Publications by authors named "Gavin Kelsey"

92 Publications

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline.

Clin Epigenetics 2021 Feb 3;13(1):27. Epub 2021 Feb 3.

Department of Animal Reproduction, INIA, Madrid, Spain.

Background: Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming.

Results: Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage.

Conclusions: Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.
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http://dx.doi.org/10.1186/s13148-021-01003-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860200PMC
February 2021

Cell-cell coupling and DNA methylation abnormal phenotypes in the after-hours mice.

Epigenetics Chromatin 2021 Jan 6;14(1). Epub 2021 Jan 6.

Genetics and Epigenetics of Behaviour (GEB) Laboratory, Istituto Italiano Di Tecnologia, via Morego, 30, 16163, Genova, Italy.

Background: DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period.

Methods: In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression.

Results: We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3.

Conclusions: The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.
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http://dx.doi.org/10.1186/s13072-020-00373-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789812PMC
January 2021

Identification of a unique epigenetic profile in women with diminished ovarian reserve.

Fertil Steril 2021 Mar 4;115(3):732-741. Epub 2020 Dec 4.

Department of Obstetrics and Gynaecology, Department of Reproductive Medicine, Hospital Herlev, Copenhagen University, Copenhagen, Denmark.

Objective: To investigate whether epigenetic profiles of mural granulosa cells (MGC) and leukocytes from women with diminished ovarian reserve (DOR) differ from those of women with normal or high ovarian reserve.

Design: Prospectively collected material from a multicenter cohort of women undergoing fertility treatment.

Setting: Private and university-based facilities for clinical services and research.

Patient(s): One hundred and nineteen women of various ages and ovarian reserve status (antimüllerian hormone level) who provided blood samples and MGC.

Intervention(s): None.

Main Outcome Measure(s): Measures of epigenetic aging rates from whole-genome methylation array data: DNA methylation variability, age acceleration, DNA methylation telomere length estimator (DNAmTL), and accumulation of epimutations.

Result(s): Comparison of DOR or high ovarian reserve samples to controls (normal ovarian reserve) showed differential methylation variability between DOR and normal samples at 4,199 CpGs in MGC, and 447 between high and normal (false-discovery rate < 0.05). Variable sites in MGC from DOR were enriched in regions marked with the repressive histone modification H3K27me3, and also included genes involved in folliculogenesis, such as insulin growth factor 2 (IGF2) and antimüllerian hormone (AMH). Regardless of ovarian reserve, very few signals were detected in leukocytes, and no overlaps with those in MGC were found. Furthermore, we found a higher number of epimutations in MGC from women with DOR (Kruskal-Wallis test, difference in mean = 3,485).

Conclusion(s): The somatic cells of human ovarian follicles have a distinctive epigenetic profile in women with DOR. A high frequency of epimutations suggests premature aging. Ovarian reserve status was not reflected in the leukocyte epigenetic profile.
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http://dx.doi.org/10.1016/j.fertnstert.2020.09.009DOI Listing
March 2021

Increased transcriptome variation and localised DNA methylation changes in oocytes from aged mice revealed by parallel single-cell analysis.

Aging Cell 2020 12 17;19(12):e13278. Epub 2020 Nov 17.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub-populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young-like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post-transcriptional basis for most age-related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age-related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age-related phenotypes in offspring.
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http://dx.doi.org/10.1111/acel.13278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744954PMC
December 2020

The role of ZFP57 and additional KRAB-zinc finger proteins in the maintenance of human imprinted methylation and multi-locus imprinting disturbances.

Nucleic Acids Res 2020 11;48(20):11394-11407

Imprinting and Cancer group, Bellvitge Institute for Biomedical Research, Gran via, L'Hospitalet de Llobregat, Barcelona, Spain.

Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation that is resistant to embryonic reprogramming, resulting in parental origin-specific monoallelic gene expression. A subset of individuals affected by imprinting disorders (IDs) displays multi-locus imprinting disturbances (MLID), which may result from aberrant establishment of imprinted differentially methylated regions (DMRs) in gametes or their maintenance in early embryogenesis. Here we investigated the extent of MLID in a family harbouring a ZFP57 truncating variant and characterize the interactions between human ZFP57 and the KAP1 co-repressor complex. By ectopically targeting ZFP57 to reprogrammed loci in mouse embryos using a dCas9 approach, we confirm that ZFP57 recruitment is sufficient to protect oocyte-derived methylation from reprogramming. Expression profiling in human pre-implantation embryos and oocytes reveals that unlike in mice, ZFP57 is only expressed following embryonic-genome activation, implying that other KRAB-zinc finger proteins (KZNFs) recruit KAP1 prior to blastocyst formation. Furthermore, we uncover ZNF202 and ZNF445 as additional KZNFs likely to recruit KAP1 to imprinted loci during reprogramming in the absence of ZFP57. Together, these data confirm the perplexing link between KZFPs and imprint maintenance and highlight the differences between mouse and humans in this respect.
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http://dx.doi.org/10.1093/nar/gkaa837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672439PMC
November 2020

Landscape of Genome-Wide DNA Methylation of Colorectal Cancer Metastasis.

Cancers (Basel) 2020 Sep 22;12(9). Epub 2020 Sep 22.

Laboratory of Integrative Biology (LIBi), Centro de Excelencia en Medicina Traslacional (CEMT), Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4810296, Chile.

Colorectal cancer is a heterogeneous disease caused by both genetic and epigenetics factors. Analysing DNA methylation changes occurring during colorectal cancer progression and metastasis formation is crucial for the identification of novel epigenetic markers of patient prognosis. Genome-wide methylation sequencing of paired samples of colon (normal adjacent, primary tumour and lymph node metastasis) showed global hypomethylation and CpG island (CGI) hypermethylation of primary tumours compared to normal. In metastasis we observed high global and non-CGI regions methylation, but lower CGI methylation, compared to primary tumours. Gene ontology analysis showed shared biological processes between hypermethylated CGIs in metastasis and primary tumours. After complementary analysis with The Cancer Genome Atlas (TCGA) cohort, , , , and genes were found associated with poor survival. We mapped the methylation landscape of colon normal tissues, primary tumours and lymph node metastasis, being capable of identified methylation changes throughout the genome. Furthermore, we found five genes with potential for methylation biomarkers of poor prognosis in colorectal cancer patients.
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http://dx.doi.org/10.3390/cancers12092710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564781PMC
September 2020

Profiling DNA Methylation Genome-Wide in Single Cells.

Methods Mol Biol 2021 ;2214:221-240

Epigenetics Programme, The Babraham Institute, Cambridge, UK.

Single-cell bisulfite sequencing (scBS-seq) enables profiling of DNA methylation at single-nucleotide resolution and across all genomic features. It can explore methylation differences between cells in mixed cell populations and profile methylation in very rare cell types, such as mammalian oocytes and cells from early embryos. Here, we outline the scBS-seq protocol in a 96-well plate format applicable to studies of moderate throughput.
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http://dx.doi.org/10.1007/978-1-0716-0958-3_15DOI Listing
March 2021

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

Int J Mol Sci 2020 Aug 25;21(17). Epub 2020 Aug 25.

Section of Human Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and β-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.
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http://dx.doi.org/10.3390/ijms21176128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504606PMC
August 2020

Imprints in the history of epigenetics.

Authors:
Gavin Kelsey

Nat Rev Mol Cell Biol 2020 10;21(10):566-567

Epigenetics Programme, The Babraham Institute, Cambridge, UK.

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http://dx.doi.org/10.1038/s41580-020-00289-8DOI Listing
October 2020

Correction to: DNA methylation changes during preimplantation development reveal interspecies differences and reprogramming events at imprinted genes.

Clin Epigenetics 2020 Jun 29;12(1):96. Epub 2020 Jun 29.

Physiology of Reproduction Group, Departamento de Fisiología, Universidad de Murcia, Campus Mare Nostrum, 30100, Murcia, Spain.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s13148-020-00887-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322833PMC
June 2020

LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function.

