26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

2009 ◽  
Vol 21 (1) ◽  
pp. 113 ◽  
Author(s):  
C. Couldrey ◽  
M. P. Green ◽  
D. N. Wells ◽  
R. S. F. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cpg Island ◽  
Somatic Cells ◽  
Cpg Sites ◽  
Donor Cells ◽  
Genomic Regions ◽  
Methylation Patterns

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P < 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

Genome-Wide DNA Methylation Analysis Shows Enrichment of Differential Methylation in “Open Seas” and Enhancers and Reveals Hypomethylation in DNMT3A Mutated Cytogenetically Normal AML (CN-AML)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 653-653 ◽  
Author(s):  
Ying Qu ◽  
Andreas Lennartsson ◽  
Verena I. Gaidzik ◽  
Stefan Deneberg ◽  
Sofia Bengtzén ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Differential Methylation ◽  
Methylation Analysis ◽  
Cpg Sites ◽  
Genome Wide ◽  
Genomic Regions ◽  
Methylation Patterns

Abstract Abstract 653 DNA methylation is involved in multiple biologic processes including normal cell differentiation and tumorigenesis. In AML, methylation patterns have been shown to differ significantly from normal hematopoietic cells. Most studies of DNA methylation in AML have previously focused on CpG islands within the promoter of genes, representing only a very small proportion of the DNA methylome. In this study, we performed genome-wide methylation analysis of 62 AML patients with CN-AML and CD34 positive cells from healthy controls by Illumina HumanMethylation450K Array covering 450.000 CpG sites in CpG islands as well as genomic regions far from CpG islands. Differentially methylated CpG sites (DMS) between CN-AML and normal hematopoietic cells were calculated and the most significant enrichment of DMS was found in regions more than 4kb from CpG Islands, in the so called open sea where hypomethylation was the dominant form of aberrant methylation. In contrast, CpG islands were not enriched for DMS and DMS in CpG islands were dominated by hypermethylation. DMS successively further away from CpG islands in CpG island shores (up to 2kb from CpG Island) and shelves (from 2kb to 4kb from Island) showed increasing degree of hypomethylation in AML cells. Among regions defined by their relation to gene structures, CpG dinucleotide located in theoretic enhancers were found to be the most enriched for DMS (Chi χ2<0.0001) with the majority of DMS showing decreased methylation compared to CD34 normal controls. To address the relation to gene expression, GEP (gene expression profiling) by microarray was carried out on 32 of the CN-AML patients. Totally, 339723 CpG sites covering 18879 genes were addressed on both platforms. CpG methylation in CpG islands showed the most pronounced anti-correlation (spearman ρ =-0.4145) with gene expression level, followed by CpG island shores (mean spearman rho for both sides' shore ρ=-0.2350). As transcription factors (TFs) have shown to be crucial for AML development, we especially studied differential methylation of an unbiased selection of 1638 TFs. The most enriched differential methylation between CN-AML and normal CD34 positive cells were found in TFs known to be involved in hematopoiesis and with Wilms tumor protein-1 (WT1), activator protein 1 (AP-1) and runt-related transcription factor 1 (RUNX1) being the most differentially methylated TFs. The differential methylation in WT 1 and RUNX1 was located in intragenic regions which were confirmed by pyro-sequencing. AML cases were characterized with respect to mutations in FLT3, NPM1, IDH1, IDH2 and DNMT3A. Correlation analysis between genome wide methylation patterns and mutational status showed statistically significant hypomethylation of CpG Island (p<0.0001) and to a lesser extent CpG island shores (p<0.001) and the presence of DNMT3A mutations. This links DNMT3A mutations for the first time to a hypomethylated phenotype. Further analyses correlating methylation patterns to other clinical data such as clinical outcome are ongoing. In conclusion, our study revealed that non-CpG island regions and in particular enhancers are the most aberrantly methylated genomic regions in AML and that WT 1 and RUNX1 are the most differentially methylated TFs. Furthermore, our data suggests a hypomethylated phenotype in DNMT3A mutated AML. Disclosures: No relevant conflicts of interest to declare.

