Epigenetic Modification of Cloned Embryos ImprovesNanogReprogramming in Pigs

Abstract

Incomplete reprogramming of pluripotent genes in cloned embryos is associated with low cloning efficiency. Epigenetic modification agents have been shown to enhance the developmental competence of cloned embryos; however, the effect of the epigenetic modification agents on pluripotent gene reprogramming remains unclear. Here, we investigated Nanog reprogramming and the expression patterns of pluripotent transcription factors during early embryo development in pigs. We found that compared with fertilized embryos, cloned embryos displayed higher methylation in the promoter and 5¢-untranslated region and lower methylation in the first exon of Nanog. When 5-aza-2¢-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of porcine cloned embryos, Nanog methylation reprogramming was also improved, similar to that detected in fertilized counterparts. Furthermore, our results showed that the epigenetic modification agents improved the expression levels of Oct4 and Sox2 and effectively promoted Nanog transcription in cloned embryos. In conclusion, our results demonstrated that the epigenetic modification agent 5-aza-dC or TSA improved Nanog methylation reprogramming and the expression patterns of pluripotent transcription factors, thereby resulting in the enhanced expression of Nanog and high development of porcine cloned embryos. This work has important implications in the improvement of cloning efficiency.

Expression of pluripotency‐related genes is highly dependent on trichostatin A‐assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos

“…Moreover, the use of the artificial modifiers of epigenomically conditioned gene expression, gives rise to the inhibition of both chromatin condensation and transcriptional repression of the genomic DNA of cultured somatic cells that provide a source of donor nuclei for the reconstruction of enucleated oocytes and subsequent generation of cloned embryos (Saini et al, ; Su et al, ; Wang, Zhang, et al, ; Wang, Su, et al, ). On the one hand, those epigenetic modifiers represent not only the subclass of highly specific extrinsic HMT inhibitors (HMTi) such as G9A (H3K9) HMTi, whose pivotal member is diazepin‐quinazolin‐amine derivative termed BIX‐01294 (Cao et al, ; Huang et al, ), but also the subclass of ectopic non‐specific DNMT inhibitors, whose most important members are: (a) 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, , ; Ning et al, ); and (b) zebularine (a nucleoside analog of cytidine; Diao et al, ). On the other hand, they represent the subclass of ectopic non‐selective HDAC inhibitors (HDACi), whose main members are (a) TSA (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, ; Opiela et al, ; Samiec et al, ); (b) scriptaid (SCPT) (Liang, Zhao, Choi, Kim, & Cui, ; Xu et al, ; Zhang et al, ); (c) oxamflatin (Hou et al, ; Mao et al, ); (d) sodium butyrate (NaBu) (Kumar et al, ; Liu et al, ); (e) m ‐carboxycinnamic acid bis hydroxamide (CBHA) (Song et al, ); (f) panobinostat, also known as LBH589 (Jin et al, ); (g) abexinostat, also termed PCI‐24781 (Jin et al, ); (h) quisinostat, also called JNJ‐26481585 (Jin, Guo, et al, ); and (i) dacinostat, also named as LAQ824 (Jin, Lee, Taweechaipaisankul, Kim, & Lee, ).…”

“…On the one hand, those epigenetic modifiers represent not only the subclass of highly specific extrinsic HMT inhibitors (HMTi) such as G9A (H3K9) HMTi, whose pivotal member is diazepin‐quinazolin‐amine derivative termed BIX‐01294 (Cao et al, ; Huang et al, ), but also the subclass of ectopic non‐specific DNMT inhibitors, whose most important members are: (a) 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, , ; Ning et al, ); and (b) zebularine (a nucleoside analog of cytidine; Diao et al, ). On the other hand, they represent the subclass of ectopic non‐selective HDAC inhibitors (HDACi), whose main members are (a) TSA (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, ; Opiela et al, ; Samiec et al, ); (b) scriptaid (SCPT) (Liang, Zhao, Choi, Kim, & Cui, ; Xu et al, ; Zhang et al, ); (c) oxamflatin (Hou et al, ; Mao et al, ); (d) sodium butyrate (NaBu) (Kumar et al, ; Liu et al, ); (e) m ‐carboxycinnamic acid bis hydroxamide (CBHA) (Song et al, ); (f) panobinostat, also known as LBH589 (Jin et al, ); (g) abexinostat, also termed PCI‐24781 (Jin et al, ); (h) quisinostat, also called JNJ‐26481585 (Jin, Guo, et al, ); and (i) dacinostat, also named as LAQ824 (Jin, Lee, Taweechaipaisankul, Kim, & Lee, ). The initiation of chromatin decondensation and gene transcriptional activity is triggered both via highly specific and transient inactivation of G9A (H3K9) HMTs by BIX‐01294 (Cao et al, ; Huang et al, ) and via non‐specifically blocking the biocatalytic activity of either DNMTs by 5‐aza‐dC and zebularine (Diao et al, ; Saini et al, ) or HDACs by TSA, SCPT, oxamflatin, NaBu, CBHA, panobinostat, abexinostat, quisinostat and dacinostat (Jin, Lee, et al, ; Jin et al, , ; Jin, Guo, et al, ; Kumar et al, ; Song et al, ; Zhang et al, ).…”

“…Such epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or SCNT‐derived embryos appears to enhance the reprogrammability of donor cell nuclei that have been transferred into a host cytoplasm of enucleated oocytes. As a consequence, they undergo the faithful recapitulation of the scenario inevitable for switching off somatic cell‐inherited epigenetic memory and accompanying cessation of their tissue‐specific gene expression, followed by correct switching on the epigenetically dependent program of transcriptional activity, characteristic for preimplantation development of cloned embryos (Huan, Wang, et al, ; Huan, Wu, et al, ; Huang et al, ; Jin, Lee, et al, ; Samiec & Skrzyszowska, ; Wang et al, ).…”

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

“…In cloned embryos, however, the genome undergoes incomplete epigenetic reprogramming (Blelloch et al, 2006;Bourc'his et al, 2001;Huan et al, 2015;Kang et al, 2001;Lee et al, 2006;Morgan et al, 2005;Santos et al, 2003), which is considered to be a potential contributor to the overall low cloning efficiency (Dean et al, 2001;Li et al, 2008;Peat and Reik, 2012). In recent studies, to correct or relieve the incomplete epigenetic reprogramming of cloned embryos, different cell types were used as the nuclear donor for pig SCNT, such as fetal fibroblasts (FFs; Onishi et al, 2000), preadipocytes (Tomii et al, 2005), adult mesenchymal stem cells (MSCs; Faast et al, 2006), recloned pig somatic cells (Cho et al, 2007), and induced pluripotent stem cells (iPSCs; Fan et al, 2013).…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Transcriptional profiling to identify the mechanism underlying this abnormal epigenetic reprogramming has been performed in an attempt to improve the developmental competence of SCNT (Oxamflatin) Park et al, 2012) and trichostatin A (TSA) (Huan et al, 2014;Zhao et al, 2010) to improve pig cloning efficiency. It is thought that hyperacetylation will enhance transcription and reprogramming by opening the chromatin structure and allowing RNA polymerase II to access transcription start sites more easily (Van Thuan et al, 2009 ).…”