145 THE METHYLATION PATTERNS OF POTENTIAL DIFFERENTIALLY METHYLATED REGIONS IN BOVINE Xist, Impact, NDN, AND H19 GENES

2007 ◽  
Vol 19 (1) ◽  
pp. 190
Author(s):  
N. T. D'Cruz ◽  
K. J. Wilson ◽  
M. K. Holland
Keyword(s):  
Genomic Dna ◽  
Bisulfite Sequencing ◽  
Cpg Island ◽  
Methylation Pattern ◽  
Imprinted Genes ◽  
Aberrant Methylation ◽  
Differentially Methylated Regions ◽  
Methylation Patterns ◽  
The Impact

Clinical and laboratory-assisted reproductive techniques such as ICSI have recently been associated with an increased incidence of several syndromes associated with defects in genomic imprinting. Genomically imprinted genes are expressed from only one parental allele and act to regulate growth of the fetus and placenta and brain development/ function. Imprinted genes are controlled by differentially methylated regions (DMRs), whereby one parental allelle (i.e. either maternal or paternal) is epigenetically silenced via methylation. Studies conducted in vitro suggest that culture of embryos and embryo manipulations may perturb the imprinting process. In the current study, the genomic DNA methylation patterns of CpG islands within bovine H19 (27 CpGs analyzed), Impact (36 CpGs), NDN (22 CpGs), and Xist (21 CpGs) were analyzed by bisulfite sequencing. Genomic DNA from a female fibroblast cell line and sperm were chosen for analysis. Potential DMRs for the 4 genes were identified, and semi-nested PCR primers were designed surrounding those regions. Second-round PCR products (2 separate reactions) were mixed, subcloned, and sequenced (n ≥ 10). The fibroblast methylation pattern of the Xist DMR showed consistent methylation in 50% of sequenced clones, with no methylation observed in sperm. The H19 DMR in fibroblast DNA also showed consistent methylation in 25% of sequenced clones, with sperm DNA fully methylated. These results confirm previous studies showing that Xist and H19 are imprinted in cattle. Sequencing of the putative Impact DMR clones indicated no methylation in either cell type, suggesting no imprinting in cattle, tissue-specific imprinting, or that this CpG island (15 bp post ATG) is not the DMR that controls imprinted expression of the Impact gene. The NDN DMR (500 bp post ATG) in sperm was not methylated, whereas the fibroblast cells had a variable methylation pattern. This may be for the same reasons suggested for Impact, but the variability within the CpG island may also be due to in vitro culture conditions resulting in aberrant methylation. This possible culture effect is currently being confirmed through bisulfite sequencing of the gene in an adult tissue. The investigation of methylation patterns in oocytes is also underway. Together, the information gathered will be used to determine the imprinting status of several bovine genes and, in the future, whether any of these imprinted genes are responsible for the increased pregnancy loss and calf abnormalities associated with advanced reproductive technologies.

Impaired Hydroxylation of 5-Methylcytosine In TET2 mutated Patients with Myeloid Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1-1
Author(s):  
Anna Jankowska ◽  
Myunggon Ko ◽  
Yun Huang (equal contribution) ◽  
Utz J. Pape ◽  
Hadrian Szpurka ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Genomic Dna ◽  
Conflicts Of Interest ◽  
Methylation Pattern ◽  
Myeloid Malignancies ◽  
Cpg Sites ◽  
Low Levels ◽  
Methylation Patterns ◽  
The Impact

Abstract Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β>0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells

1993 ◽  
Vol 13 (9) ◽  
pp. 5538-5548
Author(s):  
Y C Choi ◽  
C B Chae
Keyword(s):  
Embryonal Carcinoma ◽  
Cpg Island ◽  
Cpg Islands ◽  
High Density ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cpg Dinucleotides ◽  
F9 Embryonal Carcinoma Cells ◽  
Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

Stage-differentiated modelling of DNA methylation landscapes uncovers salient biomarkers and prognostic signatures in colorectal cancer progression

2021 ◽  
Author(s):  
Sangeetha Muthamilselvan ◽  
Abirami Raghavendran ◽  
Ashok Palaniappan
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Cpg Island ◽  
Early Stage ◽  
P Value ◽  
Consensus Analysis ◽  
One Stage ◽  
Differentially Methylated Genes ◽  
Methylation Patterns ◽  
The Impact

