Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine "Federico II" University of Naples Naples Italy.
Institute of Genetics and Biophysics "Adriano Buzzati Traverso" National Research Council of Italy Napoli Italy.
J Am Heart Assoc. 2024 Mar 19;13(6):e030460. doi: 10.1161/JAHA.123.030460. Epub 2024 Mar 8.
REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na/Caexchanger-1 () transcription in neurons through the binding of RE1 site on brain promoter after stroke. We identified a new putative RE1 site in heart promoter sequence (-RE1) that participates in neuronal transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in transcriptional regulation after stroke.
Luciferase experiments performed in SH-SY5Y cells demonstrated that activity was selectively decreased by REST, whereas activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of RE1 prevented REST-dependent downregulation of . Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to and downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at <0.05.
If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of in the brain, ameliorating stroke damage.
REST（阻遏物元件 1 [RE1]-沉默转录因子）通过结合脑启动子上的 RE1 位点，在中风后抑制神经元中的 Na/Ca 交换器-1 () 转录。我们在 心脏启动子 序列中鉴定了一个新的假定的 RE1 位点 (-RE1)，它参与神经元 的转录。由于 REST 可招募 DNA-甲基转移酶-1 (DNMT1) 和 MeCP2 (甲基-CpG 结合蛋白 2) 到不同的神经元基因上，我们研究了该复合物在中风后对 转录调控的作用。
在 SH-SY5Y 细胞中进行的荧光素酶实验表明， 活性被 REST 选择性降低，而 活性被 DNMT1、MeCP2 和 REST 降低。值得注意的是，RE1 位点的定向突变阻止了 REST 依赖性的 下调。此外，在 8 周龄雄性野生型小鼠（C57BL/6）的颞顶叶皮质中，短暂性大脑中动脉闭塞后，DNMT1、MeCP2 和 REST 与 启动子的结合增加，导致启动子 DNA 高甲基化。侧脑室注射 siREST 可防止 DNMT1/MeCP2 与 结合和 下调，从而减少中风引起的损伤。一致地，在经历氧葡萄糖剥夺加再氧合的皮质神经元中， 敲低抵消了去甲基化剂 5-氮杂胞苷诱导的神经元保护作用。对于两个实验组之间的比较，使用了 Student's 检验，而对于超过两个实验组，使用了单因素方差分析，然后是 Tukey 或 Newman Keuls 检验。统计学意义设定为 <0.05。
如果本研究的结果在人类中得到证实，可以断言，DNMT1/MeCP2/REST 复合物的破坏可能是一种新的药理学策略，可减少大脑中 的 DNA 甲基化，改善中风损伤。
