helps to maintain the expression of TSC-relevant genes. Our work shows that the first developmental cell fate decision results in distinct chromatin conformation patterns establishing lineage-specific contexts involving both repressive and active interactions.
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http://dx.doi.org/10.1038/s41467-018-06666-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180096PMC
October 2018

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

Genome Biol 2018 03 15;19(1):33. Epub 2018 Mar 15.

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

Background: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

Results: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.

Conclusions: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.
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http://dx.doi.org/10.1186/s13059-018-1408-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856372PMC
March 2018

SETDB1 prevents TET2-dependent activation of IAP retroelements in naïve embryonic stem cells.

Genome Biol 2018 01 19;19(1). Epub 2018 Jan 19.

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, E1 2AT, UK.

Background: Endogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET-dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos.

Results: We use naïve ESCs to investigate the role of SETDB1 in ERV regulation and its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We find that in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these retrotransposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming.

Conclusions: Our results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation.
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http://dx.doi.org/10.1186/s13059-017-1376-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775534PMC
January 2018

TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells.

Nucleic Acids Res 2017 Sep;45(17):10002-10017

Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908 L'Hospitalet de Llobregat, Barcelona, Spain.

The plasticity of myeloid cells is illustrated by a diversity of functions including their role as effectors of innate immunity as macrophages (MACs) and bone remodelling as osteoclasts (OCs). TET2, a methylcytosine dioxygenase highly expressed in these cells and frequently mutated in myeloid leukemias, may be a key contributor to this plasticity. Through transcriptomic and epigenomic analyses, we investigated 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and gene expression changes in two divergent terminal myeloid differentiation processes, namely MAC and OC differentiation. MACs and OCs undergo highly similar 5hmC and 5mC changes, despite their wide differences in gene expression. Many TET2- and thymine-DNA glycosylase (TDG)-dependent 5mC and 5hmC changes directly activate the common terminal myeloid differentiation programme. However, the acquisition of differential features between MACs and OCs also depends on TET2/TDG. In fact, 5mC oxidation precedes differential histone modification changes between MACs and OCs. TET2 and TDG downregulation impairs the acquisition of such differential histone modification and expression patterns at MAC-/OC-specific genes. We prove that the histone H3K4 methyltransferase SETD1A is differentially recruited between MACs and OCs in a TET2-dependent manner. We demonstrate a novel role of these enzymes in the establishment of specific elements of identity and function in terminal myeloid differentiation.
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http://dx.doi.org/10.1093/nar/gkx666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622316PMC
September 2017

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells.

Nat Struct Mol Biol 2017 Jun 24;24(6):515-524. Epub 2017 Apr 24.

Epigenetic Regulation and Chromatin Architecture Group, Berlin Institute for Medical Systems Biology, Max Delbrück Centre for Molecular Medicine, Berlin, Germany.

Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.
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http://dx.doi.org/10.1038/nsmb.3402DOI Listing
June 2017

Complex multi-enhancer contacts captured by genome architecture mapping.

Nature 2017 03 8;543(7646):519-524. Epub 2017 Mar 8.

Epigenetic Regulation and Chromatin Architecture Group, Berlin Institute for Medical Systems Biology, Max-Delbrück Centre for Molecular Medicine, Robert-Rössle Straße, Berlin-Buch 13125, Germany.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.
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http://dx.doi.org/10.1038/nature21411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366070PMC
March 2017

TET-dependent regulation of retrotransposable elements in mouse embryonic stem cells.

Genome Biol 2016 11 18;17(1):234. Epub 2016 Nov 18.

Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, E1 2AT, UK.

Background: Ten-eleven translocation (TET) enzymes oxidise DNA methylation as part of an active demethylation pathway. Despite extensive research into the role of TETs in genome regulation, little is known about their effect on transposable elements (TEs), which make up nearly half of the mouse and human genomes. Epigenetic mechanisms controlling TEs have the potential to affect their mobility and to drive the co-adoption of TEs for the benefit of the host.