Abstract Background: Aberrant DNA methylation acts epigenetically to skew the gene transcription rate up or down, with causative roles in the etiology of cancers. However research on the role of DNA methylation in driving the progression of cancers is limited. In this study, we have developed a comprehensive computational framework for the stage-differentiated modelling of DNA methylation landscapes in colorectal cancer (CRC), and unravelled significant stagewise signposts of CRC progression. Methods: The methylation β - matrix was derived from the public-domain TCGA data, converted into M-value matrix, annotated with AJCC stages, and analysed for stage-salient genes using multiple approaches involving stage-differentiated linear modelling of methylation patterns and/or expression patterns. Differentially methylated genes (DMGs) were identified using a contrast against controls (adjusted p-value <0.001 and |log fold-change of M-value| >2). These results were filtered using a series of all possible pairwise stage contrasts (p-value <0.05) to obtain stage-salient DMGs. These were then subjected to a consensus analysis, followed by Kaplan–Meier survival analysis to evaluate the impact of methylation patterns of consensus stage-salient biomarkers on disease prognosis.Results: We found significant genome-wide changes in methylation patterns in cancer cases relative to controls agnostic of stage. Our stage-differentiated analysis yielded the following stage-salient genes: one stage-I gene (FBN1), one stage-II gene (FOXG1), one stage-III gene (HCN1) and four stage-IV genes (NELL1, ZNF135, FAM123A, LAMA1). All the biomarkers were hypermethylated, indicating down-regulation and signifying a CpG island Methylator Phenotype (CIMP) manifestation. A significant prognostic signature consisting of FBN1 and FOXG1 survived all the steps of our analysis pipeline, and represents a novel early-stage biomarker. Conclusions: We have designed a workflow for stage-differentiated consensus analysis, and identified stage-salient diagnostic biomarkers and an early-stage prognostic biomarker panel. Our studies further yield a novel CIMP-like signature of potential clinical import underlying CRC progression.

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 Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells.

1993 ◽  
Vol 13 (9) ◽  
pp. 5538-5548 ◽  
Author(s):  
Y C Choi ◽  
C B Chae
Keyword(s):  
Embryonal Carcinoma ◽  
Cpg Island ◽  
Cpg Islands ◽  
High Density ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cpg Dinucleotides ◽  
F9 Embryonal Carcinoma Cells ◽  
Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

scholarly journals SUN-715 IIM May Influence Matured Oocytes’ DNA Methylation of PCOS Patients

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Congru Li ◽  
Yang Yu
Keyword(s):  
Dna Methylation ◽  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Embryo Transfer ◽  
Cpg Island ◽  
Reproductive Technologies ◽  
Childbearing Age ◽  
Vitro Fertilization ◽  
The Impact

Abstract Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of childbearing age and is the main cause of anovulatory infertility. To increase the number of oocytes obtained, controlled ovarian stimulation (COS) has become a routine choice for in vitro fertilization-embryo transfer (IVF-ET), which is one of the common assisted reproductive technologies for PCOS patients. However, for these patients, there is a high risk of ovarian hyperstimulation syndrome (OHSS). Obtaining in vitro maturation (IVM) of immature oocytes, and then in vitro fertilization and embryo transfer of mature oocytes provides a possible way for people to solve the above problems. Since the IVM technology will expose oocytes to in vitro conditions for a longer period of time, theoretically increasing the risk of the oocytes being affected by the culture environment, further research and explorations are needed for study in gene programming, epigenetics, etc. Therefore, to explore the impact of IVM operation on embryonic development is of great significance for further clarifying assisted reproductive safety and improving IVM operation conditions. Here we focused on DNA methylation reprogramming process which was essential for embryonic development. We tested the DNA methylation of sperm, IVM oocytes and IVM generated early stage embryos including pronucleus, 4cell, 8cell, morula, inner cell mass, trophoectoderm (TE) as well as six-week embryos by Nimble Gen Human DNA Methylation 3x729K CpG Island Plus RefSeq Promoter Array and compared the data with our published genome-wide DNA methylomes of human gametes and early embryos generated from in vivo maturation oocytes. We showed that IVM embryos show abnormal DNA methylation reprogramming pattern. By analyzing the abnormally reprogrammed promoters, we further found that IVM may affect the functions of demethylation related genes. Oocytes from IVM manipulation were tested with higher DNA methylation levels, and their abnormal methylated promoters mainly enriched in immune and metabolism pathways. Furthermore, we investigated the DNA methylation of TE, which was directly related with implantation process and revealed the abnormal methylated promoters were related with metabolism pathway too. Our data support that IVM may influence the DNA methylome of oocytes, which in turn affects the methylome of their embryos. However, due to the limited number of samples and the inability of the chip to cover all CpG sites, the results of this study require further research and validation.

