Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

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

Cellular Physiology and Biochemistry ◽

10.1159/000494598 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1376-1397 ◽

Cited By ~ 5

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.

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Aberrant Epigenetic Reprogramming in the First Cell Cycle of Bovine Somatic Cell Nuclear Transfer Embryos

Cellular Reprogramming ◽

10.1089/cell.2020.0079 ◽

2021 ◽

Vol 23 (2) ◽

pp. 99-107

Author(s):

LiJun Wang ◽

LiXiu Liu ◽

YongSheng Wang ◽

Nan Li ◽

HongLi Zhu ◽

...

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Epigenetic Reprogramming

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Lessons Learned from Somatic Cell Nuclear Transfer

International Journal of Molecular Sciences ◽

10.3390/ijms21072314 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2314 ◽

Cited By ~ 1

Author(s):

Chantel Gouveia ◽

Carin Huyser ◽

Dieter Egli ◽

Michael S. Pepper

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Donor Cell ◽

Lessons Learned ◽

Epigenetic Reprogramming ◽

Area Of Interest ◽

Legal Implications ◽

Low Efficiency

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

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Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear - mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination

Reproduction Fertility and Development ◽

10.1071/rd04115 ◽

2005 ◽

Vol 17 (2) ◽

pp. 69 ◽

Cited By ~ 36

Author(s):

Stefan Hiendleder ◽

Valeri Zakhartchenko ◽

Eckhard Wolf

Keyword(s):

Mitochondrial Dna ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Dna Recombination ◽

Epigenetic Reprogramming ◽

Research Activities ◽

Mitochondrial Functions ◽

New Findings ◽

Mitochondrial Dna Recombination

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear–mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.

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15 Combination of RepSox with histone deacetylation inhibitors on invitro development competence of porcine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab15 ◽

2020 ◽

Vol 32 (2) ◽

pp. 133

Author(s):

Z.-B. Luo ◽

M.-F. Xuan ◽

Z.-Y. Li ◽

X.-J. Yin ◽

J.-D. Kang

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Histone Deacetylase Inhibitors ◽

Formation Rate ◽

Epigenetic Reprogramming ◽

Histone Deacetylation ◽

Abnormal Development ◽

Control Group ◽

Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). In this study, we compared histone deacetylase inhibitors combined with the pluripotency inducer RepSox on invitro development of porcine embryos produced via SCNT. Porcine embryos were treated with valproic acid (VPA), mocetinostat, M344 and panobinostat (LBH589) after SCNT, respectively. The porcine embryo invitro-development competence, histone modification level, and pluripotency-related genes expression were analysed. The results showed that LBH589 significantly increased the blastocyst formation rate compared with mocetinostat, M344, and control. In addition, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2 vs. 67.0%; P<0.05). Furthermore, cotreatment with 12.5mM RepSox and 50 nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9 vs. 8.5%, respectively; P<0.05). Moreover, RepSox + LBH589 improved epigenetic reprogramming by histone acetylation and methylation. The expression of pluripotency-related genes NANOG and SOX2 was found to be significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming, and improves the invitro development of porcine SCNT embryos.

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Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning

Reproduction ◽

10.1530/rep-07-0397 ◽

2008 ◽

Vol 135 (2) ◽

pp. 151-163 ◽

Cited By ~ 153

Author(s):

Heiner Niemann ◽

X Cindy Tian ◽

W Allan King ◽

Rita S F Lee

Keyword(s):

Somatic Cell ◽

Cell Nucleus ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Epigenetic Reprogramming ◽

