转录因子p53、Sox2的翻译后修饰调控研究

【作者】 赵海涌；

【导师】 张业； 沈珝琲；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 在个体发育过程中以及应对外界刺激的时候,细胞内发生了一系列的基因转录事件。决定一个基因是否转录的因素主要包括调节区域染色质的开放状态,转录因子的局部丰度。组蛋白以及转录因子的翻译后修饰是调节基因转录的重要方面。DNA缠绕在组蛋白八聚体上形成核小体,一个个核小体构成串珠样的染色质纤维,并可以进一步折叠包装成更高级的结构。染色质的结构愈紧密,转录因子就愈加难以接近其特异的DNA元件。组蛋白的共价修饰可以直接或间接地影响染色质结构。如H3K4的三甲基化可以募集专司激活的蛋白质复合物,对组蛋白进行乙酰化,打开核小体间的聚合状态,减弱组蛋白和DNA间的相互作用,并有依赖ATP水解能量的蛋白复合物同时对染色质结构进行物理重塑,最终使DNA充分暴露,形成开放的染色质状态；与之相反,H3K27的三甲基化则募集使组蛋白去乙酰化的复合物,在各种蛋白复合物的配合下将核小体装配成更高级的异染色质样结构,DNA被紧密包裹,转录因子不能靠近。转录因子的翻译后修饰如多聚泛素化可引起转录因子的降解,磷酸化乙酰化甲基化可能引起转录因子亚细胞分布变化,或者改变转录因子与DNA以及其它大分子的相互作用,最终影响其转录活性。转录因子的各种共价修饰不是孤立存在的,往往互相促进或拮抗,所以共价修饰实际上是各种上级信号的输入,经转录因子整合后输出为转录因子结合调节元件及持续调节靶基因转录的能力。我们分别以p53调控细胞凋亡的过程和全反式维甲酸(RA)诱导的P19细胞神经分化过程为对象,就翻译后修饰对蛋白质功能的调节作用进行了一些研究。第一部分翻译后修饰与p53功能利用放射性同位素氚标记的甲基供体进行in vivo甲基标记实验,我们发现p53核心结构域在细胞内发生了甲基化；利用特异识别甲基化精氨酸的抗体,我们确定p53核心结构域在细胞内发生了精氨酸的甲基化。文献报道：免疫沉淀得到的内源p53蛋白经质谱鉴定,第110、209、213位精氨酸有甲基化形式存在。我们将第110、209、213位精氨酸同时突变为赖氨酸后,p53的转录活性被严重抑制。在蛋白质的精氨酸上还可以发生PAD家族催化的肽酰脱氨基修饰。PAD4是PAD家族唯一能够进行核定位的成员,能够在细胞内修饰组蛋白。我们想知道PAD4是否能够直接修饰p53并调节其活性。已经有文献报道了p53可以和PAD4相互作用,并能募集PAD4到靶基因启动子区修饰组蛋白引起染色质结构变化,导致靶基因的表达受到限制,所以我们直接检测了p53是否为PAD4的底物。我们发现p53可以在in vivo和in vitro水平上被PAD4脱氨基化,脱氨基作用使得p53的电泳迁移率发生变化。在in vitro的乙酰化反应实验中,PAD4的脱氨基作用抑制p53乙酰化。在不能表达内源p53的H1299细胞中进行的双荧光素酶报告基因实验显示,随着过表达PAD4蛋白量的增加,p53驱动报告基因表达的能力逐渐降低。根据这些结果,我们认为PAD4可以修饰p53,并引起p53转录活性的降低。在HEK293A细胞中,我们发现过表达PAD4诱导内源p53表达的显著上调。结论：PAD4对p53的调节是多方面的,可以通过直接修饰p53或被p53募集修饰染色质抑制p53活性,同时也能通过其它途径引起细胞应激状态激活p53表达。第二部分翻译后修饰与P19细胞多能性维持及神经分化为了研究蛋白质甲基化对P19细胞多能性维持及其神经分化过程的影响,我们用甲基转移酶抑制剂腺苷二醛(AdOx)来处理细胞。我们发现：1.在RA诱导时加入AdOx处理抑制P19神经分化早期转录因子Mashl和Ngnl的上调。2. AdOx处理后的P19细胞再经RA诱导,不能向神经外胚层方向分化,说明抑制蛋白质甲基化会限制P19细胞的多能性,我们对其具体机制进行了一些研究,发现：a. AdOx处理引起P19细胞周期阻滞和形态变化。b. AdOx处理抑制多种组蛋白甲基化修饰,且程度各不相同。c. AdOx处理上调多能性相关基因Utf1、Fgf4、Nanog、Sox2的转录。d. AdOx处理抑制多能性维持的关键转录因子之一Sox2的转录活性,改变Sox2的亚细胞定位,限制Sox2与染色质的动态结合能力。e.精氨酸甲基转移酶Carm1能够与Sox2相互作用,并在in vivo和invitro水平上甲基化Sox2。f. Carm1甲基化Sox2的主要位点是113位精氨酸。g.Sox2的正常转录活性需要Carm1,过表达Carm1促进Sox2转录活性。113,115,116位精氨酸同时突变使得AdOx对Sox2转录活性的抑制能力减弱。h.Sox2能被p300乙酰化,Carm1和p300能共同促进Sox2的转录活性；Akt介导的磷酸化也参与Sox2活性调节；Carm1过表达可以抑制发生在Sox2上的位点特异性蛋白酶切割。结论：蛋白质甲基化对于P19细胞多能性的维持以及神经分化能力至关重要；精氨酸甲基化参与Sox2活性调节；Sox2活性受多种翻译后修饰的共同调节。更多还原

