Effects of DNMT1 and HDAC inhibitors on gene-specific methylation reprogramming during porcine somatic cell nuclear transfer.
Effects of DNMT1 and HDAC inhibitors on gene-specific methylation reprogramming during porcine somatic cell nuclear transfer.
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DOI：
10.1371/journal.pone.0064705
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Wu Z
中科院分区：
文献类型：
--
作者：
Xu W;Li Z;Yu B;He X;Shi J;Zhou R;Liu D;Wu Z
体细胞核移植（SCNT）在哺乳动物克隆中的应用目前仍然效率低下。在克隆胚胎发育过程中，供体核不完全或错误的表观遗传重编程导致基因表达异常被普遍认为是导致SCNT效率低下的主要原因。利用小分子重编程修饰剂来辅助体细胞核模拟受精衍生受精卵核中自然发生的DNA甲基化和染色质重塑，已被广泛尝试以提高克隆效率。然而，这些小修饰因子对基因特异性甲基化动力学的影响及其对印迹基因甲基化的潜在影响很少被追踪。在此，我们尝试了两种相对较新的DNMT1抑制剂（DNMTi）和组蛋白去乙酰化酶抑制剂（HDACi） scriptaid和RG108，并展示了它们对猪SCNT胚胎基因特异性DNA甲基化和转录动力学的影响。我们发现scriptaid和RG108在SCNT期间至少部分通过抑制8细胞克隆胚胎中过表达的MBD3来挽救H19的甲基化印记。此外，我们首次在XIST基因5 '侧区发现了差异甲基化区（DMRs），并发现scriptaid单独及其与RG108的组合以不同的方式改变克隆胚胎的转录和DNA甲基化水平的动态。此外，我们发现scriptaid单独使用及与RG108联合使用均能显著促进克隆胚胎中NANOG的转录，增强其着床前发育能力。本研究结果将有助于揭示辅助小分子在提高哺乳动物克隆效率中的表观遗传重编程机制。
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.
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