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1.  Single-Cell Genome-Wide Bisulfite Sequencing for Assessing Epigenetic Heterogeneity 
Nature methods  2014;11(8):817-820.
We report a single-cell bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 48.4% of CpGs. We observed that ESCs grown in serum or 2i both display epigenetic heterogeneity, with “2i-like” cells present in serum cultures. In silico integration of 12 individual mouse oocyte datasets largely recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.
doi:10.1038/nmeth.3035
PMCID: PMC4117646  PMID: 25042786
2.  Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo 
Background
DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.
Results
We compared DHBS maps of repetitive elements in the developing zygote, the early embryo, and primordial germ cells (PGCs) at defined stages of development. In the zygote, we observed distinct effects in paternal and maternal chromosomes. A significant loss of paternal DNA methylation was linked to replication and to an increase in continuous and dispersed hemimethylated CpG dyad patterns. Overall methylation levels at maternal copies remained largely unchanged, but showed an increased level of dispersed hemi-methylated CpG dyads. After the first cell cycle, the combined DHBS patterns of paternal and maternal chromosomes remained unchanged over the next three cell divisions. By contrast, in PGCs the DNA demethylation process was continuous, as seen by a consistent decrease in fully methylated CpG dyads over consecutive cell divisions.
Conclusions
The main driver of DNA demethylation in germ cells and in the zygote is partial impairment of maintenance of symmetric DNA methylation at CpG dyads. In the embryo, this passive demethylation is restricted to the first cell division, whereas it continues over several cell divisions in germ cells. The dispersed patterns of CpG dyads in the early-cleavage embryo suggest a continuous partial (and to a low extent active) loss of methylation apparently compensated for by selective de novo methylation. We conclude that a combination of passive and active demethylation events counteracted by de novo methylation are involved in the distinct reprogramming dynamics of DNA methylomes in the zygote, the early embryo, and PGCs.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-8935-8-1) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-8935-8-1
PMCID: PMC4304184  PMID: 25621012
DNA methylation reprogramming; Pre-implantation development; DNA methylation pattern; Deep hairpin bisulfite sequencing
3.  Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human 
Cell  2014;158(6):1254-1269.
Summary
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Graphical Abstract
Highlights
•Transcription factor circuitry is rewired in human pluripotent stem cells•Transcriptome and metabolism are similar to mouse ground-state embryonic stem cells•Genome-wide hypomethylation in reset cells indicates global epigenetic erasure•Reset human cells can incorporate into mouse preimplantation epiblast
Invoking the transcription factor circuitry that defines mouse embryonic stem cell identity converts human pluripotent cells to a more naive state, characterized by altered transcriptional and metabolic activity, absence of lineage priming, and global DNA hypomethylation.
doi:10.1016/j.cell.2014.08.029
PMCID: PMC4162745  PMID: 25215486
4.  Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution 
PLoS ONE  2014;9(7):e97754.
Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated.
doi:10.1371/journal.pone.0097754
PMCID: PMC4098905  PMID: 25025377
5.  Reprogramming the Methylome: Erasing Memory and Creating Diversity 
Cell Stem Cell  2014;14(6):710-719.
The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian development. Epigenetic reprogramming results in global hypomethylation of the genome together with a profound loss of memory, which underlies naive pluripotency. Such global reprogramming occurs in primordial germ cells, early embryos, and embryonic stem cells where reciprocal molecular links connect the methylation machinery to pluripotency. Priming for differentiation is initiated upon exit from pluripotency, and we propose that epigenetic mechanisms create diversity of transcriptional states, which help with symmetry breaking during cell fate decisions and lineage commitment.
This Perspective discusses synergistic mechanisms that result in epigenetic memory loss in PGCs, the early embryo, and naive ESCs. Links between pluripotency and loss of epigenetic memory and connections between heterogeneous DNA methylation and lineage priming are also explored.
doi:10.1016/j.stem.2014.05.008
PMCID: PMC4051243  PMID: 24905162
6.  Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine 
Nature protocols  2013;8(10):1841-1851.
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.
doi:10.1038/nprot.2013.115
PMCID: PMC3919000  PMID: 24008380
7.  Etoposide Induces Nuclear Re-Localisation of AID 
PLoS ONE  2013;8(12):e82110.
