【作者】 李明；

【导师】 魏民； 冯云鹏；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2018， 硕士

【摘要】 基因组DNA的完整、准确与高效复制是生命个体正常生长和发育的前提和基础。由于DNA复制的整个过程是在染色质环境中进行的,染色质结构的调整对于复制的顺利进行至关重要。除了染色质重塑因子和修饰酶,包括乙酰化和甲基化在内的组蛋白翻译后修饰也被认为会在DNA的复制过程中发挥重要作用。近年研究显示,组蛋白也可以发生O-GlcNAc修饰,然而,确切的修饰位点以及其在DNA复制中的功能仍然未知。在本研究中,我们首先利用抑制剂处理分别上调和下调人骨肉瘤细胞(U2OS)中蛋白质的O-GlcNAc修饰水平,而后结合对复制细胞的标记和流式细胞术分析,检测了细胞内基因组DNA复制效率。实验结果显示,抑制剂处理导致的O-GlcNAc修饰水平下降,可以明显地抑制DNA的复制。考虑到DNA复制与细胞周期密切相关,我们随后又分析了O-GlcNAc修饰水平变化对S期进程的影响。利用细胞周期同步化(cell synchronization)和流式细胞术检测,我们发现抑制剂引发的O-GlcNAc修饰下降明显减缓了细胞的S期进程。以上结果说明,细胞中O-GlcNAc修饰水平的变化可以直接影响DNA的复制效率,并改变细胞的S期进程。与已有报道相一致,我们利用化学点击(click-i T?O-GlcNAc enzymatic labeling)和免疫印迹分析证实了组蛋白H3和H4上O-GlcNAc修饰的存在,并进一步针对可能的O-GlcNAc修饰位点进行了定点突变。通过外源转染突变体质粒及O-GlcNAc修饰水平分析实验,我们将H3T32和H4S47分别确定为H3和H4上的O-GlcNAc修饰位点。重要的是,在表达H3T32和H4S47突变体的细胞中,基因组DNA的复制受到了明显地抑制。综合以上,H3T32和H4S47分别是H3和H4上的O-GlcNAc修饰位点,它们很有可能在DNA的复制过程中发挥重要作用。本研究不仅可以为解析组蛋白O-GlcNAc修饰在DNA复制中的功能奠定实验基础,还将为阐释组蛋白翻译后修饰在基因组功能调控中的作用提供有价值的研究模型。更多还原

【Abstract】 The accurate and efficient duplication of the entire genome is fundamentally important for the development of diverse organisms.Since DNA is replicated in the context of chromatin,the organization and re-organization of chromatin structure are thus constantly needed throughout the replication process.Besides chromatin remodelers and modifiers,histone modifications including acetylation and methylation have been shown to be important for DNA replication.Interestingly,recent work has demonstrated that O-GlcNAc modification can also take place on histones.However,O-GlcNAcylation sites on histones and their responsible function in DNA replication remain unclear.In this study,we first used inhibitors to up-regulate or down-regulate the level of O-GlcNAc modification in human osteosarcoma U2 OS cells,which were pulse-labeled by Ed U prior to the analysis of DNA replication efficiency by flow cytometry.It appeared that the down-regulation of O-GlcNAcylation reduced replication efficiency(Ed U positive cells).Under the same condition,S phase progression was delayed revealed by our synchronization assay.These results suggest that O-GlcNAcylation of proteins in cells is required for efficient DNA replication as well as S phase progression.In agreement with previous studies,we also observed clear O-GlcNAc modification in histone H3 and H4 by using a click-i T?O-GlcNAc enzymatic labeling system.Furthermore,we performed site-directed mutagenesis to mutate potential O-GlcNAc modification sites on histone H3 and H4,which were implicated by mass spectrometry data,for the characterization of O-GlcNAc modification site(s).Interestingly,the level of O-GlcNAcylation on H3 and H4 was reduced,when H3T32 and H4S47 were mutated.Importantly,DNA replication was impaired in cells expressing H3T32 and H4S47 as compare to control cells.We thus conclude that H3T32 and H4S47 are the O-GlcNAc modifications sites and probably play an important role in DNA replication.Together,our work will contribute to the functional dissection of histone O-GlcNAcylation in DNA replication,and provide a useful tool for the mechanistic investigation of post-translational modifications on histones in the regulation of genome stability.更多还原