Department of Neurobiology and Anatomy, Xuzhou Key Laboratory of Neurobiology, Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, China.
Clin Epigenetics. 2020 Mar 17;12(1):47. doi: 10.1186/s13148-020-00835-3.
Glial cell line-derived neurotrophic factor (GDNF) is highly expressed in glioblastoma (GBM) and blocking its expression can inhibit the initiation and development of GBM. GDNF is a dual promoter gene, and the promoter II with two enhancers and two silencers plays a major role in transcription initiation. We had previously reported that histone hyperacetylation and DNA hypermethylation in GDNF promoter II region result in high transcription of GDNF in GBM cells, but the mechanism remains unclear. In this study, we investigated whether these modifications synergistically regulate high GDNF transcription in GBM.
Cyclic AMP response element binding protein (CREB) expression and phosphorylation at S133 were significantly increased in human GBM tissues and GBM cell lines (U251 and U343). In U251 GBM cells, high expressed CREB significantly enhanced GDNF transcription and promoter II activity. CREB regulated GDNF transcription via the cyclic AMP response elements (CREs) in enhancer II and silencer II of GDNF promoter II. However, the two CREs played opposite regulatory roles. Interestingly, hypermethylation of CRE in silencer II occurred in GBM tissues and cells which led to decreased and increased phosphorylated CREB (pCREB) binding to silencer II and enhancer II, respectively. Moreover, pCREB recruited CREB binding protein (CBP) with histone acetylase activity to the CRE of GDNF enhancer II, thereby increasing histone H3 acetylation and RNA polymerase II recruitment there and at the transcription start site (TSS), and promoted GDNF high transcription in U251 cells. The results indicated that high GDNF transcription was attributable to DNA hypermethylation in CRE of GDNF silencer II increasing pCREB binding to CRE in enhancer II, which enhanced CBP recruitment, histone H3 acetylation, and RNA polymerase II recruitment there and at the TSS.
Our results demonstrate that pCREB-induced crosstalk between DNA methylation and histone acetylation at the GDNF promoter II enhanced GDNF high transcription, providing a new perspective for GBM treatment.
胶质细胞源性神经营养因子（GDNF）在胶质母细胞瘤（GBM）中高度表达，阻断其表达可以抑制 GBM 的发生和发展。GDNF 是一个双启动子基因，具有两个增强子和两个沉默子的启动子 II 在转录起始中起主要作用。我们之前报道过，GDNF 启动子 II 区的组蛋白乙酰化和 DNA 高甲基化导致 GBM 细胞中 GDNF 的高转录，但机制尚不清楚。在这项研究中，我们研究了这些修饰是否协同调节 GBM 中 GDNF 的高转录。
在人 GBM 组织和 GBM 细胞系（U251 和 U343）中，环磷酸腺苷反应元件结合蛋白（CREB）的表达和 S133 磷酸化显著增加。在 U251 GBM 细胞中，高表达的 CREB 显著增强了 GDNF 的转录和启动子 II 的活性。CREB 通过 GDNF 启动子 II 中的增强子 II 和沉默子 II 中的环磷酸腺苷反应元件（CRE）调节 GDNF 转录。然而，这两个 CRE 发挥相反的调节作用。有趣的是，在 GBM 组织和细胞中，沉默子 II 中的 CRE 发生高甲基化，导致磷酸化 CREB（pCREB）与沉默子 II 和增强子 II 的结合分别减少和增加。此外，pCREB 将具有组蛋白乙酰转移酶活性的 CREB 结合蛋白（CBP）募集到 GDNF 增强子 II 的 CRE 上，从而增加那里和转录起始位点（TSS）的组蛋白 H3 乙酰化和 RNA 聚合酶 II 的募集，并促进 U251 细胞中 GDNF 的高转录。结果表明，高 GDNF 转录归因于 GDNF 沉默子 II 中 CRE 的 DNA 高甲基化增加了 pCREB 与增强子 II 中 CRE 的结合，从而增强了 CBP 的募集、组蛋白 H3 乙酰化和那里以及 TSS 的 RNA 聚合酶 II 募集。
我们的结果表明，pCREB 诱导的 GDNF 启动子 II 上的 DNA 甲基化与组蛋白乙酰化之间的串扰增强了 GDNF 的高转录，为 GBM 的治疗提供了新的视角。