BMC Genomics 2020 Jun 3;21(1):385. Epub 2020 Jun 3.

Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, 750 07, Sweden.

Background: Lipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 μg/mL LPS for 24 h.

Results: DNA samples extracted at 0 h and 24 h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24 h to time 0 h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24 h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r = - 0.41 for gene promoter, r = - 0.22 for gene body regions, p < 0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17.

Conclusions: The present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.
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http://dx.doi.org/10.1186/s12864-020-06777-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268755PMC
June 2020

DNA methylation changes during preimplantation development reveal inter-species differences and reprogramming events at imprinted genes.

Clin Epigenetics 2020 05 11;12(1):64. Epub 2020 May 11.

Physiology of Reproduction Group, Departamento de Fisiología, Universidad de Murcia, Campus Mare Nostrum, 30100, Murcia, Spain.

Preimplantation embryos experience profound resetting of epigenetic information inherited from the gametes. Genome-wide analysis at single-base resolution has shown similarities but also species differences between human and mouse preimplantation embryos in DNA methylation patterns and reprogramming. Here, we have extended such analysis to two key livestock species, the pig and the cow. We generated genome-wide DNA methylation and whole-transcriptome datasets from gametes to blastocysts in both species. In oocytes from both species, a distinctive bimodal methylation landscape is present, with hypermethylated domains prevalent over hypomethylated domains, similar to human, while in the mouse the proportions are reversed.An oocyte-like pattern of methylation persists in the cleavage stages, albeit with some reduction in methylation level, persisting to blastocysts in cow, while pig blastocysts have a highly hypomethylated landscape. In the pig, there was evidence of transient de novo methylation at the 8-16 cell stages of domains unmethylated in oocytes, revealing a complex dynamic of methylation reprogramming. The methylation datasets were used to identify germline differentially methylated regions (gDMRs) of known imprinted genes and for the basis of detection of novel imprinted loci. Strikingly in the pig, we detected a consistent reduction in gDMR methylation at the 8-16 cell stages, followed by recovery to the blastocyst stage, suggesting an active period of imprint stabilization in preimplantation embryos. Transcriptome analysis revealed absence of expression in oocytes of both species of ZFP57, a key factor in the mouse for gDMR methylation maintenance, but presence of the alternative imprint regulator ZNF445. In conclusion, our study reveals species differences in DNA methylation reprogramming and suggests that porcine or bovine models may be closer to human in key aspects than in the mouse model.
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http://dx.doi.org/10.1186/s13148-020-00857-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216732PMC
May 2020

Leptin Resistance in the Ovary of Obese Mice is Associated with Profound Changes in the Transcriptome of Cumulus Cells.

Cell Physiol Biochem 2020 Apr;54(3):417-437

Institute of Animal Reproduction and Food Research of PAS, Department of Reproductive Immunology and Pathology, Olsztyn, Poland,

Background/aims: Obesity is associated with infertility, decreased ovarian performance and lipotoxicity. However, little is known about the aetiology of these reproductive impairments. Here, we hypothesise that the majority of changes in ovarian physiology in diet-induced obesity (DIO) are a consequence of transcriptional changes downstream of altered leptin signalling. Therefore, we investigated the extent to which leptin signalling is altered in the ovary upon obesity with particular emphasis on effects on cumulus cells (CCs), the intimate functional companions of the oocyte. Furthermore, we used the pharmacological hyperleptinemic (LEPT) mouse model to compare transcriptional profiles to DIO.

Methods: Mice were subjected to DIO for 4 and 16 weeks (wk) and leptin treatment for 16 days, to study effects in the ovary in components of leptin signalling at the transcript and protein levels, using Western blot, Real-time PCR and immunostaining. Furthermore, we used low-cell RNA sequencing to characterise changes in the transcriptome of CCs in these models.

