Department of Neurosurgery, Zhongnan Hospital of Wuhan University, Wuhan, China.
Neuroreport. 2021 Sep 8;32(13):1147-1152. doi: 10.1097/WNR.0000000000001705.
This study aimed to explore the epigenetic regulation of activity-dependent and exon-specific brain-derived neurotrophic factor (BDNF) expression under KCl depolarization in primary cortical neurons.
We investigated BDNF exon I, exon IV and the growth arrest and DNA damage-inducible protein 45 alpha (Gadd45α) transcription levels under KCl-induced neuronal activation in postmitotic neurons. Gadd45α occupancy at BDNF I and IV promoter was measured by chromatin immunoprecipitation (ChIP) followed by quantitative PCR; DNA methylation level was checked by methylated DNA immunoprecipitation (MeDIP) followed by qPCR. In addition, lentiviral shRNA targeting Gadd45α was used to knockdown Gadd45α expression.
BDNF exon I and IV mRNA expressions were both highly induced by KCl depolarization. However, ChIP analysis demonstrated a significantly increased Gadd45α occupancy only at the BDNF P1 promotor, but not P4, which is associated with reducing DNA methylation within BDNF P1 promoter. Furthermore, after the lentiviral-mediated knockdown of Gadd45α, the increased Gadd45α occupancy at the BDNF P1 was inhibited, which was accompanying the complete blocking of the demethylation effect at P1. Nonetheless, the induction of BDNF exon I mRNA by KCl was only partially prevented by Gadd45α shRNA, indicting other mechanisms involved in regulating BDNF exon I expression.
DNA demethylation mediated by Gadd45α protein involves promoting the regulation of activity-dependent BDNF exon I expression in neurons.
本研究旨在探索原代皮质神经元在 KCl 去极化诱导的激活状态下，活性依赖和外显子特异性脑源性神经营养因子（BDNF）表达的表观遗传调控机制。
我们研究了 KCl 诱导的有丝分裂后神经元激活下，BDNF 外显子 I、IV 和生长停滞和 DNA 损伤诱导蛋白 45α（Gadd45α）的转录水平。通过染色质免疫沉淀（ChIP）结合定量 PCR 检测 Gadd45α 在 BDNF I 和 IV 启动子上的占有率；通过甲基化 DNA 免疫沉淀（MeDIP）结合 qPCR 检测 DNA 甲基化水平。此外，还使用靶向 Gadd45α 的慢病毒 shRNA 敲低 Gadd45α 的表达。
BDNF 外显子 I 和 IV 的 mRNA 表达均被 KCl 去极化高度诱导。然而，ChIP 分析显示，仅在 BDNF P1 启动子上，Gadd45α 的占有率显著增加，而在 P4 上则没有，这与 BDNF P1 启动子内的 DNA 甲基化减少有关。此外，在 Gadd45α 被慢病毒介导敲低后，BDNF P1 上 Gadd45α 占有率的增加被抑制，同时 P1 上的去甲基化作用也被完全阻断。然而，KCl 对 BDNF 外显子 I mRNA 的诱导仅部分被 Gadd45α shRNA 阻断，提示其他机制也参与了 BDNF 外显子 I 表达的调控。
Gadd45α 蛋白介导的 DNA 去甲基化参与了神经元中活性依赖的 BDNF 外显子 I 表达的调控。
