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.

中文翻译：

---

DNA甲基化和组蛋白乙酰化之间的串扰触发胶质母细胞瘤细胞中GDNF高转录。

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，阻断其表达可抑制GBM的启动和发育。GDNF是双重启动子基因，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。我们以前曾报道过，GDNF启动子II区的组蛋白超乙酰化和DNA超甲基化导致GBM细胞中GDNF的高转录，但机制尚不清楚。在这项研究中，我们调查了这些修饰是否协同调节GBM中的高GDNF转录。在人类GBM组织和GBM细胞系（U251和U343）中，S133处的环AMP反应元件结合蛋白（CREB）的表达和磷酸化显着增加。在U251 GBM单元中，高表达的CREB显着增强GDNF转录和启动子II活性。CREB通过GDNF启动子II的增强子II和沉默子II中的环AMP响应元件（CRE）调节GDNF转录。但是，这两个CRE发挥了相反的监管作用。有趣的是，沉默子II中CRE的甲基化程度较高，发生在GBM组织和细胞中，分别导致与沉默子II和增强子II结合的磷酸化CREB（pCREB）减少和增加。此外，pCREB募集了具有组蛋白乙酰化酶活性的CREB结合蛋白（CBP）到GDNF增强子II的CRE，从而增加了组蛋白H3乙酰化和RNA聚合酶II募集的位置以及在转录起始位点（TSS），并促进了GDNF在U251中的高转录细胞。结果表明高GDNF转录可归因于GDNF沉默II的CRE中的DNA超甲基化，增加pCREB与增强子II中CRE的结合，从而增强了CBP募集，组蛋白H3乙酰化和RNA聚合酶II募集以及在TSS处的募集。我们的结果表明，pCREB诱导的GDNF启动子II的DNA甲基化和组蛋白乙酰化之间的串扰增强了GDNF高转录，为GBM治疗提供了新的视角。