Results: In the DIO model, obesity led to establishment of ovarian leptin resistance after 16 wk high fat diet (HFD), as evidenced by increases in the feedback regulator suppressor of cytokine signalling 3 (SOCS3) and decreases in the positive effectors phosphorylation of tyrosine 985 of leptin receptor (ObRb-pTyr985) and Janus kinase 2 (pJAK2). Transcriptome analysis of the CCs revealed a complex response to DIO, with large numbers and distinct sets of genes deregulated at early and late stages of obesity; in addition, there was a striking correlation between body weight and global transcriptome profile of CCs. Further analysis indicated that the transcriptome profile in 4 wk HFD CCs resembled that of LEPT CCs, in the upregulation of cellular trafficking and impairment in cytoskeleton organisation. Conversely, after 16 wk HFD CCs showed expression changes indicative of augmented inflammatory responses, cell morphogenesis, and decreased metabolism and transport, mainly as a consequence of the physiological changes of obesity.

Conclusion: Obesity leads to ovarian leptin resistance and major time-dependent changes in gene expression in CCs, which in early obesity may be caused by increased leptin signalling in the ovary, whereas in late obesity are likely to be a consequence of metabolic changes taking place in the obese mother.
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http://dx.doi.org/10.33594/000000228DOI Listing
April 2020

The enigma of DNA methylation in the mammalian oocyte.

F1000Res 2020 25;9. Epub 2020 Feb 25.

Epigenetics Programme, The Babraham Institute, Cambridge, UK.

The mammalian genome experiences profound setting and resetting of epigenetic patterns during the life-course. This is understood best for DNA methylation: the specification of germ cells, gametogenesis, and early embryo development are characterised by phases of widespread erasure and rewriting of methylation. While mitigating against intergenerational transmission of epigenetic information, these processes must also ensure correct genomic imprinting that depends on faithful and long-term memory of gamete-derived methylation states in the next generation. This underscores the importance of understanding the mechanisms of methylation programming in the germline. methylation in the oocyte is of particular interest because of its intimate association with transcription, which results in a bimodal methylome unique amongst mammalian cells. Moreover, this methylation landscape is entirely set up in a non-dividing cell, making the oocyte a fascinating model system in which to explore mechanistic determinants of methylation. Here, we summarise current knowledge on the oocyte DNA methylome and how it is established, focussing on recent insights from knockout models in the mouse that explore the interplay between methylation and chromatin states. We also highlight some remaining paradoxes and enigmas, in particular the involvement of non-nuclear factors for correct methylation.
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http://dx.doi.org/10.12688/f1000research.21513.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043116PMC
August 2020

Correction to: Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity.

Clin Epigenetics 2020 Jan 27;12(1):18. Epub 2020 Jan 27.

Follicle Biology Laboratory (FOBI), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan, Brussels, Belgium.

After publication of the original article [1], we were notified that.
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http://dx.doi.org/10.1186/s13148-020-0812-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983974PMC
January 2020

The genetic landscape of Arab Population, Chechens and Circassians subpopulations from Jordan through HV1 and HV2 regions of mtDNA.

Gene 2020 Mar 26;729:144314. Epub 2019 Dec 26.

Department of Biology and Biotechnology, The Hashemite University, Zarqa 13133, Jordan; Radcliffe Institute for Advanced Studies, Harvard University, Cambridge, MA 02138, USA; Jepson School of Leadership, Richmond University, 221 Richmond Way, Richmond, VA 23173, USA.

Mitochondrial DNA (mtDNA) is widely used in several fields including medical genetics, forensic science, genetic genealogy, and evolutionary anthropology. In this study, mtDNA haplotype diversity was determined for 293 unrelated subjects from Jordanian population (Circassians, Chechens, and the original inhabitants of Jordan). A total of 102 haplotypes were identified and analyzed among the populations to describe the maternal lineage landscape. Our results revealed that the distribution of mtDNA haplotype frequencies among the three populations showed disparity and significant differences when compared to each other. We also constructed mitochondrial haplotype classification trees for the three populations to determine the phylogenetic relationship of mtDNA haplotype variants, and we observed clear differences in the distribution of maternal genetic ancestries, especially between Arab and the minority ethnic populations. To our knowledge, this study is the first, to date, to characterize mitochondrial haplotypes and haplotype distributions in a population-based sample from the Jordanian population. It provides a powerful reference for future studies investigating the contribution of mtDNA variation to human health and disease and studying population history and evolution by comparing the mtDNA haplotypes to other populations.
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http://dx.doi.org/10.1016/j.gene.2019.144314DOI Listing
March 2020

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity.

