Dimethylated histone H3 lysine 9 is dispensable for the interaction between developmental pluripotency-associated protein 3 (Dppa3) and ten-eleven translocation 3 (Tet3) in somatic cells

2019 ◽  
Vol 31 (2) ◽  
pp. 347 ◽  
Author(s):  
Qian-Qian Wang ◽  
Yu-Mei Zhang ◽  
Xia Zhong ◽  
Jian-Wei Li ◽  
Xiao-Rong An ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Hela Cells ◽  
Somatic Cells ◽  
Histone H3 ◽  
Early Embryogenesis ◽  
Maternal Factors ◽  
Nih3t3 Cells ◽  
Mouse Zygote ◽  
Mediated Oxidation

Both developmental pluripotency-associated protein 3 (Dppa3/Stella/PGC7) and dioxygenase ten-eleven translocation 3 (Tet3) are maternal factors that regulate DNA methylation reprogramming during early embryogenesis. In the mouse zygote, dimethylated histone H3 lysine 9 (H3K9me2) attracts Dppa3 to prevent Tet3-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we addressed the interplay between Dppa3 and Tet3 or H3K9me2 in somatic cells. In mouse NIH3T3 cells, the exogenously expressed Dppa3 preferentially accumulated in the cytoplasm and had no effect on Tet3-mediated 5hmC generation. In HeLa cells, the expressed Dppa3 was predominantly localised in the nucleus and could partially suppress Tet3-induced 5hmC accumulation, but this suppressive function was not correlated with H3K9me2. Co-immunoprecipitation assays further revealed an interaction of Dppa3 with Tet3 but not with H3K9me2 in HeLa cells. In cloned zygotes from somatic cells, Dppa3 distribution and 5hmC accumulation in nuclei were not affected by H3K9me2 levels. Taken together, these results suggest that H3K9me2 is not functionally associated with Dppa3 and Tet3 in somatic cells or somatic cell cloned embryos.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

29 COMPARISON OF DNA METHYLATION RATES BETWEEN CLONED CATS AND DOMESTIC CATS

2007 ◽  
Vol 19 (1) ◽  
pp. 133
Author(s):  
S. J. Cho ◽  
S. H. Kang ◽  
S. G. Cho ◽  
I. H. Bae ◽  
C. J. Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Satellite Dna ◽  
Genomic Dna ◽  
Mammalian Species ◽  
Methylation Status ◽  
Somatic Cells ◽  
Genomic Region ◽  
Domestic Cats ◽  
Placental Cells

Many mammalian species have recently been successfully cloned using somatic cells. We have produced cloned kittens by somatic cell nuclear transfer (SCNT). The cloning of mammals by SCNT requires epigenetic reprogramming of the differentiated state of donor cells. We recently observed that feline catus satellite DNA regions exhibit DNA methylation in the donor and somatic cells of a cloned cat. The cause of variation could lie in alterations occurring at different steps of the cloning procedure or incomplete reestablishment of DNA methylation patterns. This study was conducted to determine the methylation status of the feline catus satellite DNA region in the somatic cells of cloned cats and domestic normal cats by using a bisulfite sequencing method. Satellite sequences are heavily methylated in somatic cell chromosomes and are associated with dense heterochromatin where bundles of silent genes are wrapped up together. For the analysis of the cat satellite sequence, a 400-bp segment of the satellite genomic region, which has highly conserved 22 CpG sites, was amplified by PCR from bisulfite-treated genomic DNA, and the resulting PCR products were individually cloned and sequenced. For methylation analysis, genomic DNA was first isolated from somatic and placental cells of cloned and domestic normal cats. The DNA methylation status of the satellite DNA region was not significantly different among donor (using NT) cells (70.6, 77.0, and 79.1% in Donors A, B, and C, respectively), somatic cells of cloned cats (88.9, 82.2, and 85.9% in cloned A-1, 2, and 3; 78.0% in cloned B-1; 88.1, 78.9, 80.3, 83.2, 74.6, and 82.3% in cloned C-1, 2, 3, 4, 5, and 6, all respectively), and domestic cats (88.0, 81.6, and 81.8% in NC 1, 2, and 3, respectively). Also, the DNA methylation status of satellite DNA regions in the placenta was significantly different between cloned cats (80.5, 76.7, and 76.8% in cloned C-2, 5, and 7, respectively) and normal domestic cats (64.2, 66.7, and 74.9% in NC 1, 2, and 3, respectively). In conclusion, all of the somatic cells were highly methylated. The methylation status of the somatic cells was not different among groups, but that of placental cells was significantly different. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

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 < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 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.

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.

scholarly journals Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

2021 ◽  
Author(s):  
Diljeet Gill ◽  
Aled Parry ◽  
Fátima Santos ◽  
Irene Hernando-Herraez ◽  
Thomas M. Stubbs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Cell ◽  
Human Fibroblasts ◽  
Somatic Cells ◽  
Dermal Fibroblasts ◽  
List Type ◽  
Cell Identity ◽  
Mature Adult ◽  
Maturation Phase

AbstractAgeing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Somatic cell reprogramming, the process of converting somatic cells to induced pluripotent stem cells (iPSCs), can reverse these age-associated changes. However, during iPSC reprogramming somatic cell identity is lost, and can be difficult to reacquire as re-differentiated iPSCs often resemble foetal rather than mature adult cells. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of reprogramming, which suggests full iPSC reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first “maturation phase transient reprogramming” (MPTR) method, where reprogramming factors are expressed until this rejuvenation point followed by withdrawal of their induction. Using dermal fibroblasts from middle age donors, we found that cells reacquire their fibroblast identity following MPTR, possibly as a result of persisting epigenetic memory at enhancers. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome, including H3K9me3 histone methylation levels and the DNA methylation ageing clock, was rejuvenated to a similar extent. The magnitude of rejuvenation instigated by MTPR is substantially greater than that achieved in previous transient reprogramming protocols. MPTR fibroblasts produced youthful levels of collagen proteins, suggesting functional rejuvenation. Overall, our work demonstrates that it is possible to separate rejuvenation from pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies.HighlightsWe developed a novel method by which human fibroblasts are reprogrammed until the maturation phase of iPSCs and are then returned to fibroblast identityDNA methylation memory in fibroblast enhancers may allow recovery of cell identity when fibroblast gene expression programmes are already extinctMolecular measures of ageing including transcriptome and DNA methylation clocks and H3K9me3 levels reveal robust and substantial rejuvenationFunctional rejuvenation of fibroblasts by MPTR is suggested by reacquisition of youthful levels of collagen proteins

scholarly journals Chromatin remodeling in somatic cells injected into mature pig oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1037-1049 ◽  
Author(s):  
Hong-Thuy Bui ◽  
Nguyen Van Thuan ◽  
Teruhiko Wakayama ◽  
Takashi Miyano
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Histone H3 ◽  
Chromosome Morphology ◽  
Chromosome Condensation ◽  
Cell Nuclei ◽  
H3 Phosphorylation ◽  
Histone H3 Methylation ◽  
Histone H3 Phosphorylation

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3–S10) and serine 28 (H3–S28), and methylated at lysine 9 (H3–K9), but was not acetylated at lysine 9, 14 and 18 (H3–K9, H3–K14 and H3–K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3–S10 and H3–S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3–K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

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