Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs

Song, Xin; Z, Liu; He, Hongbin; Wang, Yunlong; Li, Huatao; Li, Jingyu; Li, Fangzheng; Jiang, Zhongling; Ye, Huan

doi:10.18632/oncotarget.16507

Cited by 23 publications

(24 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The abnormal expression of DNMTs in donor cells results in DNA methylation defects and aberrant embryo development in pigs [33]. Piccolo et al reported that TET1 and TET2 respectively mediate the demethylation of imprinted genes and pluripotent genes, respectively, during reprogramming [34].…”

Section: Discussionmentioning

confidence: 99%

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

Zhai

Zhang

et al. 2018

Cell Physiol Biochem

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Zhai

Zhang

et al. 2018

Cell Physiol Biochem

View full text Add to dashboard Cite

show abstract

“…These modifications are added and removed by specific enzymes, whose abundance can influence development. For example, removing DNMT1 from pig donor cells facilitated genome methylation and promoted cloned embryo production between zygotic gene activation and the blastocyst stage (Song et al, ). Alternatively, the efficiency of SCNT embryo production was improved by augmenting the level of H3K9Ac in pig donor cells—either by increasing chromatin access (Nebendahl et al, ) or by reducing the level of H3K9me3 (Kim et al, ).…”

Section: Discussionmentioning

confidence: 99%

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

Zhai

Zhang

et al. 2017

Molecular Reproduction Devel

View full text Add to dashboard Cite

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow-derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear-transfer (BMSC-NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC-NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF-NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC-NT embryos (30.0%, 9.8%, respectively) than those in FF-NT embryos (34.2%, 28.0%, respectively). We also found that BMSC-NT embryos had more H3K9Ac and less H3K9me3 and 5-methylcytosine than FF-NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.

show abstract

“…reprogramming, leading to a developmental stop in SCNT embryos (Song et al, 2017). Moreover, its removal allows for an efficient activation of genes essential for embryonic development, thus increasing SCNT efficiency (Song et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The difficulty in achieving efficient methylation reprogramming in SCNT embryos may be a consequence of the resistance of tissue-specific epigenetic patterns to be reprogrammed by the oocyte (Sepulveda-Rincon et al, 2016). Studies have reported that embryo development can be improved by reducing DNMT expression using epigenetic modifier agents and siRNA technology (Song et al, 2017). It has also been found that SCNT embryo development can be significantly improved by strategies that stimulate changes in DNA methylation and histone modifications, such as the use of 5-Aza-2-deoxycytidine (ZdC) (Christman, 2002;Patra and Bettuzzi, 2009;Tsuji et al, 2009), procaine (para-aminobenzoyl-diethylamino-ethanol) (Villar-Garea et al, 2003;Li et al, 2018), S-adenosyl-l-homocysteine (SAH) ( Jeon et al, 2008;Zhang et al, 2014), and scriptaid (Zhang et al, 2014), during cell culture.…”

Section: Introductionmentioning

confidence: 99%

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status ofIn VitroCultured Bovine Skin Fibroblasts

Schumann

Mendonça

Silveira

et al. 2020

DNA and Cell Biology

View full text Add to dashboard Cite

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH ( p = 0.0495) and by the combination of SAH and procaine ( p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH ( p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine ( p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine ( p = 0.0007), SAH ( p = 0.0060), and SAH/procaine ( p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine ( p = 0.0291), SAH ( p = 0.0373), and procaine/SAH ( p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

show abstract

Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs

Cited by 23 publications

References 30 publications

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

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

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status ofIn VitroCultured Bovine Skin Fibroblasts

Contact Info

Product

Resources

About