Clin Epigenetics 2019 12 19;11(1):197. Epub 2019 Dec 19.

Follicle Biology Laboratory (FOBI), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan, Brussels, Belgium.

Background: In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).

Results: In this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development.

Conclusions: Our observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.
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http://dx.doi.org/10.1186/s13148-019-0794-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923880PMC
December 2019

A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation.

Genome Med 2019 12 17;11(1):84. Epub 2019 Dec 17.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Background: Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address.

Methods: Single-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed.

Results: High-coverage scBS-seq libraries were obtained from five KHDC3L oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation.

Conclusions: We show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches.
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http://dx.doi.org/10.1186/s13073-019-0694-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918611PMC
December 2019

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Nature 2019 12 11;576(7787):487-491. Epub 2019 Dec 11.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
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http://dx.doi.org/10.1038/s41586-019-1825-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924995PMC
December 2019

The role and mechanisms of DNA methylation in the oocyte.

Essays Biochem 2019 12;63(6):691-705

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, U.K.

Epigenetic information in the mammalian oocyte has the potential to be transmitted to the next generation and influence gene expression; this occurs naturally in the case of imprinted genes. Therefore, it is important to understand how epigenetic information is patterned during oocyte development and growth. Here, we review the current state of knowledge of de novo DNA methylation mechanisms in the oocyte: how a distinctive gene-body methylation pattern is created, and the extent to which the DNA methylation machinery reads chromatin states. Recent epigenomic studies building on advances in ultra-low input chromatin profiling methods, coupled with genetic studies, have started to allow a detailed interrogation of the interplay between DNA methylation establishment and chromatin states; however, a full mechanistic description awaits.
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http://dx.doi.org/10.1042/EBC20190043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923320PMC
December 2019

Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues.

Genome Biol 2019 10 29;20(1):225. Epub 2019 Oct 29.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Background: Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored.

Results: We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast.

Conclusions: This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements.
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http://dx.doi.org/10.1186/s13059-019-1833-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819472PMC
October 2019

A DNMT3A PWWP mutation leads to methylation of bivalent chromatin and growth retardation in mice.

Nat Commun 2019 04 23;10(1):1884. Epub 2019 Apr 23.

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

DNA methyltransferases (DNMTs) deposit DNA methylation, which regulates gene expression and is essential for mammalian development. Histone post-translational modifications modulate the recruitment and activity of DNMTs. The PWWP domains of DNMT3A and DNMT3B are posited to interact with histone 3 lysine 36 trimethylation (H3K36me3); however, the functionality of this interaction for DNMT3A remains untested in vivo. Here we present a mouse model carrying a D329A point mutation in the DNMT3A PWWP domain. The mutation causes dominant postnatal growth retardation. At the molecular level, it results in progressive DNA hypermethylation across domains marked by H3K27me3 and bivalent chromatin, and de-repression of developmental regulatory genes in adult hypothalamus. Evaluation of non-CpG methylation, a marker of de novo methylation, further demonstrates the altered recruitment and activity of DNMT3A at bivalent domains. This work provides key molecular insights into the function of the DNMT3A-PWWP domain and role of DNMT3A in regulating postnatal growth.
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http://dx.doi.org/10.1038/s41467-019-09713-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478690PMC
April 2019

Genomic Imprinting and Physiological Processes in Mammals.

Cell 2019 02;176(5):952-965

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.
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http://dx.doi.org/10.1016/j.cell.2019.01.043DOI Listing
February 2019

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

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

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

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

Epigenetic regulation in development: is the mouse a good model for the human?

