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 O6D.2 Evidence of dna methylation changes by carbon nanotubes in a translational study design

2019 ◽  
Vol 76 (Suppl 1) ◽  
pp. A57.2-A57
Author(s):  
Manosij Ghosh ◽  
Deniz Öner ◽  
Lode Godderis ◽  
Peter Hoet
Keyword(s):  
Dna Methylation ◽  
Cross Sectional Study ◽  
Methylation Pattern ◽  
Global Dna Methylation ◽  
Limited Information ◽  
Cross Sectional ◽  
Translational Study ◽  
Exposed Workers

IntroductionWhile studies have addressed genotoxic effects of CNT, only limited information are available on epigenetic effects. We designed a study to investigate DNA methylation alterations in vitro, in vivo and in occupationally exposed workers.Material and methodsIn vitro studies were performed in 16-HBE and THP-1 cells. For the in vivo study, BALB/c mice were administered intratracheally with single-wall CNT (SWCNTs) and multi-wall (MWCNTs) at high (2.5 mg/kg) and low (0.25 mg/kg) doses. For the cross sectional study, 24 workers exposed to aggregates of MWCNT of 500 nm–100 µm with concentrations of 4.6–42.6 µg/m3 and 43 unexposed referents were recruited. Global DNA methylation and demethylation patterns were analysed by LC-MS/MS. Methylation of specific genes was measured by Pyromark 24® (Qiagen). Genome-wide assessment of DNA methylation was performed with Infinium HumanMethylation450 BeadChip Array.ResultsIn general, we did not find global DNA methylation alteration for both CNTs. In 16-HBE cells, differentially methylated and expressed genes (MWCNTs>SWCNTs) from p53 signalling, DNA damage repair and cell cycle pathways were observed. In THP-1 cells, CNTs induced promoter-specific methylation of genes involved in several signaling cascade, vascular endothelial growth factor and platelet activation pathways. In lungs of BALB/c mice CNTs affected methylation of ATM gene. Finally, analysis of gene-specific DNA methylation in exposed workers revealed significant changes for DNMT1, ATM, SKI, and HDAC4 promoter CpGs.ConclusionsEpigenetic changes seem to occur at sub cyto-genotoxic concentrations in vitro. Alteration in DNA methylation pattern could be a natural reaction of cells but could also silence critical genes and reprogram cellular functions.

1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 109 ◽  
Author(s):  
A. Bonk ◽  
M. Samuel ◽  
L. Lai ◽  
Y. Hao ◽  
R. Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
The Other ◽  
Specific Gene ◽  
Aberrant Dna Methylation

Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts. This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 125
Author(s):  
R. S. Deshmukh ◽  
O. Oestrup ◽  
E. Oestrup ◽  
M. Vejlsted ◽  
H. Niemann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Epifluorescence Microscopy ◽  
Preimplantation Embryos ◽  
Parthenogenetic Activation

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

scholarly journals Hypermethylation involved in DNA profiles of lung cancer specific tumour suppressor genes and epigenetic modification caused by an ultra-highly diluted homeopathic drug, Condurango 30C, in vitro and in vivo

2021 ◽  
Vol 13 (47) ◽  
pp. 99-99
Author(s):  
Anisur Rahman Khuda-Bukhsh ◽  
Sourav Sidkar
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Epigenetic Modification ◽  
Tumour Suppressor ◽  
Tumour Suppressor Genes ◽  
Dna Hypermethylation ◽  
Suppressor Genes ◽  
Homeopathic Drug

