氧化应激诱导细胞早衰过程中DNMT1介导的DNA甲基化与NSun2介导的mRNA甲基化对共同靶基因的协同调控研究

DNA methylation as well as RNA methylation has been reported to play an essential role in regulating aging-related genes. Nonetheless, the interplay between methylation of DNA and mRNA is thus far unexplored in celluar senescence. Here, we found that (1) DNA methyltransferase DNMT1 decreased concomitant with an overt increase of RNA methyltransferase NSun2 in oxidation-induced premature cellular senescence. (2) DNMT1 methylated NSun2 promoter, while NSun2 methylated DNMT1 mRNA. DNMT1 and NSun2 repressed each other expression reciprocally. Based on these results, Specific aging-related gene expression is supposed to be promoted to the greatest extent through dampened promoter methylation combined with enhanced mRNA methylation, both of which are coupled by the associated alterations of DNMT1 and NSun2. Thus, our study attempts to elaborate on the mechanism of regulation between DNMT1 and NSun2, and further to define the aging-related genes synergistically modulated by the coupled methylation of DNA and mRNA. Moreover, the integrated influence of DNMT1/NSun2-mediated DNA/mRNA methylation of differed genes on premature cellular senescence would be unveiled. Taken together, this work should shed new light on the epigenetics of hierarchy in regulating aging-related genes and also provide more support in fine-tuning gene expression patterns during cellular senescence.

DNA甲基化与mRNA甲基化是细胞衰老过程中关键衰老相关基因表达调控的重要因素，但二者之间是否相互关联，如何关联均未见诸文献报道。前期工作发现，（1）在氧化应激诱导的细胞早衰过程中，DNA甲基化酶DNMT1表达下降，RNA甲基化酶NSun2表达上调；（2）DNMT1甲基化NSun2启动子，NSun2甲基化DNMT1mRNA，二者相互抑制对方表达。据此，我们假设，氧化应激诱导细胞早衰过程中DNMT1与NSun2的表达改变可在降低特定衰老相关基因启动子甲基化的同时增高其mRNA甲基化，从而最大程度促进特定基因表达。本课题拟进一步探讨DNMT1与NSun2相互调控的机制，筛选并确认二者的共同靶标，探讨二者对这些共同靶标的“加合”调控在氧化应激诱导细胞早衰过程中的作用。这些研究将部分解析细胞衰老中不同层次表观修饰调节之间的关系，为进一步揭示细胞衰老过程中相关基因的表达调控机制提供实验依据。

胞嘧啶第5位甲基化（m5C）修饰共同存在于DNA和RNA分子中，分别在不同表观遗传水平调控基因表达及功能，但DNA与RNA m5C修饰在相应调控过程中是否关联，如何关联均未见诸文献报道。前期工作发现，在氧化应激诱导细胞早衰过程中，DNA甲基化酶DNMT1表达下降，RNA甲基化酶NSun2表达上调；DNMT1甲基化NSun2启动子，抑制NSun2表达。然而，利用NSun2突变体高通量富集筛选的DNMT1和NSun2的共同靶基因AXL、NOTCH2以及YAP1，在细胞早衰过程中表达变化并不明显。据此，我们认为这些基因并非DNMT1和NSun2分别介导不同层次m5C修饰共同调控细胞早衰的关键效应基因。我们注意到，这些靶基因在细胞抗凋亡调节中却发挥着重要作用。进一步，在顺铂（Cisplatin, Cpt）或阿霉素（Doxorubicin, Dox）诱导骨肉瘤细胞凋亡过程中，DNMT1表达上调，NSun2表达下降。DNMT1经DNA甲基化抑制NSun2、AXL、NOTCH2以及YAP1转录，NSun2则经mRNA甲基化促进AXL、NOTCH2和YAP1翻译，二者分别介导DNA和mRNA胞嘧啶甲基化，并以耦联的方式加合调控抗凋亡基因AXL、NOTCH2和YAP1表达。上述不同核酸分子m5C修饰之间的关联调控，对药物诱导骨肉瘤细胞凋亡过程中抗凋亡相关基因的表达至关重要，可影响骨肉瘤细胞的化疗抗性。这些发现将为基因表达过程中不同层次表观遗传调节之间的关系提供补充，也可为未来骨肉瘤治疗提供思路和靶点。

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