Publications by authors named "Ferdinand von Meyenn"

30 Publications

  • Page 1 of 1

Human adipose tissue-derived stem cell paracrine networks vary according metabolic risk and after TNFα-induced death: An analysis at the single-cell level.

Metabolism 2021 03 15;116:154466. Epub 2020 Dec 15.

Department of Endocrinology and Nutrition, Virgen de la Victoria Hospital (IBIMA), Malaga University, Malaga, Spain; Biomedical Research Networking Center for Physiopathology of Obesity and Nutrition (CIBEROBN), Institute of Health Carlos III (ISCIII), Malaga, Spain.

Objective: Adipose tissue-derived stem cells (ASCs) might play an important role in adipose microenvironment remodelling during tissue expansion through their response to hypoxia. We examined the cytokine profiles of hypoxic visceral ASCs (hypox-visASCs) from subjects with different metabolic risk, the interactions between cytokines as well as the impact of TNFα-induced death in the behavior of surviving hypoxic subcutaneous ASCs (hypox-subASCs) both at bulk population and single-cell level.

Materials/methods: Visceral adipose tissue was processed to isolate the ASCs from 33 subjects grouped into normal weight, obese with and without metabolic syndrome. Multiplex assay was used to simultaneously measure multiple inflammatory, anti-inflammatory and angiogenic cytokines in hypox-visASCs from these patients and to elucidate cytokine profiles of hypox-subASCs upon stimulation with IL1β or TNFα and after TNFα-induced death. qPCR and single-cell RNA-sequencing were also performed to elucidate transcriptional impact in surviving hypox-subASCs after TNFα-induced apoptosis.

Results: Hypox-visASCs from subjects without metabolic syndrome showed greater secretion levels of inflammatory, anti-inflammatory and angiogenic cytokines compared with those from patients with metabolic syndrome. While IL-1β stimulation was sufficient to increase the secretion levels of these cytokines in hypox-subASCs, TNFα-induced apoptosis also increased their levels and impacted on the expression levels of extracellular matrix proteins, acetyl-CoA producing enzymes and redox-balance proteins in surviving hypox-subASCs. TNFα-induced apoptosis under different glucose concentrations caused selective impoverishment of cell clusters and differentially influenced gene expression profiles of surviving hypox-subASCs.

Conclusions: Immunoregulatory and angiogenic functions of hypox-visASCs from patients with metabolic syndrome could be insufficient to promote healthy adipose tissue expansion. TNFα-induced apoptosis may impact on functionality of hypox-subASC populations, whose differential metabolic sensitivity to death could serve to manipulate individual populations selectively in order to elucidate their role in shaping adipose heterogeneity and treating metabolic disorders.
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http://dx.doi.org/10.1016/j.metabol.2020.154466DOI Listing
March 2021

Exercise promotes satellite cell contribution to myofibers in a load-dependent manner.

Skelet Muscle 2020 07 9;10(1):21. Epub 2020 Jul 9.

Department Health Sciences and Technology, Laboratory of Exercise and Health, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.

Background: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown.

Results: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise.

Conclusions: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.
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http://dx.doi.org/10.1186/s13395-020-00237-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346400PMC
July 2020

IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted regions.

Nucleic Acids Res 2020 09;48(16):e92

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

Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
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http://dx.doi.org/10.1093/nar/gkaa567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498334PMC
September 2020

Deletion of fibroblast activation protein provides atheroprotection.

Cardiovasc Res 2021 Mar;117(4):1060-1069

Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, CH-8952 Schlieren, Switzerland.

Aims: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice.

Methods And Results: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries.

Conclusions: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.
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http://dx.doi.org/10.1093/cvr/cvaa142DOI Listing
March 2021

Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions.

Nat Commun 2020 03 23;11(1):1528. Epub 2020 Mar 23.

The Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.
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http://dx.doi.org/10.1038/s41467-020-15271-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089967PMC
March 2020

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.

Stem Cell Reports 2019 12 7;13(6):1083-1098. Epub 2019 Nov 7.

Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address:

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2019.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915845PMC
December 2019

Environmental and Nutritional Effects Regulating Adipose Tissue Function and Metabolism Across Generations.

Adv Sci (Weinh) 2019 Jun 16;6(11):1900275. Epub 2019 Apr 16.

Department of Health Science and Technologies ETH Zürich Schorenstrasse 16 Schwerzenbach CH-8603 Switzerland.

The unabated rise in obesity prevalence during the last 40 years has spurred substantial interest in understanding the reasons for this epidemic. Studies in mice and humans have demonstrated that obesity is a highly heritable disease; however genetic variations within specific populations have so far not been able to explain this phenomenon to its full extent. Recent work has demonstrated that environmental cues can be sensed by an organism to elicit lasting changes, which in turn can affect systemic energy metabolism by different epigenetic mechanisms such as changes in small noncoding RNA expression, DNA methylation patterns, as well as histone modifications. These changes can directly modulate cellular function in response to environmental cues, however research during the last decade has demonstrated that some of these modifications might be transmitted to subsequent generations, thus modulating energy metabolism of the progeny in an inter- as well as transgenerational manner. In this context, adipose tissue has become a focus of research due to its plasticity, which allows the formation of energy storing (white) as well as energy wasting (brown/brite/beige) cells within the same depot. In this Review, the effects of environmental induced obesity with a particular focus on adipose tissue are discussed.
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http://dx.doi.org/10.1002/advs.201900275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548959PMC
June 2019

Defined conditions for propagation and manipulation of mouse embryonic stem cells.