During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.
doi:10.1371/journal.pone.0082110
PMCID: PMC3852760  PMID: 24324754
8.  Active demethylation in mouse zygotes involves cytosine deamination and base excision repair 
Background
DNA methylation in mammals is an epigenetic mark necessary for normal embryogenesis. During development active loss of methylation occurs in the male pronucleus during the first cell cycle after fertilisation. This is accompanied by major chromatin remodelling and generates a marked asymmetry between the paternal and maternal genomes. The mechanism(s) by which this is achieved implicate, among others, base excision repair (BER) components and more recently a major role for TET3 hydroxylase. To investigate these methylation dynamics further we have analysed DNA methylation and hydroxymethylation in fertilised mouse oocytes by indirect immunofluorescence (IF) and evaluated the relative contribution of different candidate factors for active demethylation in knock-out zygotes by three-dimensional imaging and IF semi-quantification.
Results
We find two distinct phases of loss of paternal methylation in the zygote, one prior to and another coincident with, but not dependent on, DNA replication. TET3-mediated hydroxymethylation is limited to the replication associated second phase of demethylation. Analysis of cytosine deaminase (AID) null fertilised oocytes revealed a role for this enzyme in the second phase of loss of paternal methylation, which is independent from hydroxymethylation. Investigation into the possible repair pathways involved supports a role for AID-mediated cytosine deamination with subsequent U-G mismatch long-patch BER by UNG2 while no evidence could be found for an involvement of TDG.
Conclusions
There are two observable phases of DNA demethylation in the mouse zygote, before and coincident with DNA replication. TET3 is only involved in the second phase of loss of methylation. Cytosine deamination and long-patch BER mediated by UNG2 appear to independently contribute to this second phase of active demethylation. Further work will be necessary to elucidate the mechanism(s) involved in the first phase of active demethylation that will potentially involve activities required for early sperm chromatin remodelling.
doi:10.1186/1756-8935-6-39
PMCID: PMC4037648  PMID: 24279473
Epigenetic reprogramming; DNA methylation; Hydroxymethylation; AID; BER; UNG2; TET3
9.  Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation 
BioTechniques  2012;53(6):10.2144/000113962.
We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400–500 ng of sheared DNA for the library preparation.
doi:10.2144/000113962
PMCID: PMC3808810  PMID: 23227987
Pacific Biosciences; PacBio; next-generation DNA Sequencing; single molecule real time; library prep
10.  A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation 
Genome Biology  2013;14(10):R119.
Background
DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.
Results
We compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex).
Conclusions
0ur screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.
doi:10.1186/gb-2013-14-10-r119
PMCID: PMC4014808  PMID: 24156278
11.  Nanog-dependent function of Tet1 and Tet2 in establishment of pluripotency 
Nature  2013;495(7441):370-374.
Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein Nanog1–2, which plays an essential role in establishing ground state pluripotency during somatic cell reprogramming3–4. While the genomic occupancy of Nanog has been extensively investigated, comparatively little is known about Nanog-associated proteins5 and their contribution to the Nanog-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identified 27 high-confidence protein interaction partners of Nanog in mouse ES cells. These consist of 19 novel partners of Nanog that have not been reported before including the Ten eleven translocation (Tet) family methylcytosine hydroxylase Tet1. We confirmed physical association of Nanog with Tet1, and demonstrated that Tet1, in synergy with Nanog, enhances the efficiency of reprogramming. We also found physical association and reprogramming synergy of Tet2 with Nanog, and demonstrated that knockdown of Tet2 abolishes the reprogramming synergy of Nanog with a catalytically deficient mutant of Tet1 (Tet1Mut). These results indicate that the physical interaction between Nanog and Tet1/2 proteins facilitates reprogramming in a manner that is dependent on Tet1/2's catalytic activity. Tet1 and Nanog co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in ES cells, and Tet1 binding is reduced upon Nanog depletion. Co-expression of Nanog and Tet1 results in expression priming of and increased 5hmC levels at top ranked common targets Esrrb and Oct4 before reprogramming to naïve pluripotency. We propose that Tet1 is recruited by Nanog to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of Nanog and uncover a novel role for 5mC hydroxylases in the establishment of naïve pluripotency.
doi:10.1038/nature11925
PMCID: PMC3606645  PMID: 23395962
Nanog; Tet1; Tet2; pluripotency; reprogramming; self-renewal; epigenetics
12.  FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency 
Cell Stem Cell  2013;13(3):351-359.
Summary
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.