【Abstract】 For a eukaryotic gene, its transcription need an active transcription factor and an "open" chromatin confirmation in its regulatory region. Nucleosomes composed of core histones octamer and surrounded DNA are the fundamental repeating units of the eukaryotic "beads-on-a string" chromatin fibre. Chromatin fibre can be compacted into more complex spatial organizations. Modification of core histones play a key role in structure regulation of chromatin. Different modifications integratively affect the interactions among nuceosomes and affinity of core histones to DNA. Compacted chromatin exhibits reduced accessibility for transcription factors. Post-translational modifications of transcription factors also modulate transcription. Poly-ubiquitination leads to transcription factors degradation; phosphorylation, acetylation and methylation sometimes affect transcription factors subcellular distribution, affinity to DNA elements or other macromolecules. Different modifications may regulate transcription factor synergistically or exclusively, all kinds of upstream signals are integrated into modulated transcription activities.We investigated covalent modifications mediated p53 activity regulation, we also explored the roles of post-tranlational modifications in pluripotency maintenance and retinoic acid induced neural differentiation of P19 cells.Part I post-translational modifications and p53 regulation.By performing [3H-methyl] labeling in vivo, methylation of p53 core domain was detected; with a specific antibody, we found arginine methylation was involved in modifications of p53 core domain. P53 R110,209,213 had been reported to be methylated in vivo by mass spectrometry analysis. In the dual-luciferase reporter assays, transcription activity of p53 with tri-valent mutation of R110,209,213 to lysines was severely reduced.Peptidyl-deimination by PAD family is another post-translational modification of arginine residues. PAD4, the only PAD resides in nucleus, deiminates histones and is implicated in chromatin regulation. We sought to figure out whether PAD4 deiminates p53 and regulates its activity. It had been reported that p53 interacts with PAD4 and recruits PAD4 to target gene promoters to modify histones, as a result target gene expressions are restrained. At first we detected whether p53 is a substrate for PAD4.We found that PAD4 deiminates p53 in vitro and in vivo, causes changed mobility of p53 in SDS-PAGE.PAD4 deimination reduces p53 acetylation in vitro.PAD4 represses p53 transcription activity in H1299 cells, a cell line doesn’t express endogenous p53.PAD4 induces endogenous p53 expression in HEK293A cells.Taken together, arginine methylation takes part in p53 regulation. PAD4 regulates p53 in three possible ways:direct deimination of p53; deimination of histones in p53 target promoters; induction of endogenous p53 expression.Part II To get a insight into the contribution of protein methylation to pluripotency maintenance and neural differentiation, we treated P19 cells with a methyltransferase inhibitor AdOx. Results are listed below.1. AdOx reduces P19 neural differentiation.2. AdOx impaires P19 cells pluripotency maintenance.3. AdOx causes P19 cells morphological change and blocked cell cycle.4. AdOx differentially inhibits histone methylations.5. AdOx upregulates transcription of pluripotency related genes(Utfl, Fgf4, Sox2, Nanog).6. AdOx reduces Sox2 transcription activity, alters Sox2 subcellular distribution, inhibits the dynamic association of Sox2 with chromatin.7. Protein arginine methyltransferase Carm1 interacts and methylates Sox2 in vitro and in vivo.8. Carml methylates Sox2 preferentially at R113.9. Carml enhances Sox2 transcription activity. R113,115,116K tri-valent mutation reduces the inhibition of Sox2 activity by AdOx. 10. p300 interacts and acetylates Sox2, stimulations of Sox2 activity by Carml and p300 are compatible; Akt negatively regulates Sox2 activity; Carml can prevent Sox2 from site-specific proteolytic cleavage.Taken together:protein methylation is essential for P19 cells pluripotency maintenance and neural differentiation; arginine methylations modulate Sox2 transcription activity; different post-translational modifications jointly regulate Sox2.更多还原