Development 2019 03 26;146(6). Epub 2019 Mar 26.

Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK

The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.
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http://dx.doi.org/10.1242/dev.173146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451320PMC
March 2019

Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution.

Cell Rep 2019 01;26(4):815-824.e4

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK. Electronic address:

Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood. Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific. We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution.
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http://dx.doi.org/10.1016/j.celrep.2018.12.099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344340PMC
January 2019

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

PLoS Biol 2019 01 10;17(1):e3000107. Epub 2019 Jan 10.

Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
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http://dx.doi.org/10.1371/journal.pbio.3000107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343928PMC
January 2019

Publisher Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 Nov;24(11):1777

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

In the version of this article originally published, the bars in the mean temperature graph in Fig. 1a were incorrectly aligned. The left-most bar should have been aligned with the Apr label on the projected month of conception axis. The error has been corrected in the print, PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41591-018-0163-yDOI Listing
November 2018

Author Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 Nov;24(11):1776

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

In the version of this article originally published, the months on the axis labeled projected month of conception in Fig. 1a were out of order. April and March should have been the first and last months listed, respectively. The error has been corrected in the print, PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41591-018-0162-zDOI Listing
November 2018

Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 09 9;24(9):1372-1383. Epub 2018 Jul 9.

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.
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http://dx.doi.org/10.1038/s41591-018-0102-yDOI Listing
September 2018

An endosiRNA-Based Repression Mechanism Counteracts Transposon Activation during Global DNA Demethylation in Embryonic Stem Cells.

Cell Stem Cell 2017 Nov;21(5):694-703.e7

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; University of Cambridge, The Old Schools, Trinity Lane, Cambridge CB2 1TN, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. Electronic address:

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.
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http://dx.doi.org/10.1016/j.stem.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678422PMC
November 2017

A lncRNA fine tunes the dynamics of a cell state transition involving , and DNA methylation.

Elife 2017 08 18;6. Epub 2017 Aug 18.

Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) hereafter (), that modulates the dynamics of exit from naïve pluripotency. deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of , expression is reduced which results in persistence of the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. deletion retards ES cell transition, correlating with delayed promoter methylation and phenocopying loss of or . The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. illustrates how lncRNAs may introduce species-specific network modulations.
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http://dx.doi.org/10.7554/eLife.23468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562443PMC
August 2017

Epigenetic resetting of human pluripotency.

Development 2017 08;144(15):2748-2763

Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR, UK

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, -induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.
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http://dx.doi.org/10.1242/dev.146811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560041PMC
August 2017

Multi-tissue DNA methylation age predictor in mouse.

Genome Biol 2017 04 11;18(1):68. Epub 2017 Apr 11.

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

Background: DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse.

Results: We have generated a comprehensive set of genome-scale base-resolution methylation maps from multiple mouse tissues spanning a wide range of ages. Many CpG sites show significant tissue-independent correlations with age which allowed us to develop a multi-tissue predictor of age in the mouse. Our model, which estimates age based on DNA methylation at 329 unique CpG sites, has a median absolute error of 3.33 weeks and has similar properties to the recently described human epigenetic clock. Using publicly available datasets, we find that the mouse clock is accurate enough to measure effects on biological age, including in the context of interventions. While females and males show no significant differences in predicted DNA methylation age, ovariectomy results in significant age acceleration in females. Furthermore, we identify significant differences in age-acceleration dependent on the lipid content of the diet.

Conclusions: Here we identify and characterise an epigenetic predictor of age in mice, the mouse epigenetic clock. This clock will be instrumental for understanding the biology of ageing and will allow modulation of its ticking rate and resetting the clock in vivo to study the impact on biological age.
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http://dx.doi.org/10.1186/s13059-017-1203-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389178PMC
April 2017

DNA methylation homeostasis in human and mouse development.

Curr Opin Genet Dev 2017 Apr 2;43:101-109. Epub 2017 Mar 2.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. Electronic address:

The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals. An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species.
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http://dx.doi.org/10.1016/j.gde.2017.02.003DOI Listing
April 2017

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms.

Proc Natl Acad Sci U S A 2016 10 11;113(43):12202-12207. Epub 2016 Oct 11.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, United Kingdom; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.
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http://dx.doi.org/10.1073/pnas.1608679113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086989PMC
October 2016

Comparative Principles of DNA Methylation Reprogramming during Human and Mouse In Vitro Primordial Germ Cell Specification.

