DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

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.

Download Full-text

Identification of Animals Produced by Somatic Cell Nuclear Transfer Using DNA Methylation in the Retrotransposon-Like 1 Promoter

Cellular Reprogramming ◽

10.1089/cell.2014.0063 ◽

2014 ◽

Vol 16 (6) ◽

pp. 411-417

Author(s):

Christine Couldrey ◽

Paul Maclean ◽

David N. Wells

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer

Download Full-text

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos

Cellular Reprogramming ◽

10.1089/cell.2014.0084 ◽

2015 ◽

Vol 17 (5) ◽

pp. 404-414 ◽

Cited By ~ 5

Author(s):

Hongliang Sun ◽

Fenghua Lu ◽

Peng Zhu ◽

Xiaohua Liu ◽

Mingming Tian ◽

...

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Histone Acetylation ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer

Download Full-text

Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

Molecular Reproduction and Development ◽

10.1002/mrd.20786 ◽

2007 ◽

Vol 75 (2) ◽

pp. 250-264 ◽

Cited By ~ 28

Author(s):

Aaron J. Bonk ◽

Rongfeng Li ◽

Liangxue Lai ◽

Yanhong Hao ◽

Zhonghua Liu ◽

...

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Aberrant Dna Methylation

Download Full-text

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab45 ◽

2019 ◽

Vol 31 (1) ◽

pp. 148

Author(s):

Y. H. Zhai ◽

X. L. An ◽

Z. R. Zhang ◽

S. Zhang ◽

Z. Y. Li

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Chromatin Modification ◽

Blastocyst Stage ◽

Imprinted Genes ◽

Cell Stage ◽

Genomic Methylation

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P<0.05). The imprinted genes were significantly up-regulated (P<0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P<0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P<0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P<0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

Download Full-text

Antioxidant treatment during manipulation procedures prevents mitochondrial and DNA damage and enhances nuclear reprogramming of bovine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd14027 ◽

2015 ◽

Vol 27 (7) ◽

pp. 1088 ◽

Cited By ~ 7

Author(s):

Hyo-Kyung Bae ◽

In-Sun Hwang ◽

Ji-Ye Kim ◽

Sung-Young Lee ◽

Choon-Keun Park ◽

...

Keyword(s):

Dna Methylation ◽

Dna Damage ◽

Membrane Potential ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Methylation Status ◽

Nuclear Reprogramming ◽

Antioxidant Treatment ◽

Treatment Groups ◽

H2o2 Level

We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P < 0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P < 0.05) in the antioxidant treatment groups (30.5 ± 2.5 to 30.6 ± 1.6%) versus the control (23.0 ± 1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P < 0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

Download Full-text