Laboratory of Epileptogenesis, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland.
Laboratory of Bioinformatics, Neurobiology Center, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland.
Sci Rep. 2016 Sep 21;6:33736. doi: 10.1038/srep33736.
The aim of the present study was to examine involvement of MBD3 (methyl-CpG-binding domain protein 3), a protein involved in reading DNA methylation patterns, in epileptogenesis and epilepsy. We used a well-characterized rat model of temporal lobe epilepsy that is triggered by status epilepticus, evoked by electrical stimulation of the amygdala. Stimulated and sham-operated animals were sacrificed 14 days after stimulation. We found that MBD3 transcript was present in neurons, oligodendrocytes, and astrocytes in both control and epileptic animals. We detected the nuclear localization of MBD3 protein in neurons, mature oligodendrocytes, and a subpopulation of astrocytes but not in microglia. Amygdala stimulation significantly increased the level of MBD3 immunofluorescence. Immunoprecipitation followed by mass spectrometry and Western blot revealed that MBD3 in the adult brain assembles the NuRD complex, which also contains MTA2, HDAC2, and GATAD2B. Using chromatin immunoprecipitation combined with deep sequencing, we observed differences in the occupancy of DNA regions by MBD3 protein between control and stimulated animals. This was not followed by subsequent changes in the mRNA expression levels of selected MBD3 targets. Our data demonstrate for the first time alterations in the MBD3 expression and DNA occupancy in the experimental model of epilepsy.
本研究旨在探讨 MBD3（甲基化CpG 结合域蛋白 3）在癫痫发生和癫痫中的作用，MBD3 是一种参与读取 DNA 甲基化模式的蛋白质。我们使用了一种经过充分表征的颞叶癫痫大鼠模型，该模型由杏仁核电刺激诱发癫痫持续状态引起。刺激和假手术动物在刺激后 14 天被处死。我们发现 MBD3 转录本存在于对照和癫痫动物的神经元、少突胶质细胞和星形胶质细胞中。我们检测到 MBD3 蛋白在神经元、成熟少突胶质细胞和星形胶质细胞的一个亚群中的核定位，但不在小胶质细胞中。杏仁核刺激显著增加了 MBD3 免疫荧光的水平。免疫沉淀后进行质谱分析和 Western blot 显示，成年大脑中的 MBD3 组装了 NuRD 复合物，该复合物还包含 MTA2、HDAC2 和 GATAD2B。使用染色质免疫沉淀结合深度测序，我们观察到对照和刺激动物之间 MBD3 蛋白对 DNA 区域的占有率存在差异。这并没有导致随后所选 MBD3 靶标 mRNA 表达水平的变化。我们的数据首次证明了在癫痫实验模型中 MBD3 表达和 DNA 占有率的改变。