30 OXYGEN DEPRIVATION DOES NOT FURTHER AUGMENT MITOCHONDRIAL MEMBRANE POTENTIAL IN PHARMACOLOGICALLY TREATED FIBROBLASTS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER

2017 ◽  
Vol 29 (1) ◽  
pp. 122
Author(s):  
B. R. Mordhorst ◽  
S. N. Bogue ◽  
K. D. Wells ◽  
J. A. Green ◽  
R. S. Prather
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitochondrial Membrane ◽  
Somatic Cells ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Donor Cells

Somatic cells commonly used in nuclear transfer primarily utilise the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively, the metabolism of somatic cells contrasts that of cells within early embryos, which predominantly use glycolysis and exhibit Warburg Effect (WE)-like characteristics. We hypothesised that fibroblast cells can become more blastomere-like if driven either pharmacologically or by oxygen constraint and could result in improved in vitro embryonic development after somatic cell nuclear transfer (SCNT). The pharmaceuticals used (PS48 and CPI-613) should decrease mitochondrial use of the tricarboxylic acid (TCA) cycle and promote the PI3K pathway, respectively. Furthermore, we hypothesised that oxygen constraint (1.3%) would hinder TCA cycle activity and promote glycolysis. The goal was to achieve a WE-like effect in donor cells before nuclear transfer (NT) by treating Day 35 porcine fetal fibroblasts with CPI-613 (100 µM), PS48 (10 µM), both drugs combined (MIX), or as controls (CON, 0 µM) for 7 days under stepwise oxygen constraint (OC; 1.3%) or under normal conditions (ON; 5%). Three biological replicates were collected and data were analysed for main effect of treatment via GLM procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). To determine if our treatments affected mitochondria respiratory capacity (thereby TCA cycle capability) within embryos, we measured mitochondrial membrane potential (Δψm) using JC-10, a biphasic cationic dye. Mitotracker green (MTG) was used to estimate mitochondrial quantity. The percentage of cells with low Δψm was increased (P = 0.02) with any CPI or MIX treatment (treatments ≥ 95%) compared with OC-PS48 and both control (ON and OC) treatments (treatments ≥ 77.4%), whereas ON-PS48 had an intermediate level (90.4%; error = 4.9%). Contrary to our prediction, MTG intensity was lower across all ON treatments compared with OC treatments (NO treatments ≤ 736 AU v. OC treatments ≥ 872 AU; error = 23 AU; P < 0.01). Regardless of oxygen level, controls and PS48 treatments yielded the highest percentages of viable cells (treatments ≥ 94%) and OC-CPI and NO-MIX the lowest (treatments ≤ 86%) with NO-CPI and OC-MIX being intermediate (treatments ≥ 90%; error = 3%; P < 0.01). Oxygen constraint did not promote a reduction in mitochondrial membrane potential in pharmacologically treated fibroblasts. Additionally, intensity of MTG was increased in fibroblasts cultured under oxygen constraint compared with those cultured in 5% oxygen. Our results warrant further investigation of the mitochondrial changes occurring with oxygen deprivation in donor-cells. Experiments are underway to determine if gene expression in cells treated pharmacologically and with oxygen constraint are augmented, and whether these treatments will result in better development after SCNT. This study was funded by Food for the 21 st Century and NIH R01HD080636.

200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 198
Author(s):  
T. Xiang ◽  
S. Walker ◽  
K. Gregg ◽  
W. Zhou ◽  
V. Farrar ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Donor Cells

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 130
Author(s):  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. Y. Won ◽  
H. Shim
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Somatic Cells ◽  
Dna Demethylation ◽  
Term Survival ◽  
Cpg Sites ◽  
Fetal Fibroblasts

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P &lt; 0.05), and DNA demethylation occurred in embryos from both EGCNT (P &lt; 0.05) and SCNT (P &lt; 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P &lt; 0.01) and ICSI (P &lt; 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.

scholarly journals S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 815-828 ◽  
Author(s):  
Byeong-Gyun Jeon ◽  
Gianfranco Coppola ◽  
Steven D Perrault ◽  
Gyu-Jin Rho ◽  
Dean H Betts ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
X Chromosome ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Telomerase Activity ◽  
X Chromosome Inactivation ◽  
Dna Demethylation ◽  
Chromosome Inactivation ◽  
Donor Cells

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment withS-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (∼1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes,ANT3,MECP2,XIAP,XIST, andHPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.

