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 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.

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 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 Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences in gut and papOperons

1998 ◽  
Vol 180 (22) ◽  
pp. 5913-5920 ◽  
Author(s):  
Marjan van der Woude ◽  
W. Bradley Hale ◽  
David A. Low
Keyword(s):  
Dna Methylation ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Published Data ◽  
Catabolite Gene Activator Protein ◽  
A Site ◽  
Methylation Patterns ◽  
Gatc Site

ABSTRACT Most of the adenine residues in GATC sequences in theEscherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepap GATC-I and GATC-II sites.

Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1586-1586
Author(s):  
Christine Richardson ◽  
Rodwell Mabaera ◽  
Christopher H. Lowrey
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Globin Promoter ◽  
Methylation Patterns ◽  
Adult Bone

Abstract Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.

scholarly journals Analysis of DNA Methylation Patterns Associated with In Vitro Propagated Globe Artichoke Plants Using an EpiRADseq-Based Approach

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 263 ◽  
Author(s):  
Elisa Cerruti ◽  
Cinzia Comino ◽  
Alberto Acquadro ◽  
Gianpiero Marconi ◽  
Anna Maria Repetto ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Status ◽  
Transcriptional Analysis ◽  
Globe Artichoke ◽  
Epigenetic Alterations ◽  
Coding Regions ◽  
Methylation Patterns ◽  
Reproductive Processes ◽  
Photosynthesis Regulation

Globe artichoke represents one of the main horticultural species of the Mediterranean basin, and ‘Spinoso sardo’ is the most widespread and economically relevant varietal type in Sardinia, Italy. In the last decades, in vitro culture of meristematic apices has increased the frequency of aberrant plants in open-field production. These off-type phenotypes showed highly pinnate-parted leaves and late inflorescence budding, and emerged from some branches of the true-to-type ‘Spinoso sardo’ plants. This phenomenon cannot be foreseen and is reversible through generations, suggesting the occurrence of epigenetic alterations. Here, we report an exploratory study on DNA methylation patterns in off-type/true-to-type globe artichoke plants, using a modified EpiRADseq technology, which allowed the identification of 2,897 differentially methylated loci (DML): 1,998 in CG, 458 in CHH, and 441 in CHG methylation contexts of which 720, 88, and 152, respectively, were in coding regions. Most of them appeared involved in primary metabolic processes, mostly linked to photosynthesis, regulation of flower development, and regulation of reproductive processes, coherently with the observed phenotype. Differences in the methylation status of some candidate genes were integrated with transcriptional analysis to test whether these two regulation levels might interplay in the emergence and spread of the ‘Spinoso sardo’ non-conventional phenotype.

scholarly journals Role of DNA Methylation and CpG Sites in the Viral Telomerase RNA Promoter during Gallid Herpesvirus 2 Pathogenesis

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Srđan Pejaković ◽  
André Claude Mbouombouo Mfossa ◽  
Laëtitia Wiggers ◽  
Ahmed Kheimar ◽  
Damien Coupeau ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Telomerase Activity ◽  
Tumor Formation ◽  
Telomerase Rna ◽  
Viral Promoter ◽  
Cpg Dinucleotides ◽  
Cpg Sites ◽  
Infected Cells ◽  
Methylation Patterns

ABSTRACT Gallid herpesvirus type 2 (GaHV-2) is an oncogenic alphaherpesvirus that induces malignant T-cell lymphoma in chicken. GaHV-2 encodes a viral telomerase RNA subunit (vTR) that plays a crucial role in virus-induced tumorigenesis, enhances telomerase activity, and possesses functions independent of the telomerase complex. vTR is driven by a robust viral promoter, highly expressed in virus-infected cells, and regulated by two c-Myc response elements (c-Myc REs). The regulatory mechanisms involved in controlling vTR and other genes during viral replication and latency remain poorly understood but are crucial to understanding this oncogenic herpesvirus. Therefore, we investigated DNA methylation patterns of CpG dinucleotides found in the vTR promoter and measured the impact of methylation on telomerase activity. We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within c-Myc REs showed specific changes in methylation after in vitro reactivation and in infected animals over time. Promoter reporter assays indicated that one of the c-Myc REs is involved in regulating vTR transcription, and that methylation strongly influenced vTR promoter activity. To study the importance of the CpG sites found in c-Myc REs in virus-induced tumorigenesis, we generated recombinant virus containing mutations in CpG sites of c-Myc REs together with the revertant virus by two-step Red-mediated mutagenesis. Introduced mutations in the vTR promoter did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication of the mutant virus in infected chickens was severely impaired, and tumor formation completely abrogated. Our data provides an in-depth characterization of c-Myc oncoprotein REs and the involvement of DNA methylation in transcriptional regulation of vTR. IMPORTANCE Previous studies demonstrated that telomerase RNAs possess functions that promote tumor development independent of the telomerase complex. vTR is a herpesvirus-encoded telomerase RNA subunit that plays a crucial role in virus-induced tumorigenesis and is expressed by a robust viral promoter that is highly regulated by the c-Myc oncoprotein binding to the E-boxes. Here, we demonstrated that the DNA methylation patterns in the functional c-Myc response elements of the vTR promoter change upon reactivation from latency, and that demethylation strongly increases telomerase activity in virus-infected cells. Moreover, the introduction of mutation in the CpG dinucleotides of the c-Myc binding sites resulted in decreased vTR expression and complete abrogation of tumor formation. Our study provides further confirmation of the involvement of specific DNA methylation patterns in the regulation of vTR expression and vTR importance for virus-induced tumorigenesis.

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