Background and objectives: DNA hyper-methylation is an important aspect involved in carcinogenesis and cancer progression, which affects mainly CpG islands of DNA and causes inactivation of tumour suppressor genes. Therefore DNA hypermethylation status of the genomic DNA in both the transformed cancerous cell lines and in carcinogen-induced lung cancer was ascertained by analysis of expressions of certain major lung cancer specific tumour suppressor genes. The other objective was to examine if ultra highly diluted homeopathic drug, Condurango 30C, had ability to modulate DNA methylation. Methods: DNA methylation activity, if any, has been ascertained in H460-NSCLC cells in vitro and in BaP-induced lung cancer of rats in vivo, in respect of tumour suppressor genes like p15, p16, p18 and p53 by using PCR-SSCP analyses. The ability of modulation of DNA methylation, if any, by Condurango 30C was also verified against placebo control in a blinded manner. Results: Condurango 30C-treated DNA showed significant decrease in band-intensity of p15 and p53 genes especially in methylated condition, in vitro, at the IC50 dose (2.43µl/100µl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also supported ability of Condurango 30C to modulate methylation state, in vitro. Inhibition of p15 hypermethylation was observed after post cancer treatment of rat with Condurango 30C. SSCP results gave a better indication of differences in band-position and single strand separation of p15 and p53 in Condurango 30C treated samples. Conclusion: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation but placebos could not.

scholarly journals Analysis of epigenetic aging in vivo and in vitro: Factors controlling the speed and direction

2020 ◽  
Vol 245 (17) ◽  
pp. 1543-1551 ◽  
Author(s):  
Mieko Matsuyama ◽  
Arne Søraas ◽  
Sarah Yu ◽  
Kyuhyeon Kim ◽  
Evi X Stavrou ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Biological Significance ◽  
Methylation Pattern ◽  
Tissue Type ◽  
Epigenetic Clock ◽  
Whole Genome ◽  
Aging Research ◽  
Epigenetic Aging

The mechanism of aging is not yet fully understood. It has been recognized that there are age-dependent changes in the DNA methylation pattern of the whole genome. To date, there are several DNA methylation-based estimators of the chronological age. A majority of the estimators use the DNA methylation data from a single tissue type, such as blood. In 2013, for the first time, Steve Horvath reported the DNA methylation-based age estimator (353 CpGs were used) that could be applied to multiple tissues. A refined, more sensitive version that uses 391 CpGs was subsequently developed and validated in human cells, including fibroblasts. In this review, the age predicted by DNA methylation-based age estimator is referred to as DNAmAge, and the biological process controlling the progression of DNAmAge is referred to as the epigenetic aging in this minireview. The concepts of DNAmAge and epigenetic aging provide us opportunities to discover previously unrecognized biological events controlling aging. In this article, we discuss the frequently asked questions regarding DNAmAge and the epigenetic aging by introducing recent studies of ours and others. We focus on addressing the following questions: (1) Is there any synchronization of DNAmAge between cells in a human body?, (2) Can we use in vitro (cell culture) systems to study the epigenetic aging?, (3) Is there an age limit of DNAmAge?, and (4) Is it possible to change the speed and direction of the epigenetic aging? We describe our current understandings to these questions and outline potential future directions. Impact statement Aging is associated with DNA methylation (DNAm) changes. Recent advancement of the whole-genome DNAm analysis technology allowed scientists to develop DNAm-based age estimators. A majority of these estimators use DNAm data from a single tissue type such as blood. In 2013, a multi-tissue age estimator using DNAm pattern of 353 CpGs was developed by Steve Horvath. This estimator was named “epigenetic clock”, and the improved version using DNAm pattern of 391 CpGs was developed in 2018. The estimated age by epigenetic clock is named DNAmAge. DNAmAge can be used as a biomarker of aging predicting the risk of age-associated diseases and mortality. Although the DNAm-based age estimators were developed, the mechanism of epigenetic aging is still enigmatic. The biological significance of epigenetic aging is not well understood, either. This minireview discusses the current understanding of the mechanism of epigenetic aging and the future direction of aging research.