22 OXAMFLATIN TREATMENT ENHANCES NUCLEAR REPROGRAMMING BY INHIBITING XIST EXPRESSION AND REDUCING DNA METHYLATION IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
J. Mao ◽  
M. T. Zhao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
K. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Cell ◽  
Real Time ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Xist Gene ◽  
Nuclear Reprogramming ◽  
Xist Expression ◽  
Donor Cells

Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) enhances developmental potential by alteration of epigenetic status. Oxamflatin is one of the potent HDACi. In our previous study, development to Day 7 blastocysts was enhanced when the porcine somatic cell nuclear transfer (SCNT) embryos were treated with oxamflatin for 16 h. The objective of the present study was to investigate the effect of oxamflatin treatment on XIST gene expression and DNA methylation of XIST gene and centromeric repeat element in Day 7 SCNT blastocysts. Somatic cell nuclear transfer was performed on enucleated metaphase II oocytes using a transgene female cell line. Cloned embryos were electrically fused and activated, treated with 150 nM oxamflatin for 16 h and cultured in PZM3 under 5% CO2, 5% oxygen, and 90% N2 for 7 days. Clones without Oxamflatin treatment were used as controls. For XIST methylation, IVF blastocysts at Day 7 were used as controls. Blastocysts at Day 7 were pooled from each treatment group and processed for methylation analysis by bisulfite sequencing and gene expression by quantitative real-time PCR. This experiment was replicated 4 times. The percent of CpG methylation in donor cells before SCNT was also determined. Data were analysed by using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). In donor cells, 45.3 ± 5.8% of CpGs in a centromeric repeat element (9 CpGs in GenBank Z75640) were methylated. In the SCNT embryos, oxamflatin treatment reduced methylation from 27.3 ± 3.1% in the control to 18.2 ± 3.2% (P < 0.05). The average methylation in XIST (11 CpGs in GenBank KC149530.1) in donor cells was 42.4 ± 6.4%. This CpG island had 2 sites that were not methylated in any of the samples. However, the remaining 9 CpGs were methylated in 8 of 15 samples; for example, showing a parental imprint of ~50%. This implied that the CpG island studied represented the real-time status of the XIST locus in the cell and provides a good marker for reprogramming studies. XIST methylation level in Day 7 blastocysts was not different between oxamflatin (11.8 ± 3.2%) and control (11.8 ± 3.2%). However, XIST methylation in SCNT embryos was higher than in the same age IVF blastocysts (11.7 ± 1.7 v. 0.6 ± 2.4%; P < 0.01). Oxamflatin treatment tended to decrease XIST expression in Day 7 blastocysts compared with controls (18.8 ± 0.8 v. 21.7 ± 0.8; P < 0.1) as measured by real-time PCR. Interestingly, XIST gene expression was positively correlated with its methylation (P < 0.05). In conclusion, these results indicate that during nuclear reprogramming there was a dramatic decrease in DNA methylation from donor cells to Day 7 SCNT embryos. The higher methylation of XIST in SCNT embryos compared with IVF embryos suggests that the reprogramming of donor cells was not completed, which may be a contributor to low cloning efficiency. Oxamflatin treatment of SCNT embryos may enhance nuclear reprogramming by inhibiting XIST expression and reducing DNA methylation, resulting in better embryo development.

scholarly journals Epigenetic abnormalities associated with somatic cell nuclear transfer

Reproduction ◽  
2021 ◽  
Author(s):  
Atsuo Ogura ◽  
Shogo Matoba ◽  
Kimiko Inoue
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Genome ◽  
Imprinted Genes ◽  
Donor Genome ◽  
Genome Wide ◽  
Donor Cells ◽  
Genomic Reprogramming

Twenty-five years have passed since the birth of Dolly the sheep, the first mammalian clone produced by adult somatic cell nuclear transfer (SCNT). During that time, the main thrust of SCNT-related research has been the elucidation of SCNT-associated epigenetic abnormalities and their correction, with the aim of improving the efficiency of cloned animal production. Through these studies, it has become clear that some epigenomic information can be reprogrammed by the oocyte, while some cannot. Now we know that the imprinting memories in the donor genome, whether canonical (DNA-methylation-dependent) or noncanonical (H3K27me3-dependent), are not reprogrammed by SCNT. Thus, SCNT-derived embryos have the normal canonical imprinting and the erased noncanonical imprinting, both being inherited from the donor cells. The latter can cause abnormal phenotypes in SCNT-derived placentas arising from biallelic expressions of noncanonically imprinted genes. By contrast, repressive epigenomic information, such as DNA methylation and histone modifications, might be more variably reprogrammed, leaving room for technical improvements. Low-input analytical technologies now enable us to analyze the genome of gametes and embryos in a high-throughput, genome-wide manner. These technologies are being applied rapidly to the SCNT field, providing evidence for incomplete reprogramming of the donor genome in cloned embryos or offspring. Insights from the study of epigenetic phenomena in SCNT are highly relevant for our understanding of the mechanisms of genomic reprogramming that can induce totipotency in the mammalian genome.

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