Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Pil-Soo Jeong,Tsevelmaa Nanjidsuren,Hyo-Gu Kang,Deog-Bon Koo,Sanghoon Lee,Sun-Uk Kim,Bong-Seok Song,Min Ju Kim,Bo-Woong Sim,Soo-Hyun Park

doi:10.3390/ijms21144836

Abstract

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Highlights

The pig is a useful animal model because of its physiological and anatomical similarities to humans [1,2]
We investigated the effects of chaetocin on the H3K9me3 level and global DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos, and confirmed the effect of chaetocin on autophagic activity and the levels of maternal mRNAs in porcine SCNT embryos using immunofluorescence and quantitative real-time polymerase chain reaction
SCNT embryos showed a significantly higher level of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilization (IVF) embryos, whereas there were no differences at the morula and blastocyst stages (Figure 1E)

Summary

Introduction

The pig is a useful animal model because of its physiological and anatomical similarities to humans [1,2]. Incomplete reprogramming in SCNT embryos results in a low cloning efficiency [7]. Histone H3 lysine 9 trimethylation (H3K9me3) is enriched in reprogramming resistant regions of SCNT embryos and blocks donor cell reprogramming, leading to failure of preimplantation development in mice [8], humans [9], pigs [10,11], and monkeys [12]. The H3K9me3-specific methyltransferases suv39h1 and suv39h2 are highly expressed in heterochromatin regions of mammalian cells [14]. Injection of an H3 lysine9-specific demethylase (KDM) into SCNT embryos or silencing of the expression of suv39h1 and suv39h2 in donor cells modulates the H3K9me level and so increases developmental efficiency [8,15]. Previous studies reported that suv39h1 and suv39h2 interact directly with the DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, and DNMT3b, to methylate DNA with heterochromatin protein 1 (HP1), a transcriptional repressor, the end result being modulation of gene transcription [16,17]

Methods

Results

Conclusion