体细胞中核糖体基因活性对重编程的影响研究

Eukaryotic cells contain several hundred ribosomal RNA genes (rDNA), they transcript to pre-rRNA and then process to become the components of ribosome. Cell growth and proliferation requires enough ribosome to maintain protein synthesis. Only about 50% of the rDNA copies are actually expressed in somatic cells, other rDNA was gradully silenced during development and differentiation. Up to now, reprogramming of somatic cells, such as nuclear transfer cloning (SCNT) or induced pluripoential stem cells (iPS), is still an unefficient event. The reprogramming needs to eliminate the modification of somatic cells during their differentiation, and initiate the undifferentiated state. Reactivation of silenced rDNA may be as same important as reactivate the critial genes for early development, which ensure to synthesize reasonable amount of ribosome and proteins. In the present study, we are going to compare state of active rDNA in different types of cells, analyze their rRNA synthesis, processing, rDNA methylation and histone acetylation. Then we will correspond the effects of rDNA activity with the reprogramming process of SCNT and iPS. Finally we will overexpress or inhibit rDNA transcription, and try to explain whether epigenetic modification of rDNA plays key role in reprogramming of fibroblast cells. We believe our study is likely to guide us in understanding the alterations of rDNA activity in reprogramming, and eventually apply the cloning and iPS technologies into practice.

真核生物基因组中存在大量重复排列的核糖体基因（rDNA），rDNA转录成rRNA前体，再加工形成核糖体组装的成分。细胞的生长和增殖依赖足够的核糖体以维持蛋白质的合成。随着发育和分化rDNA逐渐发生沉默，在体细胞中仅50%的rDNA拷贝具有转录活性。目前无论是体细胞克隆还是诱导多潜能干细胞的重编程，效率都很低。这两种重编程过程都需要去除体细胞的分化修饰，使之转变为未分化的状态。重编程过程能否开启沉默的rDNA转录，从而产生充足的rRNA以保障核糖体和蛋白的合成，这可能和开启发育关键基因的转录同样重要。本次课题我们拟通过rRNA合成、加工、DNA甲基化、组蛋白乙酰化等多个层面比较不同细胞rDNA的转录活性，分析其对体细胞核移植及iPS建系过程中重编程的影响，探讨改变成纤维细胞rDNA活性状态对重编程效率的促进，解析重编程过程的表观调控，为克隆和iPS技术的更好应用奠定基础。

本项目旨在探讨核糖体基因（rDNA）表达对体细胞重编程的影响，核糖体基因表达对核糖体的生物合成及蛋白质的翻译具有关键作用。在分化过程中逐渐沉默的核糖体基因在体细胞重编程过程中能否充分打开，其对体细胞核移植和诱导多潜能干细胞（iPS）有什么影响，这些问题是本研究关注的重点。我们分析了不同供体细胞中rDNA的表达和其启动子区的甲基化程度，比较了不同供体细胞构建的核移植胚胎核仁的超微结构和发育特定阶段的rDNA表达，发现体细胞核移植胚胎功能核仁建立晚于对照ICSI，核移植重编程不能有效的去除rDNA启动子的甲基化；在iPS建立过程中血清饥饿同步细胞周期，促进了rDNA转录和重编程效率；利用生物信息手段比较了两种重编程过程中共同上调和下调的基因，筛选出一系列在重编程过程中起作用的重要基因；分析了甲基结合蛋白MeCP2对重编程的作用，通过过表达MeCP2促进了核移植胚胎发育，并通过染色体免疫共沉淀的方法寻找了MeCP2调控的下游基因，为下一步研究提供了方向；通过诱导自噬有效地提高了体细胞核移植胚胎发育，使囊胚率达到68.6%。通过本项目的实施，我们进一步明确了核糖体合成对重编程的影响，对rDNA转录调控的机制提出了新的观点，也进一步获得了2017年度国家自然基金面上项目的资助（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31671545）。