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

Xuexiong Song,Huatao Li,Fangzheng Li,Hongbin He,Jianyu Wang,Jingyu Li,Zhongling Jiang,Zhonghua Liu,Yanjun Huan

doi:10.18632/oncotarget.16507

Abstract

Low development of somatic cell nuclear transfer embryos could be due to the incomplete DNA methylation reprogramming, and Dnmt1s existing in donor cells may be one cause of this disrupted DNA methylation reprogramming. However, the reprogramming pattern of Dnmt1s and its effect on DNA methylation reprogramming in cloned embryos remain poorly understood. Here, we displayed that along with the significantly higher Dnmt1 expression at the zygotic gene activation stage of cloned embryos, genomic methylation level was markedly upregulated, and the arrested rate was significantly higher compared with their in vitro fertilization counterparts. Then, we demonstrated that Dnmt1s, not Dnmt1o, methylation and expression levels in cloned embryos were significantly higher from the 1-cell to 4-cell stage but markedly lower at the blastocyst stage. When Dnmt1s in donor cells was appropriately removed, more cloned embryos passed through the zygotic gene activation stage and the blastocyst rate significantly increased. Furthermore, Dnmt1s knockdown significantly improved itself and genomic methylation reconstruction in cloned embryos. Finally, we found that Dnmt1s removal significantly promoted the demethylation and expression of pluripotent genes in cloned embryos. Taken together, these data suggest that Dnmt1s in donor cells is a critical barrier to somatic cell nuclear transfer mediated DNA methylation reprogramming, impairing the development of cloned embryos.

Highlights

Three main approaches including cell fusion, somatic cell nuclear transfer (SCNT) and transcriptionfactor transduction have been developed to reprogram terminally differentiated somatic cells toward pluripotency, among which, SCNT can induce totipotency to produce cloned animals, with the great application prospect in the fields of agriculture, medicine, species conservation, etc [1,2,3]
We further demonstrated that the percentage of SCNT embryos arrested before zygotic genome activation (ZGA) was significantly higher than that of in vitro fertilization (IVF) embryos during ZGA (Figure 1A1 and 1A2, 49.68% vs 37.50%, P
SCNT embryos displayed the significantly higher genomic methylation level than IVF embryos during ZGA (Figure 1C1 and 1C2, 40.28% vs 20.56%, P

Summary

Introduction

Three main approaches including cell fusion, somatic cell nuclear transfer (SCNT) and transcriptionfactor transduction have been developed to reprogram terminally differentiated somatic cells toward pluripotency, among which, SCNT can induce totipotency to produce cloned animals, with the great application prospect in the fields of agriculture, medicine, species conservation, etc [1,2,3]. The reason for the low cloning efficiency is considered to be the incomplete epigenetic reprogramming [5], among which, DNA methylation, the most studied epigenetic modification, can reflect the epigenetic reprogramming degree of SCNT embryos [7,8,9]. It is thought that the incomplete DNA methylation reprogramming in SCNT embryos can be due www.impactjournals.com/oncotarget to that tissue specific genes in donor cells sustain their fate against nuclear reprogramming induced by oocyte factors [6]. As the most studied repressive factor, the maintenance DNA methyltransferase Dnmt is thought to be closely associated with the aberrant development of SCNT embryos, whether there is a positive relationship between the disrupted expression of Dnmt and the incomplete DNA methylation reprogramming still needs to be determined [12, 13]

Methods

Results

Conclusion