TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

RESEARCH ARTICLE TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos Zubing Cao☯, Renyun Hong☯, Biao Ding, Xiaoyuan Zuo, Hui Li, Jianping Ding, Yunsheng Li, Weiping Huang, Yunhai Zhang* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Anhui Provincial Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Cao Z, Hong R, Ding B, Zuo X, Li H, Ding J, et al. (2017) TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos. PLoS ONE 12(1): e0169092. doi:10.1371/journal.pone.0169092 Editor: Austin John Cooney, University of Texas at Austin Dell Medical School, UNITED STATES Received: August 13, 2016 Accepted: December 12, 2016 Published: January 23, 2017 Copyright: © 2017 Cao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, cotreatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only cotreatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Natural Science Foundation of China (31272442) and National Transgenic New Species Breeding Program of China (2014ZX08006-01B) and National Science Foundation of the Higher Education Institutions of Anhui Province (KJ2016A227). Competing Interests: The authors have declared that no competing interests exist. Introduction Genetically modified pigs generated by genomic editing technology are widely used as animal model for human diseases in biomedical researches and also as initial genetic resources for breeding in swine production [1]. Previous studies showed that homologous recombinationmediated gene targeting in embryonic stem cells are widely used to generate the transgenic model organisms [2]. Recently, nuclease-driven genomic editing technologies including zincfinger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and PLOS ONE | DOI:10.1371/journal.pone.0169092 January 23, 2017 1 / 15 TSA and BIX-01294 Restore Normal Methylation in SCNT Embryos CRISPR/Cas9 in zygotes are becoming more popular for the generation of mutant animals [3]. Therefore, these novel genomic editing technologies-mediated genetic modifications in somatic cells combined with somatic cell nuclear transfer (SCNT) technology may be the primary method for generating the transgenic pigs in future. SCNT is thought to be a promising and powerful biotechnology that the terminally differentiated somatic cells are successfully reprogrammed into the totipotent embryo by transplanting a donor nucleus into an enucleated oocyte [4,5]. Indeed, more than 20 different cloned species have been successfully generated by SCNT until now[6]. However, the cloning efficiency has been very low for many species, especially, porcine cloning efficiency is yet less than 5% [7], which significantly limits the biomedical and agricultural application of transgenic pigs. Increasing evidence suggests that defective epigenetic reprogramming of donor cell’s nuclear chromatins [8] and abnormal gene expression profiles [9] likely contribute to the overall poor cloning efficiency. To date, a number of strategies have been developed to improve the cloning efficiency. One of the most commonly utilized strategies involves the application of small molecule inhibitors to regulate the epigenetic modifications of cloned embryos. Histone deacetylases (HDACs) inhibitors, such as TSA [10,11], Scriptaid [12], VPA [13], m-carboxycinnamic acid bishydroxamide [14], significantly increased the early or full-term developmental efficiency of porcine SCNT embryos through improvements in histone acetylation. In addition, histone methylation might affect the reprogramming efficiency of somatic cells to pluripotent or totipotent state. Recently, H3K9me3 and H3K79me3 are identified as two negative reprogramming regulators for generating the induced pluripotent stem cells in mouse [15] and human [16]. Similarly, abnormal histone methylation profiles involving H3K9me2 and H3K9me3 are discovered during porcine early SCNT embryonic development in our previous study [17]. Based on these studies, we hypothesize that abnormal histone methylation leads to the reduction of developmental efficiency of SCNT embryos. Recent studies demonstrate that histone demethylase, either KDM4A or KDM4B overexpression-mediated reduction in H3K9me3 level dramatically improves the overall mouse cloning efficiency [18,19]. Meanwhile, G9a knockdown by RNAi or the reduction of H3K9me2 level through a small molecule inhibitor BIX-01294-mediated inhibition of G9A activity significantly promotes the cloning efficiency in mouse [20] and pig [21]. However, the molecular mechanisms underlying the enhanced cloning efficiency in pig via modulating the status of histone acetylation and methylation remain largely unknown. Therefore, in the present study we attempt to elucidate the potential mechanisms involved in the improved early development of porcine SCNT embryos exposed to TSA and BIX-01294. Materials and Methods All reagents were purchased from Sigma (Sigma-Aldrich, St Louis, MO) unless otherwise stated. Ethics statement All experiments involving animals were conducted in accordance with (...truncated)