Effects of DNMT1 and HDAC Inhibitors on Gene-Specific Methylation Reprogramming during Porcine Somatic Cell Nuclear Transfer

Weihua Xu,Bo Yu,Rong Zhou,Zicong Li,Dewu Liu,Junsong Shi,Zhenfang Wu,Xiaoyan He

doi:10.1371/journal.pone.0064705

Abstract

Somatic cell nuclear transfer (SCNT) in mammalian cloning currently remains inefficient. Incomplete or erroneous epigenetic reprogramming of specialized donor somatic nuclear and resulting aberrant gene expression during development of cloned embryos is commonly believed as the main reason that causes the low efficiency of SCNT. Use of small molecular reprogramming modifiers to assist the somatic nucleus to mimic naturally occurring DNA methylation and chromatin remodeling in nucleus of fertilization-derived zygotes, has been widely attempted to improve cloning efficiency. However, impacts of these small modifiers on gene-specific methylation dynamics and their potential effects on methylation of imprinted gene have rarely been traced. Here, we attempted two relatively novel DNMT1 inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi), scriptaid and RG108, and demonstrated their effects on dynamics of gene-specific DNA methylation and transcription of porcine SCNT embryos. We found that scriptaid and RG108 had synergetic effects on rescuing the disrupted methylation imprint of H19 during SCNT at least partially by repression over-expressed MBD3 in eight-cell cloned embryos. Furthermore, we firstly identified a differential methylation regions (DMRs) at 5′ flanking regions of XIST gene and found that scriptaid alone and its combination with RG108 modify the dynamics of both transcription and DNA methylation levels in cloned embryos, by different manners. Additionally, we found that scriptaid alone and its combination with RG108 can significantly promote the transcription of NANOG in cloned embryos and enhance their pre-implantation developmental capacity. Our results would contribute to uncovering the epigenetic reprogramming mechanisms underlying the effects of assisted small molecules on improvement of mammalian cloning efficiency.

Highlights

Applications of cloned pigs in biomedicine and agriculture by somatic cell nuclear transfers (SCNT) have been achieved greatly [1]
To determine the optimum addition of RG108, We firstly designed three levels of RG108 (100 mM, 200 mM, 400 mM) to observe potential cytotoxicity to donor adult fibroblasts and found 400 mM RG108 displayed an obvious deleterious effect on cell proliferation (Figure S1 in File S1). We compared their effects on developmental potentials and obtained the highest average total cells of blastocysts at the moderate levels of RG108 (200 mM) (P,0.05, Table S1 in File S2). 500 nM scriptaid was used in previous reports [4,11]; we set a lower level of scriptaid (100 nM) and compared its effect on embryos developmental capacity with the reported levels (500 nM)
We investigated the dynamics of transcription by quantitative PCR and found XIST was fully re-activated at the morulastage in Con-NT and RG+Scr-NT embryos, whereas shifted earlier at eight-cell stage in Scr-NT embryos (Figure 10), embryos treated with RG108 and scriptaid (RG+Scr-NT) exhibited a similar narrowed reactivation window and a minor expression peak as IVF counterparts (Figure 10), which fit well with a partial de-methylation and fully established methylation in RG+Scr-NT embryos (Figure 8 C)

Summary

Introduction

Applications of cloned pigs in biomedicine and agriculture by somatic cell nuclear transfers (SCNT) have been achieved greatly [1]. Despite these achievements, SCNT technology in pigs, as it does in most mammals, remains inefficient and cloning efficiency is usually around 1–5% of embryos transferred surviving to term [2,3,4]. To facilitate nuclear reprogramming and improve cloning efficiency, several methods, including treating the donor cells and/or early nuclear transferred embryos with DNMT1 inhibitors (DNMTi) like 5-aza-20-deoxycytidine (5-aza-dC) and histone deacetylatse inhibitors (HDACi) like TSA and scriptaid, have been tested to assist the somatic nucleus to mimic DNA methylation and chromatin remodeling[6,8,9]. In mouse, cloned embryos treated with 500 mM RG108 from the two-cell to morula/blastocyst stage, higher POU5F1 expression and more ICM cells were observed [14]

Methods

Results

Conclusion