Histone Deacetylase Inhibition Improves Activation of Ribosomal RNA Genes and Embryonic Nucleolar Reprogramming in Cloned Mouse Embryos
Histone Deacetylase Inhibition Improves Activation of Ribosomal RNA Genes and Embryonic Nucleolar Reprogramming in Cloned Mouse Embryos
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DOI：
10.1095/biolreprod.110.089474
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发表时间：
2011-11-01
影响因子：
3.6
通讯作者：
Kim, Jin-Hoi
中科院分区：
文献类型：
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作者：
Hong-Thuy Bui;Seo, Hyeon-Jeong;Kim, Jin-Hoi
本研究小组发现，用组蛋白去乙酰化酶抑制剂（HDACi）处理体细胞核移植（SCNT）克隆后的胚胎，包括trichostatin A、Scriptaid、亚eroylanilide羟肟酸和oxamflatin，可显著提高效率。尽管许多研究人员已经研究了使用HDACi治疗来提高克隆小鼠胚胎的质量，但这种治疗的机制尚未完全了解。我们认为HDACi对胚胎基因激活（EGA）的影响对克隆胚胎的正常发育具有重要意义。在本研究中，利用高灵敏度荧光原位杂交（FISH）与小鼠rDNA互补的探针，研究了Scriptaid对克隆胚胎rRNA合成起始的影响。此外，为了确定Scriptaid如何影响rDNA转录激活的SCNT胚胎中的pre-rRNA加工机制，我们通过联合评估着床前胚胎的rRNA合成和核仁蛋白分配，详细分析了功能性核仁形成。在本实验中，经过α -amanitin处理后，至少部分FISH定位的rRNA被5-溴吡啶5'-三磷酸染色取代。结果表明，在2细胞后期，许多SCNT胚胎启动转录激活，尽管两个细胞核都处于间期，但其中一个卵裂球显示rRNA转录失活，另一个卵裂球显示rRNA转录活化。此外，在一些SCNT胚胎中，同一细胞核中含有非活性和活性转录rRNA的混合物，这在胞浆内单精子注射胚胎中很少观察到。这种非同步转录导致SCNT胚胎功能核仁激活的一个细胞周期延迟。Scriptaid可以通过在EGA早期激活rRNA基因和促进核仁蛋白分配来克服胚胎基因转录及时启动的失败。
Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.