Dev Cell 2016 10;39(1):104-115

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. Electronic address:

Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster. The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline.
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http://dx.doi.org/10.1016/j.devcel.2016.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064768PMC
October 2016

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

Mol Cell 2016 06 26;62(6):848-861. Epub 2016 May 26.

Department of Molecular Biology, Faculty of Science, Radboud University, 6525GA Nijmegen, the Netherlands. Electronic address:

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.
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http://dx.doi.org/10.1016/j.molcel.2016.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914828PMC
June 2016

Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.

Stem Cell Reports 2016 Apr 3;6(4):437-446. Epub 2016 Mar 3.

Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 4BG, UK. Electronic address:

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.
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http://dx.doi.org/10.1016/j.stemcr.2016.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834040PMC
April 2016

Forget the Parents: Epigenetic Reprogramming in Human Germ Cells.

Cell 2015 Jun;161(6):1248-51

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

Epigenetic reprogramming in the germline resets genomic potential and erases epigenetic memory. Three studies by Gkountela et al., Guo et al., and Tang et al. analyze the transcriptional and epigenetic landscape of human primordial germ cells, revealing a unique transcriptional network and progressive and conserved global erasure of DNA methylation.
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http://dx.doi.org/10.1016/j.cell.2015.05.039DOI Listing
June 2015

The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes.

Nat Med 2015 Jun 18;21(6):619-27. Epub 2015 May 18.

1] Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Disease, Swiss Federal Institute of Technology, Zurich, Switzerland.

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.
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http://dx.doi.org/10.1038/nm.3862DOI Listing
June 2015

Glucagon-induced acetylation of Foxa2 regulates hepatic lipid metabolism.

Cell Metab 2013 Mar 14;17(3):436-47. Epub 2013 Feb 14.

Institute of Molecular Health Sciences, ETH Zurich, Schafmattstrasse 22, 8093 Zurich, Switzerland.

Circulating levels of insulin and glucagon reflect the nutritional state of animals and elicit regulatory responses in the liver that maintain glucose and lipid homeostasis. The transcription factor Foxa2 activates lipid metabolism and ketogenesis during fasting and is inhibited via insulin-PI3K-Akt signaling-mediated phosphorylation at Thr156 and nuclear exclusion. Here we show that, in addition, Foxa2 is acetylated at the conserved residue Lys259 following inhibition of histone deacetylases (HDACs) class I-III and the cofactors p300 and SirT1 are involved in Foxa2 acetylation and deacetylation, respectively. Physiologically, fasting states and glucagon stimulation are sufficient to induce Foxa2 acetylation. Introduction of the acetylation-mimicking (K259Q) or -deficient (K259R) mutations promotes or inhibits Foxa2 activity, respectively, and adenoviral expression of Foxa2-K259Q augments expression of genes involved in fatty acid oxidation and ketogenesis. Our study reveals a molecular mechanism by which glucagon signaling activates a fasting response through acetylation of Foxa2.
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http://dx.doi.org/10.1016/j.cmet.2013.01.014DOI Listing
March 2013

Regulation of adaptive behaviour during fasting by hypothalamic Foxa2.

Nature 2009 Dec;462(7273):646-50

The Rockefeller University, Laboratory of Metabolic Diseases, 1230 York Avenue, New York, New York 10021, USA.

The lateral hypothalamic area is considered the classic 'feeding centre', regulating food intake, arousal and motivated behaviour through the actions of orexin and melanin-concentrating hormone (MCH). These neuropeptides are inhibited in response to feeding-related signals and are released during fasting. However, the molecular mechanisms that regulate and integrate these signals remain poorly understood. Here we show that the forkhead box transcription factor Foxa2, a downstream target of insulin signalling, regulates the expression of orexin and MCH. During fasting, Foxa2 binds to MCH and orexin promoters and stimulates their expression. In fed and in hyperinsulinemic obese mice, insulin signalling leads to nuclear exclusion of Foxa2 and reduced expression of MCH and orexin. Constitutive activation of Foxa2 in the brain (Nes-Cre/+;Foxa2T156A(flox/flox) genotype) results in increased neuronal MCH and orexin expression and increased food consumption, metabolism and insulin sensitivity. Spontaneous physical activity of these animals in the fed state is significantly increased and is similar to that in fasted mice. Conditional activation of Foxa2 through the T156A mutation expression in the brain of obese mice also resulted in improved glucose homeostasis, decreased fat and increased lean body mass. Our results demonstrate that Foxa2 can act as a metabolic sensor in neurons of the lateral hypothalamic area to integrate metabolic signals, adaptive behaviour and physiological responses.
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http://dx.doi.org/10.1038/nature08589DOI Listing
December 2009

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells.

Immunobiology 2006 5;211(6-8):557-65. Epub 2006 Jul 5.

Institut für Medizinische Mikrobiologie, Immunologie and Hygiene, Technische Universität München, Trogerstrasse 30, 81675 München, Germany.

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFalpha, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFalpha and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.
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http://dx.doi.org/10.1016/j.imbio.2006.05.004DOI Listing
December 2006