PDF(Pubmed)
Abstract：
BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 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 Ncx1 transcriptional regulation after stroke.
RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. 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 Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student\'s t 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 P<0.05.
CONCLUSIONS: 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 Ht in the brain, ameliorating stroke damage.
RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. 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 Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student\'s t 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 P<0.05.
CONCLUSIONS: 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 Ht in the brain, ameliorating stroke damage.
摘要：
背景：脑卒中后，抑制因子1[RE1]-沉默转录因子通过结合脑启动子（Br）上的RE1位点抑制神经元中Na+/Ca2+交换因子-1（Ncx1）的转录。我们在Ncx1心脏启动子（Ht）序列（Ht-RE1）中确定了一个新的推定RE1位点，该位点参与神经元Ncx1转录。因为REST在不同的神经元基因上招募DNA-甲基转移酶-1（DNMT1）和MeCP2（甲基-CpG结合蛋白2），我们研究了该复合物在卒中后Ncx1转录调控中的作用。
结果：在SH-SY5Y细胞中进行的荧光素酶实验表明，Br活性被REST选择性地降低，而Ht活性被DNMT1、MeCP2和REST降低。值得注意的是,Ht-RE1的定点突变阻止了Ncx1的REST依赖性下调。此外，在短暂大脑中动脉闭塞的8周龄雄性野生型小鼠（C57BL/6）的颞顶皮层中，DNMT1、MeCP2和REST与Ht启动子的结合增加，随之而来的DNA启动子超甲基化。侧脑室注射siREST可防止DNMT1/MeCP2结合Ht和Ncx1下调，从而减少中风引起的损伤。始终如一，在接受氧和葡萄糖剥夺加复氧的皮质神经元中，Ncx1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，使用学生的t检验，而对于两个以上的实验组，使用了一元方差分析，其次是Tukey或NewmanKeuls.P<0.05时具有统计学意义。
结论：如果这项研究的结果在人类中得到证实，可以断言DNMT1/MeCP2/REST复合物破坏可能是减少大脑中HtDNA甲基化的新药理学策略，改善中风损伤。
结果：在SH-SY5Y细胞中进行的荧光素酶实验表明，Br活性被REST选择性地降低，而Ht活性被DNMT1、MeCP2和REST降低。值得注意的是,Ht-RE1的定点突变阻止了Ncx1的REST依赖性下调。此外，在短暂大脑中动脉闭塞的8周龄雄性野生型小鼠（C57BL/6）的颞顶皮层中，DNMT1、MeCP2和REST与Ht启动子的结合增加，随之而来的DNA启动子超甲基化。侧脑室注射siREST可防止DNMT1/MeCP2结合Ht和Ncx1下调，从而减少中风引起的损伤。始终如一，在接受氧和葡萄糖剥夺加复氧的皮质神经元中，Ncx1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，使用学生的t检验，而对于两个以上的实验组，使用了一元方差分析，其次是Tukey或NewmanKeuls.P<0.05时具有统计学意义。
结论：如果这项研究的结果在人类中得到证实，可以断言DNMT1/MeCP2/REST复合物破坏可能是减少大脑中HtDNA甲基化的新药理学策略，改善中风损伤。
