H3K9me3与H3K27me3对猪体细胞重编程影响机制

It has been proposed that the aberrant histone epigenetic reprogramming during early embryonic development is one of the major reason for the low efficiency of somatic cell nuclear transfer (SCNT) technique, whereas effects of decreasing the DNA methylation or increasing the histone acetylation level, on clone efficiency are limited. This prompts researchers to investigate detailed mechanism of epigenetic reprogramming. There are reports showed that aberrant H3K9me3 modification is the main reason for low efficiency of mouse and human SCNT embryos, and removal of H3K9me3 greatly improves SCNT efficiency. Our group have found that H3K9me3 level is higher at 2-cell and 4-cell stages in SCNT embryos than IVF embryos in pig. Meanwhile, our study indicates that H3K27me3 level is higher in somatic cells and SCNT embryos than IVF embryos, and the reduction of H3K27me3 level in somatic cells or SCNT embryo both can significantly improve development efficiency of cloned embryo. Therefore, the current proposed study aims to determine whether excessive H3K9me3 and H3K27me3 modification was the main reason for aberrant development of SCNT embryo. For this purpose, we will determine the abundance of key methyltransferases and demethylases of H3K9me3/H3K27me3 in preimplantation SCNT and IVF embryo. Key developmental factors or related signal pathways in preimplantation embryo which repressed by H3K9me3 and H3K27me3 will be identified and verified. The answer of this question would help clarifying the mechanism of somatic cell reprogramming in pig.

表观遗传修饰异常是体细胞核移植胚胎发育能力低下的重要原因之一，而降低DNA甲基化或提高组蛋白乙酰化水平并不能有效地解决这一问题。最近的研究表明H3K9me3的异常修饰是小鼠与人的体细胞核移植胚胎发育效率低下的主要原因，通过导入其去甲基化酶能够显著提高核移植胚胎发育效率。本课题组的前期工作发现猪体细胞核移植胚胎2细胞与4细胞期H3K9me3水平显著高于IVF胚胎；同时，核移植胚胎中H3K27me3也呈现高的修饰水平，而降低其水平后能够显著地提高核移植胚胎囊胚率。基于此，本课题拟评价H3K9me3对核移植胚胎发育的影响，探索H3K9me3与H3K27me3修饰相关因子表达模式，挖掘被H3K9me3或H3K27me3修饰调节的关键发育基因，藉此探讨H3K9me3与H3K27me3是否为猪体细胞重编程的关键修饰位点，以期加深对猪体细胞重编程机制的理解。

猪体细胞核移植技术的总体效率仍然较低。大量的研究表明表观遗传修饰异常是体细胞核移植重编程的主要障碍。在植入前的胚胎发育过程中，组蛋白修饰异常会导致胚胎发育异常。本项目对猪核移植胚胎的H3K9me3与H3K27me3水平进行了检测，结果表明H3K9me3其在2细胞及4细胞阶段修饰水平显著高于IVF胚胎，而H3K27me3在2细胞、4细胞及桑葚胚时期核移植胚胎中的修饰水平显著高于IVF胚胎。接下来我们尝试利用不同手段纠正核移植胚胎发育过程中H3K9me3与H3K27me3的异常修饰以及在降低H3K9me3与H3K27me3后评价胚胎发育效率。针对异常H3K9me3，结果显示鼠源及猪源Kdm4d的过表达均未能降低体细胞的H3K9me3水平。利用猪Kdm4a mRNA进行胚胎注射能够显著降低2细胞及4细胞时期H3K9me3修饰水平，并且胚胎发育率显著提高。另外，我们尝试利用H3K9me3相关甲基转移酶抑制剂处理来降低其修饰水平，我们评价了SUV39H1/2的抑制剂Chaetocin对H3K9me3水平及胚胎发育能力的影响。结果表明在4细胞期进行6 h的处理能够显著提高核移植胚胎囊胚率。针对H3K27me3异常修饰，我们利用体外转录的Kdm6a mRNA进行了注射，结果表明过表达Kdm6a能够显著提高体细胞核移植胚胎的囊胚率。进一步我们进行了Chaetocin处理与Kdm6a过表达进行两种修饰的擦除，结果显示同时擦除两种修饰也能够显著提高核移植胚胎发育效率。为了确认受H3K9me3影响的关键基因，我们对ZGA相关潜在Marker基因进行了检测，结果表明在核移植胚胎中Zscan4，Eif1ax，USP26，Ythdf2，Ythdc2和hnRNP A2B1等基因的表达显著低于IVF胚胎，而降低H3K9me3后显著提高了这些基因的表达水平。综上，本项目在猪上确认了H3K9me3与H3K27me3异常修饰是体细胞核移植胚胎发育的关键障碍，利用SUV39H1/2的抑制剂Chaetocin处理及过表达Kdm6a能够有效降低H3K9me3与H3K27me3的修饰水平并最终提高胚胎发育效率。

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