组蛋白变体H3.3掺入rDNA异染色质在体细胞重编程中的作用研究

Transcription of rDNA gene controls biosynthesis of the ribosome and regulates the translation of proteins in cell. Our previous studies indicated that efficiency of somatic cell reprogramming was affected by rDNA activity. And somatic cell reprogramming could be improved when rRNA transcription was increased. It is known that histone H3 variant H3.3 is related to active transcription, which incorprates into paternal chromatin is a critial factor involving in preimplantation embryonic development. Researches also demonstrated that H3.3 is an indispensable maternal factor for development of somatic cell nuclear transfer cloned embryos. DAXX mediating H3.3 incorporation into rDNA chromatin leads to opening state of the chromatin which might be the key event in early stage of somatic cell reprogramming. In the present study, we are going to investigate dynamic changes and epigenetic modification of histone variant H3.3 incorporating into rDNA chromatin during two reprogramming procesures, namely, somatic cell nuclear transfer (SCNT), and induced pluripotent stem cells (iPS) . And we will discuss effects of H3.3 incorporation into rDNA chromatin on rRNA transcription. Furthermore, we will try to improve reprogramming efficiency through regulate rRNA transcription by controling H3.3 incorportion. We believe this study will facilitate understanding of histone code and rDNA activity involving in somatic reprogramming, and will be of great significance to apply SCNT and iPS technologies into practice.

rDNA基因的转录控制着细胞核糖体的生物合成，从而调控蛋白质的翻译。我们的前期研究表明体细胞rDNA活性影响了重编程的效率，提高rRNA转录有利于体细胞重编程。H3.3是与转录激活相关的组蛋白变体，其掺入父源染色质是早期胚胎发育的关键影响因素， H3.3也是体细胞核移植胚胎发育不可或缺的母源因子。DAXX介导的H3.3掺入rDNA染色质，从而开放rRNA转录可能是体细胞重编程的早期关键事件。本次申请旨在研究体细胞在核移植和诱导多潜能干细胞两种重编程过程中，H3.3掺入rDNA染色质区的动态变化及其表观修饰的改变，并研究 H3.3掺入使rDNA染色质开放对rRNA转录的影响。进一步通过调控H3.3掺入，促进rDNA转录，从而促进体细胞重编程效率。我们的研究对理解重编程中的组蛋白密码与rDNA活性的作用,提高重编程效率，促进核移植与诱导多潜能干细胞技术的应用具有重要意义。

本课题围绕H3.3及组蛋白修饰对核糖体基因（rDNA）表达的调控展开，重点关注rDNA转录和核糖体生物合成对两种体细胞重编程过程，即体细胞核移植（SCNT）和诱导多潜能干细胞（iPSC）的影响。首先通过原位杂交和免疫荧光确定了单精注射（ICSI）和SCNT胚胎中母源H3.3及其分子伴侣DAXX整合进入父源或体细胞基因组，并与rDNA存在共定位。敲减Daxx后rDNA转录增加，但是不利于基因组的稳定和克隆胚胎发育。随后我们利用药物处理、rRNA合成分析、透射电镜观察等，确定了在核移植之前短暂抑制供体细胞的rDNA转录，可以加速体细胞的核仁退化，促进克隆胚胎功能核仁超微结构的重建，提高SCNT胚胎发育。通过分析比较ChIP-seq数据、敲除Daxx、ChIP-qPCR、ChIP-reChIP、甲基化分析等方法，我们证明了ATRX/DAXX复合体通过H3.3在沉默rDNA的启动子建立H3K9me3，且这一复合物是沉默rDNA的状态维持所必需的。在iPSC中敲除Daxx促进rDNA转录和神经发育相关基因的表达。对SCNT和iPSC这两种重编程中的抑制性组蛋白修饰H3K27me3进行了分析，发现在SCNT胚胎中过表达了其去甲基化酶Kdm6a，显著降低了SCNT胚胎中H3K27me3含量，改善了异常的X染色体失活，提高了SCNT的发育。进一步，我们发现过表达Kdm6A可促进iPSC的重编程效率，而这种促进作用依赖于其具有去甲基化酶活性的JMJC结构域。KDM6A可以通过擦除多能性以及细胞代谢相关基因上H3K27me3，从而改变细胞代谢以及基因表达模式，使之更加接近于iPSC的状态。KDM6A还可促进IL-6的表达，而高水平的IL-6也可以促进iPSC的重编程效率。此外，KDM6A还可促进PTEN的表达，从而减少p-AKT和p-mTOR的蛋白水平，进而促进体细胞重编程。.核糖体对体细胞重编程的影响是本课题组的研究方向，通过本课题的研究我们阐述了rDNA及其分子伴侣和组蛋白修饰对重编程的作用，为深入理解体细胞重编程的机制提供了理论依据。