Highlights
•Genome-wide analysis of 2i ESCs reveals demethylated genome similar to ICM and PGCs•Demethylation involves TETs, replicative loss of 5mC and 5hmC, suppression of Dnmt3s•NANOG/PRDM14 binding element in Dnmt3b enhancer is highly responsive to signaling•2i and serum epigenetic signatures exist in populations of NanogGFP ESCs and ICM
The transition to ground state pluripotency involves genome-wide DNA demethylation and oxidation of 5mC to 5hmC by Tet proteins.
doi:10.1016/j.stem.2013.06.004
PMCID: PMC3765959  PMID: 23850245
14.  Robust 3D DNA FISH Using Directly Labeled Probes 
3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.
doi:10.3791/50587
PMCID: PMC3846859  PMID: 23978815
Genetics; Issue 78; Molecular Biology; Biochemistry; Cellular Biology; Genomics; Epigenetics; Cell Nucleus; Fluorescence;  In Situ Hybridization; FISH; 3D DNA FISH; fluorescence in situ hybridization; nuclear structure; fluorescently labeled probes; visualization; imaging; DNA; chromosomes; sequencing; probes; assay
15.  Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm 
PLoS ONE  2013;8(6):e68846.
Background
DNA methylation is reprogrammed during early embryogenesis by active and passive mechanisms in advance of the first differentiation event producing the embryonic and extraembryonic lineage cells which contribute to the future embryo proper and to the placenta respectively. Embryonic lineage cells re-acquire a highly methylated genome dependent on the DNA methyltransferases (DNMTs) Dnmt3a and Dnmt3b that are required for de novo methylation. By contrast, extraembryonic lineage cells remain globally hypomethylated but the mechanisms that underlie this hypomethylation remain unknown.
Methodology/Principal Findings
We have employed an inducible system that supports differentiation between these two lineages and recapitulates the DNA methylation asymmetry generated in vivo. We find that in vitro down-regulation of Oct3/4 in ES cells recapitulates the decline in global DNA methylation associated with trophoblast. The de novo DNMTs Dnmt3a2 and Dnmt3b are down-regulated during trophoblast differentiation. Dnmt1, which is responsible for maintenance methylation, is expressed comparably in embryonic and trophoblast lineages, however importantly in trophoblast giant cells Dnmt1fails to be attracted to replication foci, thus allowing loss of DNA methylation while implicating a passive demethylation mechanism. Interestingly, Dnmt1 localization was restored by exogenous Np95/Uhrf1, a Dnmt1 chaperone required for Dnmt1-targeting to replication foci, yet DNA methylation levels remained low. Over-expression of de novo DNMTs also failed to increase DNA methylation in target sequences.
Conclusions/Significance
We propose that induced trophoblast cells may have a mechanism to resist genome-wide increases of DNA methylation, thus reinforcing the genome-wide epigenetic distinctions between the embryonic and extraembryonic lineages in the mouse. This resistance may be based on transcription factors or on global differences in chromatin structure.
doi:10.1371/journal.pone.0068846
PMCID: PMC3692478  PMID: 23825703
16.  Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers 
In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.
doi:10.1098/rstb.2011.0330
PMCID: PMC3539359  PMID: 23166394
DNA methylation; germline; reprogramming; development; hydroxymethylation; epigenetics
17.  The H19 lincRNA is a developmental reservoir of miR-675 which suppresses growth and Igf1r 
Nature cell biology  2012;14(7):659-665.
The H19 large intergenic noncoding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extraembryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded within H19’s first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extraembryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth promoting Insulin-like growth factor 1 receptor (Igf1r). Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress response RNA binding protein HuR. These results suggest that H19’s main physiological role is in limiting growth of the placenta prior to birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.
doi:10.1038/ncb2521
PMCID: PMC3389517  PMID: 22684254
18.  The Dynamics of Genome-wide DNA Methylation Reprogramming in Mouse Primordial Germ Cells 
Molecular Cell  2012;48(6):849-862.
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
doi:10.1016/j.molcel.2012.11.001
PMCID: PMC3533687  PMID: 23219530
19.  Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase 
Genome Biology  2012;13(8):R69.
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.
doi:10.1186/gb-2012-13-8-r69
PMCID: PMC3491369  PMID: 22902005
20.  Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status 
PLoS ONE  2012;7(7):e38983.
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.
doi:10.1371/journal.pone.0038983
PMCID: PMC3389011  PMID: 22802932
21.  Epigenetic reprogramming in plant and animal development 
Science (New York, N.Y.)  2010;330(6004):622-627.
Epigenetic modifications of the genome are generally stable in somatic cells of multicellular organisms. In germ cells and early embryos, however, epigenetic reprogramming occurs on a genome-wide scale, which includes demethylation of DNA and remodeling of histones and their modifications. Mechanisms of genome-wide erasure of DNA methylation are being unraveled, which involve modifications to 5-methylcytosine (5mC) and DNA repair. Epigenetic reprogramming has important roles in imprinting, the natural as well as experimental acquisition of totipotency and pluripotency, control of transposons, and epigenetic inheritance across generations. Small RNAs and inheritance of histone marks may also contribute to epigenetic inheritance and reprogramming. Reprogramming occurs in flowering plants and in mammals and the similarities and differences illuminate developmental and reproductive strategies.
doi:10.1126/science.1190614
PMCID: PMC2989926  PMID: 21030646
22.  Increased Placental Glucose Transport Rates in Pregnant Mice Carrying Fetuses with Targeted Disruption of Their Placental-Specific Igf2 Transcripts Are Not Associated with Raised Circulating Glucose Concentrations 
Experimental Diabetes Research  2011;2011:171376.