Biological Research ◽

Foetal Development ◽

Research Activities ◽

Somatic Cell Nucleus

The birth of ‘Dolly’, the first mammal cloned from an adult donor cell, has sparked a flurry of research activities to improve cloning technology and to understand the underlying mechanism of epigenetic reprogramming of the transferred somatic cell nucleus. Especially in ruminants, somatic cell nuclear transfer (SCNT) is frequently associated with pathological changes in the foetal and placental phenotype and has significant consequences for development both before and after birth. The most critical factor is epigenetic reprogramming of the transferred somatic cell nucleus from its differentiated status into the totipotent state of the early embryo. This involves an erasure of the gene expression program of the respective donor cell and the establishment of the well-orchestrated sequence of expression of an estimated number of 10 000–12 000 genes regulating embryonic and foetal development. The following article reviews the present knowledge on the epigenetic reprogramming of the transferred somatic cell nucleus, with emphasis on DNA methylation, imprinting, X-chromosome inactivation and telomere length restoration in bovine development. Additionally, we briefly discuss other approaches towards epigenetic nuclear reprogramming, including the fusion of somatic and embryonic stem cells and the overexpression of genes crucial in the formation and maintenance of the pluripotent status. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realising the great potential of SCNT for basic biological research and for various agricultural and biomedical applications.

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Effect of the Time Interval Between Fusion and Activation on Epigenetic Reprogramming and Development of Bovine Somatic Cell Nuclear Transfer Embryos

Cellular Reprogramming ◽

10.1089/cell.2012.0052 ◽

2013 ◽

Vol 15 (2) ◽

pp. 134-142 ◽

Cited By ~ 4

Author(s):

Jun Liu ◽

Yongsheng Wang ◽

Jianmin Su ◽

Lijun Wang ◽

Ruizhe Li ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Epigenetic Reprogramming ◽

Time Interval

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Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.709574 ◽

2021 ◽

Vol 9 ◽

Author(s):

Pil-Soo Jeong ◽

Hae-Jun Yang ◽

Soo-Hyun Park ◽

Min Ah Gwon ◽

Ye Eun Joo ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Histone Deacetylase Inhibitors ◽

Epigenetic Modification ◽

Trichostatin A ◽

Combined Treatment ◽

Epigenetic Reprogramming ◽

Developmental Competence ◽

Developmental Potential

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

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Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

Cellular Physiology and Biochemistry ◽

10.1159/000487579 ◽

2018 ◽

Vol 45 (4) ◽

pp. 1529-1540 ◽

Cited By ~ 13

Author(s):

Zhiren Zhang ◽

Yanhui Zhai ◽

Xiaoling Ma ◽

Sheng Zhang ◽

Xinglan An ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Epigenetic Modification ◽

Expression Patterns ◽

Epigenetic Reprogramming ◽

Preimplantation Embryos ◽

Down Regulation ◽

Low Efficiency

Background/Aims: Aberrantly high levels of H3K4me3, caused by incomplete epigenetic reprogramming, likely cause a low efficiency of somatic cell nuclear transfer (SCNT). Smal molecule inhibitors aimed at epigenetic modification can be used to improve porcine SCNT embryo development. In this study, we examined the effects of MM-102, an H3K4 histone methyltransferase inhibitor, on porcine SCNT preimplantation embryos to investigate the mechanism by which H3K4 methylation regulated global epigenetic reprograming during SCNT. Methods: MM-102 was added to the SCNT embryos culture system and the global levels of various epigenetic modifications were measured by immunofluorescence (IF) staining and were quantified by Image J software. Relative genes expression levels were detected by quantitative real-time PCR. Results: MM-102 (75 μM) treatment reduced global H3K4, H3K9 methylation and 5mC levels especially at the zygotic gene activation (ZGA) and blastocyst stages. MM-102 treatment mainly down-regulated a series of DNA and histone methyltransferases, and up-regulated a number of hitone acetyltransferases and transcriptional activators. Furthermore, MM-102 treatment positively regulated the mRNA expression of genes related to pluripotency (OCT4, NANOG, CDX2) and apoptosis (BCL2). Conclusion: Down-regulation of H3K4me3 with MM-102 rescued aberrant gene expression patterns of a series of epigenetic chromatin modification enzymes, pluripotent and apoptotic genes at the ZGA and blastocyst stages, thereby greatly improving porcine SCNT efficiency and blastocyst quality, making them more similar to in vivo embryos (IVV).

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