Results: We performed a detailed investigation of the role of TET enzymes in the regulation of TEs in mouse embryonic stem cells (ESCs). We find that TET1 and TET2 bind multiple TE classes that harbour a variety of epigenetic signatures indicative of different functional roles. TETs co-bind with pluripotency factors to enhancer-like TEs that interact with highly expressed genes in ESCs whose expression is partly maintained by TET2-mediated DNA demethylation. TETs and 5-hydroxymethylcytosine (5hmC) are also strongly enriched at the 5' UTR of full-length, evolutionarily young LINE-1 elements, a pattern that is conserved in human ESCs. TETs drive LINE-1 demethylation, but surprisingly, LINE-1s are kept repressed through additional TET-dependent activities. We find that the SIN3A co-repressive complex binds to LINE-1s, ensuring their repression in a TET1-dependent manner.

Conclusions: Our data implicate TET enzymes in the evolutionary dynamics of TEs, both in the context of exaptation processes and of retrotransposition control. The dual role of TET action on LINE-1s may reflect the evolutionary battle between TEs and the host.
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http://dx.doi.org/10.1186/s13059-016-1096-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116139PMC
November 2016

Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.

Methods Mol Biol 2016 ;1400:387-401

Blizard Institute, School of Medicine and Dentistry, QMUL, E1 2AT, London, UK.

DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.
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http://dx.doi.org/10.1007/978-1-4939-3372-3_24DOI Listing
December 2016

Maternal DNA Methylation Regulates Early Trophoblast Development.

Dev Cell 2016 Jan;36(2):152-63

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

Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms.
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http://dx.doi.org/10.1016/j.devcel.2015.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729543PMC
January 2016

Bridging genomics technology and biology.

Authors:
Miguel R Branco

Genome Biol 2013 ;14(10):312

A report on the UK Genome Science Meeting, held at the University of Nottingham, UK, 2-4 September 2013.
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http://dx.doi.org/10.1186/gb4135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054847PMC
September 2014

Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine.

Nat Protoc 2013 Oct 5;8(10):1841-51. Epub 2013 Sep 5.

Department of Chemistry, University of Cambridge, Cambridge, UK.

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.
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http://dx.doi.org/10.1038/nprot.2013.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919000PMC
October 2013

FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.

Cell Stem Cell 2013 Sep 11;13(3):351-9. Epub 2013 Jul 11.

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

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.
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http://dx.doi.org/10.1016/j.stem.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765959PMC
September 2013

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline.

Development 2012 Oct;139(19):3623-32

MRC Human Genetics Unit, IGMM, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK.

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (~E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.
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http://dx.doi.org/10.1242/dev.081661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436114PMC
October 2012

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase.

Genome Biol 2012 Aug 17;13(8):R69. Epub 2012 Aug 17.

Background: Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation.

Results: Our method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts.

Conclusions: Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.
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http://dx.doi.org/10.1186/gb-2012-13-8-r69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491369PMC
August 2012

Pairing of homologous regions in the mouse genome is associated with transcription but not imprinting status.

PLoS One 2012 3;7(7):e38983. Epub 2012 Jul 3.

Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

Although somatic homologous pairing is common in Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038983PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389011PMC
December 2012

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

PLoS Genet 2012 Jun 28;8(6):e1002750. Epub 2012 Jun 28.

Department of Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland, Saarbrücken, Germany.

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.
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http://dx.doi.org/10.1371/journal.pgen.1002750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386304PMC
June 2012

Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

Science 2012 May 26;336(6083):934-7. Epub 2012 Apr 26.

Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.
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http://dx.doi.org/10.1126/science.1220671DOI Listing
May 2012

Uncovering the role of 5-hydroxymethylcytosine in the epigenome.

Nat Rev Genet 2011 Nov 15;13(1):7-13. Epub 2011 Nov 15.

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

Just over 2 years ago, TET1 was found to catalyse the oxidation of 5-methylcytosine, a well-known epigenetic mark, into 5-hydroxymethylcytosine in mammalian DNA. The exciting prospect of a novel epigenetic modification that may dynamically regulate DNA methylation has led to the rapid accumulation of publications from a wide array of fields, from biochemistry to stem cell biology. Although we have only started to scratch the surface, interesting clues on the role of 5-hydroxymethylcytosine are quickly emerging.
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http://dx.doi.org/10.1038/nrg3080DOI Listing
November 2011

Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation.

Nature 2011 May 3;473(7347):398-402. Epub 2011 Apr 3.

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

Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.
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http://dx.doi.org/10.1038/nature10008DOI Listing
May 2011