At the beginning of the third week of pregnancy, mouse fetuses with targeted disruption of their paternally-transmitted insulin-like growth factor 2 gene placental-specific transcripts have growth-restricted placentas but normal body weights due to upregulated placental nutrient transport. We assessed whether increased placental glucose transport rates were associated with raised maternal glucose concentrations by performing intraperitoneal glucose tolerance tests (ipGTT) in pregnant mice carrying knockout pups and comparing them with mice carrying genotype-matched phenotypically wild type pups. Mean ± SD body weights of affected pups were 95 ± 8% of control values at e16 and 73 ± 7% at e18. There were no differences in areas under the maternal ipGTT curves at either e16 (mean ± SD being 99.0 ± 9.1% of control values; P = .9) or e18 (91.4 ± 13.4%; P = .3), suggesting that effects on transplacental glucose transport in these mice are not mediated through changes in maternal glucose concentrations.
doi:10.1155/2011/171376
PMCID: PMC3038613  PMID: 21331382
23.  Raised Late Pregnancy Glucose Concentrations in Mice Carrying Pups With Targeted Disruption of H19Δ13 
Diabetes  2009;59(1):282-286.
OBJECTIVE
We have hypothesized that variation in imprinted growth-promoting fetal genes may affect maternal glucose concentrations in pregnancy. To test this hypothesis we evaluated the effects of fetal disruption of murine H19Δ13 on maternal glucose concentrations in pregnancy.
RESEARCH DESIGN AND METHODS
Experimental mice were pregnant females that had inherited the disrupted H19Δ13 from their fathers and were therefore phenotypically wild type due to imprinting; approximately half of their litters were null for H19Δ13 through maternal inheritance of the disrupted gene. In control mice approximately half the litter paternally inherited the disrupted H19Δ13, so the pups were either genetically wild type or phenotypically wild type due to imprinting. Blood glucose concentrations were assessed by intraperitoneal glucose tolerance tests on days 1, 16, and 18 of pregnancy.
RESULTS
There were no differences in the glucose concentrations of control and experimental pregnant mice at day 1. However, at day 16 mothers carrying H19Δ13-null pups had a significantly higher area under the glucose tolerance test curves than controls (1,845 ± 378 vs. 1,386 ± 107 mmol · min · l−1 [P = 0.01]) in association with increasing pregnancy-related insulin resistance. Although this difference lessened toward term, overall, mothers of maternally inherited H19Δ13 mutants had significantly higher glucose concentrations during the last trimester (1,602 ± 321 [n = 17] vs. 1,359 ± 147 [n = 18] mmol · min · l−1 [P = 0.009]).
CONCLUSIONS
This study provides evidence that maternal glucose concentrations in pregnant mice can be affected by targeted disruption of fetal H19Δ13. This implies that variable fetal IGF2 expression could affect risk for gestational diabetes.
doi:10.2337/db09-0757
PMCID: PMC2797934  PMID: 19794064
24.  Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by Aid deficiency 
Nature  2010;463(7284):1101-1105.
Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency1-9. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases Aid and Apobec1 can deaminate 5-methylcytosine in vitro and in E coli, and in the mouse are expressed in tissues in which demethylation occurs10. Here we profiled DNA methylation throughout the genome by unbiased bisulfite Next Generation Sequencing11-13 (BS-Seq) in wildtype and Aid deficient PGCs at E13.5. Wildtype PGCs revealed dramatic genome-wide erasure of methylation to a level below that of methylation deficient (Np95-/-) ES cells, with female PGCs being less methylated than male ones. By contrast, Aid deficient PGCs were up to three times more methylated than wildtype ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in Aid deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. Aid deficiency interferes with genome-wide erasure of DNA methylation patterns, suggesting that Aid has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.
doi:10.1038/nature08829
PMCID: PMC2965733  PMID: 20098412
25.  Retrotransposons and germ cells: reproduction, death, and diversity 
The evolutionary success of retrotransposable elements is reflected by their abundance in mammalian genomes. To restrict their further advance, a number of defence mechanisms have been put in place by the host. These seem to be particularly effective in the germ line while somatic lineages might be more permissive to new insertions, as recent work by Kano and colleagues suggests.
doi:10.3410/B2-44
PMCID: PMC2950035  PMID: 20948789

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