SiRNA inhibition and not chemical inhibition of Suv39h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos.

Farnoosh Jafarpour,Abbas Kiani-Esfahani,M H Nasr-Esfahani,Somayyeh Ostadhosseini,Faezeh Ghazvini Zadegan,Mehdi Hajian,Peter J Hansen

doi:10.1371/journal.pone.0233880

Abstract

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.

Highlights

Extensive chromatin remodeling plays an indispensable role in different developmental processes, especially after fertilization and during somatic cell nuclear transfer (SCNT) [1,2,3]
Our results did not show any synergistic effect on the inhibition of cell proliferation (Fig 1B), suggesting that chaetocin was cytotoxic in Bovine fetal fibroblast cells (BFFs) cells only at high concentrations
Chaetocin results in global changes to H3K9 trimethylation and H3K9 acetylation in BFF cells

Summary

Introduction

Extensive chromatin remodeling plays an indispensable role in different developmental processes, especially after fertilization and during somatic cell nuclear transfer (SCNT) [1,2,3]. The outcomes of in vitro fertilization (IVF) and SCNT are dependent on adequate chromatin remodeling [3]. The efficiency of SCNT has been found to be low in the majority of mammalian species [5, 6]. Nuclear reprogramming in SCNT-derived embryos is highly error-prone and leads to inadequate early and late embryonic development [7,8,9]. While the mechanisms underlying incomplete reprogramming remain poorly understood, the epigenetic status of the donor cell is an important biological factor for determining the efficiency of SCNT [10, 11]

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