不同表观遗传修饰间协同调控胶质瘤细胞中GDNF高表达的机制研究

Different types of epigenetic modifications are closely related with tumor initiation and development. High expression of glial cell line-derived neurotrophic factor (GDNF) is an important prerequisite for glioma initiation and development, and this event is related to aberrant promoter DNA methylation and histone acetylation. Our preliminary experiments showed that the binding ratio of cAMP response element-binding protein (CREB) to GDNF enhancer II was increased by DNA methylation in glioblastoma cells. Interestingly, CREB can reportedly recruit histone acetyltransferase CBP/p300. Therefore, we hypothesized that DNA methylation in glioblastoma was coupled with histone acetylation via CREB-recruited CBP/p300 to synergistically promote RNA polymerase II recruitment, thus enhancing GDNF expression. This project will verify that DNA methylation and histone acetylation synergistically regulate GDNF expression; demonstrate that DNA methylation promotes the binding of CREB and CBP/p300 to GDNF enhancer II; and show the impact of CBP/p300 on GDNF promoter histone acetylation, chromatin openness, RNA polymerase II recruitment, and GDNF expression. Implementation of this project will reveal the mechanisms of synergistic gene regulation due to epigenetic modification and provide new information for glioma prevention and treatment.

表观遗传存在多种修饰方式，且这些修饰的异常与肿瘤发生发展关系密切。GDNF高表达是胶质瘤发生发展的重要前提，其高表达与启动子区DNA甲基化和组蛋白乙酰化修饰异常有关。预实验发现，胶质母细胞瘤中DNA甲基化修饰升高了CREB与GDNF增强子Ⅱ的结合比率。且研究证实，CREB可募集组蛋白乙酰化酶CBP/p300。故我们推测：胶质母细胞瘤中DNA甲基化经CREB募集的CBP/p300与组蛋白乙酰化修饰发生偶联，协同促进RNA聚合酶Ⅱ的募集，导致GDNF高表达。本项目拟在证实DNA甲基化与组蛋白乙酰化协同调节GDNF表达的基础上，明确DNA甲基化修饰促进CREB及CBP/p300与GDNF增强子Ⅱ的结合，探讨CBP/p300对GDNF启动子区组蛋白乙酰化、染色质开放程度、RNA聚合酶Ⅱ募集以及GDNF表达的影响。本项目的实施可揭示表观遗传修饰间协同调节基因表达的作用方式，为胶质瘤的防治提供新思路。

胶质细胞系源性神经营养因子（GDNF）与胶质母细胞瘤（GBM）发生发展关系密切。该基因在GBM细胞中的转录水平异常升高，且与GDNF启动子II区DNA甲基化以及该区域组蛋白乙酰化的异常修饰有关。研究表明，以上两种修饰间存在多种调控基因转录的协同方式。然而，GBM中两者是否及如何协同调控GDNF基因的高转录不清。本研究显示：在人GBM组织和GBM细胞系中CREB高表达，高表达的CREB显著上调了U251细胞中GDNF的转录水平及其启动子II区的转录活性。CREB经GDNF启动子II区内增强子II和沉默子II中的CRE元件促进或抑制该基因的转录。U251和U343细胞中，沉默子II内的CRE区域发生高甲基化修饰，该修饰在显著降低CREB与之结合的同时，显著升高了CREB与增强子II内CRE的结合水平。U251细胞中，S133位点磷酸化是CREB调控GDNF转录的必要条件，pCREB募集具有组蛋白乙酰化酶活性的CREB结合蛋白(CBP)至GDNF增强子II内的CRE区域，显著升高该区域和转录起始位点（TSS）区域组蛋白H3的乙酰化水平和RNA聚合酶II的募集量。结果表明：GDNF基因沉默子II内CRE区域发生高甲基化修饰，迫使pCREB升高与增强子II内CRE的结合，经CBP募集增加、增强子II内CRE和TSS区域组蛋白H3乙酰化升高、TSS区域RNA聚合酶II的募集量上调，最终导致了GBM细胞中GDNF基因的高转录。此外，发现Egr-1和RNA POL II共同参与了GBM细胞中启动子II区组蛋白高乙酰化对GDNF基因转录的促进作用。本研究进一步揭示了GDNF基因高转录的表观遗传学机制，为GBM的治疗提供了新思路。