Hum Reprod Update 2018 09;24(5):556-576

Epigenetics programme, Babraham Institute, Cambridge, UK.

Background: Over the past few years, advances in molecular technologies have allowed unprecedented mapping of epigenetic modifications in gametes and during early embryonic development. This work is allowing a detailed genomic analysis, which for the first time can answer long-standing questions about epigenetic regulation and reprogramming, and highlights differences between mouse and human, the implications of which are only beginning to be explored.

Objective And Rationale: In this review, we summarise new low-cell molecular methods enabling the interrogation of epigenetic information in gametes and early embryos, the mechanistic insights these have provided, and contrast the findings in mouse and human.

Search Methods: Relevant studies were identified by PubMed search.

Outcomes: We discuss the levels of epigenetic regulation, from DNA modifications to chromatin organisation, during mouse gametogenesis, fertilisation and pre- and post-implantation development. The recently characterised features of the oocyte epigenome highlight its exceptionally unique regulatory landscape. The chromatin organisation and epigenetic landscape of both gametic genomes are rapidly reprogrammed after fertilisation. This extensive epigenetic remodelling is necessary for zygotic genome activation, but the mechanistic link remains unclear. While the vast majority of epigenetic information from the gametes is erased in pre-implantation development, new insights suggest that repressive histone modifications from the oocyte may mediate a novel mechanism of imprinting. To date, the characterisation of epigenetics in human development has been almost exclusively limited to DNA methylation profiling; these data reinforce that the global dynamics are conserved between mouse and human. However, as we look closer, it is becoming apparent that the mechanisms regulating these dynamics are distinct. These early findings emphasise the importance of investigations of fundamental epigenetic mechanisms in both mouse and humans.

Wider Implications: Failures in epigenetic regulation have been implicated in human disease and infertility. With increasing maternal age and use of reproductive technologies in countries all over the world, it is becoming ever more important to understand the necessary processes required to establish a developmentally competent embryo. Furthermore, it is essential to evaluate the extent to which these epigenetic patterns are sensitive to such technologies and other adverse environmental exposures.
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http://dx.doi.org/10.1093/humupd/dmy021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093373PMC
September 2018

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

Nat Commun 2018 02 22;9(1):781. Epub 2018 Feb 22.

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

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.
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http://dx.doi.org/10.1038/s41467-018-03149-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823944PMC
February 2018

MLL2 conveys transcription-independent H3K4 trimethylation in oocytes.

Nat Struct Mol Biol 2018 01 1;25(1):73-82. Epub 2018 Jan 1.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.
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http://dx.doi.org/10.1038/s41594-017-0013-5DOI Listing
January 2018

Cultured bovine embryo biopsy conserves methylation marks from original embryo.

Biol Reprod 2017 Aug;97(2):189-196

Departamento de Reproducción Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.
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http://dx.doi.org/10.1093/biolre/iox077DOI Listing
August 2017

Single-cell epigenomics: Recording the past and predicting the future.

Science 2017 10;358(6359):69-75

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

Single-cell multi-omics has recently emerged as a powerful technology by which different layers of genomic output-and hence cell identity and function-can be recorded simultaneously. Integrating various components of the epigenome into multi-omics measurements allows for studying cellular heterogeneity at different time scales and for discovering new layers of molecular connectivity between the genome and its functional output. Measurements that are increasingly available range from those that identify transcription factor occupancy and initiation of transcription to long-lasting and heritable epigenetic marks such as DNA methylation. Together with techniques in which cell lineage is recorded, this multilayered information will provide insights into a cell's past history and its future potential. This will allow new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease.
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http://dx.doi.org/10.1126/science.aan6826DOI Listing
October 2017

DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).

Bioessays 2017 11 21;39(11). Epub 2017 Sep 21.

Physiology of Reproduction Group, University of Murcia, Murcia, Spain.

DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children.
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http://dx.doi.org/10.1002/bies.201700106DOI Listing
November 2017