scholarly journals Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

2013 ◽  
Vol 368 (1609) ◽  
pp. 20110330 ◽  
Author(s):  
Stefanie Seisenberger ◽  
Julian R. Peat ◽  
Timothy A. Hore ◽  
Fátima Santos ◽  
Wendy Dean ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Life Cycle ◽  
Cell Fate ◽  
Excision Repair ◽  
Primordial Germ Cells ◽  
Biological Significance ◽  
Epigenetic Reprogramming ◽  
Developmental Potential ◽  
Methylation Patterns

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro . Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

scholarly journals Specification and epigenetic resetting of the pig germline exhibit conservation with the human lineage

2020 ◽  
Author(s):  
Qifan Zhu ◽  
Fei Sang ◽  
Sarah Withey ◽  
Walfred Tang ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Large Scale ◽  
Excision Repair ◽  
Primordial Germ Cells ◽  
Epigenetic Inheritance ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Preimplantation Embryos ◽  
Gene Coding ◽  
Base Excision

SummaryInvestigations on the human germline and programming are challenging due to limited access to embryonic material. However, the pig as a model may provide insight on transcriptional network and epigenetic reprogramming applicable to both species. Here we show that during the pre- and early migratory stages pig primordial germ cells (PGCs) initiate large-scale epigenetic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and potentially base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3, as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.

scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

scholarly journals Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

2020 ◽  
Vol 21 (20) ◽  
pp. 7459
Author(s):  
María Elena González-Benito ◽  
Miguel Ángel Ibáñez ◽  
Michela Pirredda ◽  
Sara Mira ◽  
Carmen Martín
Keyword(s):  
Dna Methylation ◽  
In Situ Conservation ◽  
Plant Conservation ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Regenerated Plants ◽  
Before And After ◽  
Methylation Patterns

Epigenetic variation, and particularly DNA methylation, is involved in plasticity and responses to changes in the environment. Conservation biology studies have focused on the measurement of this variation to establish demographic parameters, diversity levels and population structure to design the appropriate conservation strategies. However, in ex situ conservation approaches, the main objective is to guarantee the characteristics of the conserved material (phenotype and epi-genetic). We review the use of the Methylation Sensitive Amplified Polymorphism (MSAP) technique to detect changes in the DNA methylation patterns of plant material conserved by the main ex situ plant conservation methods: seed banks, in vitro slow growth and cryopreservation. Comparison of DNA methylation patterns before and after conservation is a useful tool to check the fidelity of the regenerated plants, and, at the same time, may be related with other genetic variations that might appear during the conservation process (i.e., somaclonal variation). Analyses of MSAP profiles can be useful in the management of ex situ plant conservation but differs in the approach used in the in situ conservation. Likewise, an easy-to-use methodology is necessary for a rapid interpretation of data, in order to be readily implemented by conservation managers.

53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 132
Author(s):  
V. A. Michalczechen-Lacerda ◽  
F. C. Rodrigues ◽  
R. V. de Sousa ◽  
R. Rumpf ◽  
M. M. Franco
Keyword(s):  
Dna Methylation ◽  
Cell Viability ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Imprinted Genes ◽  
Demethylating Agent ◽  
Chromosome Integrity ◽  
Methylation Patterns ◽  
Number Of Cells

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

1 INSULIN TREATMENT DURING IN VITRO OOCYTE MATURATION LEADS TO DIFFERENT GENE EXPRESSION AND METHYLATION PATTERNS OF KEY GENES ASSOCIATED WITH METABOLISM AND STEROID SYNTHESIS IN THE BOVINE BLASTOCYST

2017 ◽  
Vol 29 (1) ◽  
pp. 108
Author(s):  
D. Laskowski ◽  
P. Humblot ◽  
M. A. Sirard ◽  
Y. Sjunnesson ◽  
G. Andersson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Steroid Synthesis ◽  
Developmental Potential ◽  
Increased Risk ◽  
In Vitro Oocyte Maturation ◽  
Methylation Patterns ◽  
And Oxidative Stress

Obesity and overfeeding are common causes for female infertility, leading to insulin resistance and hyperinsulinemia and associated with an increased risk for type 2 diabetes mellitus (Pasquali et al., http://dx.doi.org/10.1093/humupd/dmg024). We investigated here the effect of insulin during in vitro oocyte maturation on methylation changes in bovine Day 8 blastocysts (BC8) and focused on methylation patterns of candidate genes associated with metabolism and steroidogenesis (Day 0 = day of oocyte collection). Abattoir-derived oocytes (n = 882) were in vitro matured for 22 h with 2 different insulin concentrations, INS10 (10 µg mL−1) and INS0.1 (0.1 µg mL−1) or without insulin (INS0, control). Subsequently, IVF and IVC were performed to equal standardized conditions for all groups. Parallel genomic DNA and total RNA extraction (AllPrepDNA/RNA micro kit, cat no. 80284, Qiagen®, Valencia, CA, USA) from pools of 10 frozen (−80°C) BC8 was followed by transcriptome and epigenome analysis (Laskowski et al., http://dx.doi.org/10.1071/RD15315). An empirical Bayes moderated t-test and the ‘limma’ package in R (www.r-project.org) were used to search for differentially expressed genes between the control and the insulin groups. Analysis of the epigenome by using a specific pipeline, described by Shojaei Saadi et al. (2014 BMC Genomics 15, 451), showed that 7632 and 3914 regions were hypomethylated in the INS0.1 and INS10 v. INS0, whereas 6026 and 8504 regions were hypermethylated in INS0.1 and INS10 v. INS0. Combining epigenetic and transcriptomic data, we found that high methylation and low expression or the reverse (low methylation and high expression) were observed for a set of 14 and 11 genes for INS0.1 and INS10 respectively. Most of these genes are associated with lipid metabolism, steroid synthesis, and oxidative stress. Further investigation of the localization of differentially methylated regions (DMR) in genes showed that the conservation odds (methylation) was in general higher in coding regions and CpG islands than in noncoding regions. We observed a large overlap of DMR in the 2 insulin groups compared with controls (3233 common DMR). These numerous changes illustrate the potential unfavourable effects of elevated insulin during maturation leading to alteration of the methylation patterns of the early embryo. This model may help us better understand the mechanisms by which metabolic disorders observed pre-conception can affect embryonic development and subsequent health of the offspring. Our results based on changes in transcriptome or epigenome did show that insulin challenge during maturation leads to postponed effects associated with steroidogenesis, lipid metabolism and oxidative stress in the BC8. By this early stage, if persistent, specific changes in the expression and methylation patterns of genes associated to hyperinsulinemia may decrease the developmental potential of early embryos or could be responsible for subsequent pathologies. This study was funded by FORMAS.

DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 138-145 ◽  
Author(s):  
Ying Liang ◽  
Xiang-Wei Fu ◽  
Jun-Jie Li ◽  
Dian-Shuai Yuan ◽  
Shi-En Zhu
Keyword(s):  
Dna Methylation ◽  
Fluorescein Isothiocyanate ◽  
Methylation Pattern ◽  
Oocyte Vitrification ◽  
Developmental Potential ◽  
Mouse Oocytes ◽  
Early Embryos ◽  
Vitrification Solution ◽  
Early Mouse

SummaryThis study was conducted to investigate the pattern of DNA methylation in vitrified–thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified–thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos.

scholarly journals Influence of Mouse-Strain-Specific Factors on Position-Dependent Transgene DNA Methylation Patterns

1996 ◽  
Vol 45 (1-2) ◽  
pp. 243-244
Author(s):  
P.A. Koetsier ◽  
W. Doerfler
Keyword(s):  
Dna Methylation ◽  
Genetic Background ◽  
Promoter Activity ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Adenovirus Type ◽  
Methylation Pattern ◽  
Parent Of Origin ◽  
Methylation Patterns

In previous work from this laboratory, an inverse dependence was established for the adenovirus type 2 E2A late promoter between sequence-specific DNA methylation and promoter activity [1-5; for reviews see ref. 6, 7]. The effect of DNA methylation on promoter activity was also assessed in the transgenic mice, which were obtained from microinjections of unmethylated or in vitro HpaII-premethylated pAd2E2AL-CAT DNA [1] into F2 zygotes from B6D2F, (C57BL/6 × DBA/2) hybrid mice. In CAT assays carried out on organ extracts from the pAd2E2AL-CAT mice, the inverse relationship was confirmed [2].We studied the stability of the pAd2E2AL-CAT DNA methylation patterns in up to eight mouse generations and assessed the influence of the strain-specific genetic background. Three pAd2E2AL-CAT mouse lines were crossed with inbred DBA/2, C57BL/6 or B6D2F, mice. Parent-of-origin effects were controlled by exclusive hemizygous transgene transmission either via females or males. The founder animal of line 7-1 carried two groups of transgenes (A and B) on separate chromosomes. The transgene methylation patterns of the 7-1B transgenes and those of the lines 5-8 and 8-1 were stably transmitted.Southern blot hybridization experiments [8, 9] revealed that the 7-1A transgene methylation pattern was a cellular mosaic. In mixed-genetic-background offspring from 7-1A animals, 10% carried transgenes with HpaII-DNA methylation levels that were reduced from 40 to 10-15%. This finding suggested that in this background the factors that supported high methylation levels were dominant. When inbred DBA/2 mice were the mates, 40% of the siblings carried demethylated transgenes, whereas this ratio amounted to only 10% in C57BL/6 offspring (comparable to B6D2F1 crossings). Transgene methylation patterns were not detectably influenced by the parent-of-origin.

scholarly journals Locus-specific analysis of DNA methylation patterns in cloned and in vitro fertilized porcine embryos

2020 ◽  
Vol 66 (6) ◽  
pp. 505-514
Author(s):  
Weihua XU ◽  
Hongyi LI ◽  
Mao ZHANG ◽  
Junsong SHI ◽  
Zhengchao WANG
Keyword(s):  
Dna Methylation ◽  
Specific Analysis ◽  
Methylation Patterns

scholarly journals Global Modulation in DNA Epigenetics during Pro-Inflammatory Macrophage Activation

2019 ◽  
Author(s):  
Nikhil Jain ◽  
Tamar Shahal ◽  
Tslil Gabrieli ◽  
Noa Gilat ◽  
Dmitry Torchinsky ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Single Molecule ◽  
Transcriptional Control ◽  
Excision Repair ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Inflammatory Activation ◽  
Methylation Patterns

AbstractDNA methylation patterns create distinct gene expression profiles. These patterns are maintained after cell division, thus enabling the differentiation and maintenance of multiple cell types from the same genome sequence. The advantage of this mechanism for transcriptional control is that chemical-encoding allows to rapidly establish new epigenetic patterns “on-demand” through enzymatic methylation and de-methylation of DNA. Here we show that this feature is associated with the fast response of macrophages during their pro-inflammatory activation. By using a combination of mass spectroscopy and single-molecule imaging to quantify global epigenetic changes in the genomes of primary macrophages, we followed three distinct DNA marks (methylated, hydroxymethylated and unmethylated), involved in establishing new DNA methylation patterns during pro-inflammatory activation. The observed epigenetic modulation together with gene expression data generated for the involved enzymatic machinery, may suggest that de-methylation upon LPS-activation starts with oxidation of methylated CpGs, followed by excision-repair of these oxidized bases and their replacement with unmodified cytosine.

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