Oct4翻译后修饰调控多能干细胞自我更新和定向分化的机理研究

发布时间：2018-03-14 07:28

本文选题：多能干细胞　切入点：自我更新　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：胚胎干细胞和胚胎癌细胞因具有多向分化的潜能而被统称多能干细胞,其自我更新及多能性受各种转录因子、表观调控因子及相关信号通路共同调控。这些多能干细胞在体外培养条件下被诱导分化的分子机制与胚胎早期发育类似,已被作为模型广泛用于早期胚胎发育研究。实现对细胞分化调控机制的全面解析,将有力推动干细胞生物学、发育生物学和再生医学等的发展。OCT4是胚胎发育过程中的核心转录因子,它能调节细胞的多能性和命运决定,参与调控胚胎发育过程中三胚层的形成。Oct4的功能很大程度取决于它的蛋白水平。Oct4蛋白水平的改变会影响它与其它蛋白的互作以及与DNA的结合。有研究报道Oct4蛋白上特定位点的翻译后修饰(如SUMO化、磷酸化或泛素化)会显著影响Oct4蛋白的生物学功能,但Oct4蛋白的翻译后修饰又是受什么信号通路调控,以及这些修饰是如何影响Oct4对靶基因选择性调控的机制仍不明了。近期有研究显示蛋白激酶Akt也是调控胚胎干细胞自我更新能力的重要因子。本论文工作揭示Akt在胚胎癌细胞中能促进Oct4-T235位点磷酸化,未磷酸化的Oct4结合在AKT1启动子上抑制其转录,而Akt介导的Oct4磷酸化会激活AKT1的转录。Oct4-T235位点磷酸化修饰能促进胚胎癌细胞的自我更新,并抑制细胞向外胚层分化。Oct4-Sox2蛋白复合物是维持多能干细胞自我更新和多能性的关键因子。有研究发现Oct4蛋白在SUMO化后,Oct4-Sox2异二聚体的结合被破坏.然而 Oct4-Sox2互作的位点及该互作在细胞分化过程中的动态变化仍不清楚。本论文工作揭示了Oct4蛋白上高度保守的K156位点与D212形成分子间盐桥来稳定Oct4蛋白POU结构域的空间构象,从而加强Oct4-Sox2的结合。当K156被修饰或突变时(比如在膀胱癌患者组织样本中发现的K156N突变),K156-D212分子间盐桥的形成受阻,从而影响Oct4-Sox2异二聚体形成。在细胞中Oct4-K156N会减少Oct4与Sox2的结合,显著抑制受Oct4-Sox2二元调控的下游干性基因的转录,同时促进中内胚层分化基因的表达,并且可通过上调SNAIL家族基因的表达,来促进EMT过程。综上所述,本论文工作证明Oct4蛋白上特定位点的翻译后修饰或突变会通过调节其转录活性、蛋白互作、蛋白稳定性等,协同调控多能干细胞的自我更新、定向分化和癌变增殖等过程。
[Abstract]:Embryonic stem cells and embryonic cancer cells are commonly known as pluripotent stem cells because of their potential for multidifferentiation. Their self-renewal and pluripotency are subject to various transcription factors. These pluripotent stem cells are induced to differentiate in vitro through a molecular mechanism similar to that of early embryonic development. Has been widely used as a model for early embryonic development research. To achieve a comprehensive understanding of the mechanism of cell differentiation regulation will have the power to promote stem cell biology, Development of Developmental Biology and Regenerative Medicine .OCT4 is the core transcription factor in embryonic development, which regulates the pluripotency and fate of cells. The function of participating in regulating the formation of tridermal layer. Oct4 depends largely on its protein level. Oct4 protein level changes will affect its interaction with other proteins and binding with DNA. It has been reported that Oct4 protein. Post translational modification (E. g., SUMO) at specific sites, Phosphorylation or ubiquitin can significantly affect the biological function of Oct4 protein, but the posttranslational modification of Oct4 protein is regulated by what signaling pathway. It is still unclear how these modifications affect the selective regulation of target genes by Oct4. Recent studies have shown that protein kinase Akt is also an important factor in regulating the self-renewal ability of embryonic stem cells. This work reveals that Akt is also an important factor in the self-renewal of embryonic stem cells. In embryonic cancer cells, phosphorylation of Oct4-T235 sites is promoted. Unphosphorylated Oct4 binds to the AKT1 promoter to inhibit its transcription, while Oct4 phosphorylation mediated by Akt activates the phosphorylation of AKT1 at .Oct4-T235 site, which promotes the self-renewal of embryonic cancer cells. Inhibiting the differentiation of cells into ectoderm. Oct4-Sox2 protein complex is the key factor to maintain the self-renewal and pluripotency of pluripotent stem cells. Some studies have found that the binding of Oct4-Sox2 heterodimer of Oct4 protein was destroyed after SUMO. However, Oct4-Sox2 interacted. The dynamic changes of the site and the interaction in the process of cell differentiation are still unclear. In this work, it is revealed that the highly conserved K156 site on the Oct4 protein forms an intermolecular salt bridge with D212 to stabilize the spatial conformation of the POU domain of the Oct4 protein. When K156 is modified or mutated (such as K156N mutation found in bladder cancer tissue samples), the formation of intermolecular salt bridge of K156-D212 is blocked, which affects the formation of Oct4-Sox2 heterodimer. In cells, Oct4-K156N reduces the binding of Oct4 to Sox2. It can significantly inhibit the transcription of downstream dry genes regulated by Oct4-Sox2, promote the expression of mesodermal differentiation genes, and promote the EMT process by upregulating the expression of SNAIL family genes. In this work, we have demonstrated that the post-translational modification or mutation of specific sites of Oct4 protein may regulate its transcriptional activity, protein interaction, protein stability, and co-regulate the self-renewal, directional differentiation and carcinogenesis of pluripotent stem cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730

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