Publications by authors named "Gavin Kelsey"

102 Publications

Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice.

Nat Commun 2022 May 5;13(1):2464. Epub 2022 May 5.

Lymphocyte Development & Epigenetic Memory Groups, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.
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http://dx.doi.org/10.1038/s41467-022-30022-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9072353PMC
May 2022

Single-cell Atlas of common variable immunodeficiency shows germinal center-associated epigenetic dysregulation in B-cell responses.

Nat Commun 2022 04 1;13(1):1779. Epub 2022 Apr 1.

Epigenetics and Immune Disease Group, Josep Carreras Research Institute (IJC), Badalona, Barcelona, Spain.

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.
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http://dx.doi.org/10.1038/s41467-022-29450-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8975885PMC
April 2022

Loss of histone methyltransferase SETD1B in oogenesis results in the redistribution of genomic histone 3 lysine 4 trimethylation.

Nucleic Acids Res 2022 02;50(4):1993-2004

Epigenetics Programme, Babraham Institute, Cambridge, UK.

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.
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http://dx.doi.org/10.1093/nar/gkac051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8887468PMC
February 2022

Sex-specific chromatin remodelling safeguards transcription in germ cells.

Nature 2021 12 8;600(7890):737-742. Epub 2021 Dec 8.

MRC London Institute of Medical Sciences (LMS), London, UK.

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.
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http://dx.doi.org/10.1038/s41586-021-04208-5DOI Listing
December 2021

Leptin Signaling in the Ovary of Diet-Induced Obese Mice Regulates Activation of NOD-Like Receptor Protein 3 Inflammasome.

Front Cell Dev Biol 2021 3;9:738731. Epub 2021 Nov 3.

Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland.

Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficient (+/? and -/-) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1β were upregulated in 16 L and downregulated in Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation in . We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.
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http://dx.doi.org/10.3389/fcell.2021.738731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8595835PMC
November 2021

DNA Methylation Dynamics in the Female Germline and Maternal-Effect Mutations That Disrupt Genomic Imprinting.

Genes (Basel) 2021 08 6;12(8). Epub 2021 Aug 6.

Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030, USA.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID.
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http://dx.doi.org/10.3390/genes12081214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394515PMC
August 2021

Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos.

Int J Mol Sci 2021 Jun 15;22(12). Epub 2021 Jun 15.

Physiology of Reproduction Group, Physiology Department, Mare Nostrum Campus, University of Murcia, 30100 Murcia, Spain.

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.
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http://dx.doi.org/10.3390/ijms22126426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232708PMC
June 2021

Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes.

Clin Epigenetics 2021 06 28;13(1):132. Epub 2021 Jun 28.

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

Background: Reproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.

Results: An in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O) and hypoxia (5% oxygen, 5% O). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.

Conclusions: Our observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.
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http://dx.doi.org/10.1186/s13148-021-01116-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240245PMC
June 2021

Features and mechanisms of canonical and noncanonical genomic imprinting.

Genes Dev 2021 06;35(11-12):821-834

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, United Kingdom.

Genomic imprinting is the monoallelic expression of a gene based on parent of origin and is a consequence of differential epigenetic marking between the male and female germlines. Canonically, genomic imprinting is mediated by allelic DNA methylation. However, recently it has been shown that maternal H3K27me3 can result in DNA methylation-independent imprinting, termed "noncanonical imprinting." In this review, we compare and contrast what is currently known about the underlying mechanisms, the role of endogenous retroviral elements, and the conservation of canonical and noncanonical genomic imprinting.
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http://dx.doi.org/10.1101/gad.348422.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168557PMC
June 2021

Revisiting the Impact of Local Leptin Signaling in Folliculogenesis and Oocyte Maturation in Obese Mothers.

Int J Mol Sci 2021 Apr 20;22(8). Epub 2021 Apr 20.

Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research of PAS, Tuwima 10, 10-748 Olsztyn, Poland.

The complex nature of folliculogenesis regulation accounts for its susceptibility to maternal physiological fitness. In obese mothers, progressive expansion of adipose tissue culminates with severe hyperestrogenism and hyperleptinemia with detrimental effects for ovarian performance. Indeed, maternal obesity is associated with the establishment of ovarian leptin resistance. This review summarizes current knowledge on potential effects of impaired leptin signaling throughout folliculogenesis and oocyte developmental competence in mice and women.
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http://dx.doi.org/10.3390/ijms22084270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074257PMC
April 2021

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

Clin Epigenetics 2021 02 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 01 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 03 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
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