Epigenetic Modulation by Vidaza™ induces Apoptosis and Overcomes In Vitro Drug Resistance in Human Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5001-5001
Author(s):  
Tanyel Kiziltepe ◽  
Laurence Catley ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Hiroshi Yasui ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Epigenetic Modification ◽  
Biological Effects ◽  
Hematologic Malignancies ◽  
Aberrant Methylation ◽  
Aberrant Expression ◽  
Induced Apoptosis

Abstract Methylation of DNA is an epigenetic modification that plays an important role in the regulation of gene expression in mammalian cells. Although DNA methylation is required for normal cell development and function, aberrant methylation and the resulting aberrant expression of genes, such as tumor suppressor genes and oncogenes, contribute to the development of malignancies. Interestingly, aberrant DNA methylation has recently emerged as one of the most frequent molecular alterations in hematologic malignancies, providing a powerful rationale to use inhibitors of DNA methylation as a novel means of targeting hematologic malignancies. Vidaza™ [Pharmion Corporation] (5-azacytidine), an FDA approved drug for the treatment of myelodysplastic syndromes, is an inhibitor of DNA methylation. Multiple myeloma (MM) is currently an incurable hematological malignancy despite all the conventional and novel therapies, and we here examined the biological effects using Vidaza™ on human MM cells. We demonstrate here that Vidaza™ has significant cytotoxicity in both conventional therapy sensitive (MM1S, RPMI-8226) and resistant (MM1R, RPMI-Dox40, RPMI-LR5) MM cell lines, as well as freshly isolated patient MM tumor cells, with an IC50 of 1.25–4 mM at 72 hours in vitro. Importantly, no cytotoxic effects of Vidaza™ were detected in peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at ≤20 mM, suggesting a therapeutic index. Moreover, Vidaza™ overcame the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). Vidaza™ induced apoptosis in MM cells, as determined by flow cytometric analysis using PI and Annexin V staining. Vidaza™-induced apoptosis was associated with PARP, caspase 8 and caspase 9 cleavage. Importantly, pan-caspase inhibitor zVAD-fmk, significantly, but only partially, inhibited apoptosis induced by Vidaza™, suggesting the involvement of both caspase dependent and independent pathways. Taken together, our studies therefore demonstrate that Vidaza™ induces apoptosis and overcomes in vitro drug resistance in MM cells. Ongoing studies are delineating the mechanism of action of Vidaza™ against MM cells to further provide the preclinical rationale for clinical evaluation of Vidaza™, alone or in combination with other agents, to improve patient outcome in MM.

scholarly journals Hypermethylation-mediated Transcriptional Silencing of lncRNA-SCARF1 Promotes Progression and Metastasis of Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Boyi Liao ◽  
Peiran Huang ◽  
Xiangyu Zhang ◽  
Xinyu Wang ◽  
Kaiqian Zhou ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Methylation ◽  
Target Genes ◽  
Malignant Tumors ◽  
Aberrant Methylation ◽  
Promoter Regions ◽  
Migration Ability ◽  
Downstream Target

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths. Recent studies have demonstrated that deregulation of long noncoding RNAs (lncRNAs), such as abnormal DNA methylation of promoter, is strongly associated with development and progression of diverse malignant tumors. This study investigated the mechanisms and changes in DNA methylation levels of promoter regions of HCC-specific lncRNAs, and alterations of downstream target genes.Methods: LncRNA expression profile data of 8 human HCC tissues and matched normal tissues were obtained. LncRNAs with aberrant methylation were identified through DNA methylation microarray. The biological functions of the lncRNAs were investigated through targeted knockdown of lncRNA-SCARF1 in vitro and in vivo. Furthermore, the downstream targets of lncRNA-SCARF1 were identified through ChIRP-MS.Results: LncRNA-SCARF1 was significantly down-regulated in HCC samples. Hypermethylation in the promoter of lnc-SCARF1 induced its down-regulation in HCC. Over-expression of lnc-SCARF1 inhibited the tumor proliferation and migration ability of HCC cells in vitro and in vivo. Furthermore, CUL9 was found to be a potential downstream target of lncRNA-SCARF1.Conclusion: LncRNA-SCARF1 regulates HCC progression by interacting with CUL9 and may serve as a prognostic biomarker or an effective therapeutic target in HCC.

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

2007 ◽  
Vol 30 (4) ◽  
pp. 90
Author(s):  
Kirsten Niles ◽  
Sophie La Salle ◽  
Christopher Oakes ◽  
Jacquetta Trasler
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Chromosome 9 ◽  
Specific Gene ◽  
Global Methylation ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

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