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 Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

2019 ◽  
Author(s):  
Jes Alexander ◽  
Gregory M. Findlay ◽  
Martin Kircher ◽  
Jay Shendure
Keyword(s):  
Genome Editing ◽  
Cpg Island ◽  
Exogenous Dna ◽  
Cpg Dinucleotides ◽  
Epigenome Editing ◽  
Functional Consequences ◽  
Cpg Dinucleotide ◽  
Methylation Patterns

AbstractRecent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double stranded break (DSB), followed by NHEJ or HDR mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, i.e. to drive replacement of the DNA sequence intervening the dual cuts via NHEJ. In a proof-of-concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event, and opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

264 ANALYSIS OF DNA METHYLATION PATTERN IN PRE-IMPLANTATION-STAGE EMBRYOS DERIVED FROM NUCLEAR TRANSFER USING PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 248
Author(s):  
D.-H. Choi ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
H.-S. Kim ◽  
H.-Y. Son ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Perinatal Death ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Paternal Allele ◽  
High Birth Weight ◽  
Aberrant Methylation

Somatic cell nuclear transfer (SCNT) has been successfully used to produce live cloned offspring in various mammals. However, some studies had reported that cloned embryos by SCNT had many problems in reprogramming or epigenetic modification, such as DNA methylation. DNA methylation is an essential process in epigenetic modification for development, and aberrant methylation in cloned embryos gives rise to abortion, high birth weight, and perinatal death. In this study, embryonic germ (EG) cells were used as donor cells for nuclear transfer. EG cells may have less reprogramming or demethylation than SCNT because these are already in erased status. However, little is known about methylation state or developmental capacity of the EG cell as a donor. The objective of this study was to analyze the methylation pattern of pre-implantation embryos cloned from porcine EG cells. Two regions, PRE-1 and microsatellite (MS), were analyzed for methylation patterns of cloned embryos from porcine EG cells and compared with the pattern of mature oocytes and in vitro-fertilized (IVF) embryos as a control. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro for 44 h, followed by use for IVF and NT with porcine EG cells. The porcine EG cells were prepared from 28-day-old fetuses after mating; genital ridges were isolated from fetuses, and then transferred into a culture medium on a feeder layer. The number of embryos for analysis was 300 for matured oocytes, 50–80 for 4–8 cell embryos, 30–40 for morulae, and 20–30 for blastocysts. The genomic DNA was isolated from the embryos and treated with bisulfite solution. PCR was performed for the amplification of PRE-1 and MS regions. The PCR products were sequenced by using an automatic DNA sequencer. The methylation rates of the PRE-1 and MS regions in IVF embryos showed that the demethylation process had occurred during the pre-implantation stage, which is a typical phenomenon of in vivo counterparts (Kang et al. 2001 J. Biol. Chem. 276, 39 980). However, compared to IVF embryos, embryos derived from NT using EG cells showed differences at the morula (PRE-1) and blastocyst (MS) stage. These results indicate that porcine EG cells also have problems in reprogramming during NT. For detailed and reliable results, the methylation pattern analysis of the imprinting region, for example, H19 in maternal allele and Igf2 in paternal allele, must be examined. Table 1.Methylation of PRE-1 and MS regions in embryos derived from IVF and NT using porcine EG cells

scholarly journals Hemimethylation of CpG dyads is characteristic of secondary DMRs associated with imprinted loci and correlates with 5-hydroxymethylcytosine at paternally methylated sequences

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Julianna Nechin ◽  
Emma Tunstall ◽  
Naideline Raymond ◽  
Nicole Hamagami ◽  
Chris Pathmanabhan ◽  
...  
Keyword(s):  
Methylation Status ◽  
Differential Methylation ◽  
Imprinted Genes ◽  
Differentially Methylated Regions ◽  
High Incidence ◽  
Sequence Elements ◽  
Parent Of Origin ◽  
Methylation Patterns ◽  
Post Implantation ◽  
Control Regions

Abstract Background In mammals, the regulation of imprinted genes is controlled by differential methylation at imprinting control regions which acquire parent of origin-specific methylation patterns during gametogenesis and retain differences in allelic methylation status throughout fertilization and subsequent somatic cell divisions. In addition, many imprinted genes acquire differential methylation during post-implantation development; these secondary differentially methylated regions appear necessary to maintain the imprinted expression state of individual genes. Despite the requirement for both types of differentially methylated sequence elements to achieve proper expression across imprinting clusters, methylation patterns are more labile at secondary differentially methylated regions. To understand the nature of this variability, we analyzed CpG dyad methylation patterns at both paternally and maternally methylated imprinted loci within multiple imprinting clusters. Results We determined that both paternally and maternally methylated secondary differentially methylated regions associated with imprinted genes display high levels of hemimethylation, 29–49%, in comparison to imprinting control regions which exhibited 8–12% hemimethylation. To explore how hemimethylation could arise, we assessed the differentially methylated regions for the presence of 5-hydroxymethylcytosine which could cause methylation to be lost via either passive and/or active demethylation mechanisms. We found enrichment of 5-hydroxymethylcytosine at paternally methylated secondary differentially methylated regions, but not at the maternally methylated sites we analyzed in this study. Conclusions We found high levels of hemimethylation to be a generalizable characteristic of secondary differentially methylated regions associated with imprinted genes. We propose that 5-hydroxymethylcytosine enrichment may be responsible for the variability in methylation status at paternally methylated secondary differentially methylated regions associated with imprinted genes. We further suggest that the high incidence of hemimethylation at secondary differentially methylated regions must be counteracted by continuous methylation acquisition at these loci.

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